Background
==========

Traditional Chinese medicine has been practised by Chinese communities world-wide for many generations, and there is a wealth of literature information available related to the therapeutic use of this type of medicine. In recent years, there has been a global surge in the popularity of herbal/traditional medicine, and currently there is enormous interest in developing new pharmaceutical products from such resources.

Over the last decade, bis-benzylisoquinoline (BBI) and especially protoberberine alkaloids (e.g., berberine and jatrorrhizine; Fig. [1](#F1){ref-type="fig"}) have attracted considerable attention in this respect; protoberberines represent a structural class of organic cations and have been found to be predominantly distributed in several genera of the families *Ranunculaceae* and *Berberidaceae* (e.g., *Berberis, Mahonia, Coptis*). Berberine, a major representative of the protoberberine alkaloids, displays diverse biochemical and pharmacological actions while being relatively non-toxic to man; its antimicrobial activity has been demonstrated against many bacterial and fungal species \[[@B1]-[@B4]\]. The drug was subsequently screened for anti-cancer activity following evidence of antineoplastic properties \[[@B5]-[@B7]\]. It has also been shown that berberine exhibits the ability to induce apoptosis in promyelocytic leukemia HL-60 and 3T3 fibroblast cells \[[@B5],[@B8]\]. In addition, some protoberberines are highly effective as cytotoxic agents against several carcinoma such as HeLa, SVKO(3), Hep-2, primary culture from mouse embryo and human fibroblast cells \[[@B9],[@B10]\]; berberine showed consistently the highest cytotoxicity among the alkaloids tested. Only recently it has been reported that berberine possesses a dual topoisomerase I and II poisoning activity \[[@B11]-[@B13]\] and binds to double helical DNA with a high affinity \[[@B14]\]. Furthermore, computer modeling studies on protoberberine-DNA complexes suggest that these alkaloids are able to bind to the host DNA by both intercalative (through rings C and D; Fig. [1](#F1){ref-type="fig"}) and minor groove (rings A and B) binding modes \[[@B12],[@B13]\].

![Chemical structures of berberine and jatrorrhizine.](1472-6882-2-2-1){#F1}

All the above findings raise the possibility that protoberberines may be effective in deactivation of carcinogens and tumour promoters. For in vitro screening of antimutagens/anticarcinogens a variety of prokaryotic and eukaryotic micro-organisms have been used. In the present study, a new eukaryotic model, viz. green unicellular flagellate *Euglena gracilis* was chosen \[[@B15]\]. This micro-organism possesses a multigenomic system with nuclear, mitochondrial and chloroplast DNAs. The detection ability of this model is based on the preferential and selective sensitivity of the chloroplast genetic apparatus to xenobiotics resulting in elimination of the functional chloroplast from the cells. Antichloroplastic activity of mutagens is macroscopically manifested by an irreversible loss of the capability of cells to form green colonies (bleaching effect). Compounds with antimutagenic properties prevent such a bleaching of *E. gracilis* cells by mutagens / carcinogens. Previous studies on several standard mutagens and antimutagens revealed a high sensitivity and reliability of the model \[[@B16]-[@B19]\].

Thus, the aim of the present work was to study the possible antimutagenic effect of berberine and jatrorrhizine as well as of the BBI alkaloids fraction and of the overall *M. aquifolium* crude extract, on acridine orange (AO) -- induced mutagenicity in the *E. gracilis* assay.

Materials and methods
=====================

Plant material
--------------

The stem bark of *Mahonia aquifolium* (Pursh) Nutt. (*Berberidaceae*) was collected in October 1998 in the Arboretum Tesárske Mlyòany, Slovakia. Voucher specimens are deposited at the Herbarium of the Faculty of Pharmacy, Comenius University, Bratislava (No. Ma-108/8). Dried and powdered stem bark was macerated at room temperature with 62% aqueous EtOH (1:10 w/v) for 5 days and then filtered. The filtrate (crude *M. aquifolium* extract) was stored in a refrigerator until use in the biological assay. The BBI and the protoberberine alkaloid fractions were separated and purified using standard procedures as described previously \[[@B20]\].

Berberine \[CAS 2086-83-1\] and jatrorrhizine \[CAS 3621-38-3\], the main quaternary protoberberine alkaloids were isolated as iodides from the protoberberine fraction and identified by direct comparison of their physical and spectral properties with the literature data \[[@B20]\].

HPLC analysis of the BBI alkaloids was performed according to the procedure reported earlier \[[@B20]\]. Totally 6 tertiary BBI alkaloids (oxyacanthine, aromoline, baluchistine, berbamine, obamegine and aquifoline) were positively identified by referring to authentic compounds described earlier \[[@B20]\]. The identification was made on the basis of their UV absorption spectra and retention time.

Microorganism
-------------

*Euglena gracilis* (strain Z) was obtained from S.H. Hutner, Haskins Laboratory, Pace University, New York, NY, USA and maintained on Cramer-Myers (CM) medium \[[@B21]\] under static conditions at 27°C and with permanent lighting (16.4 W/m^2^).

Chemicals
---------

Acridine orange \[CAS 65-61-2\] was purchased from Merck, Darmstadt, Germany. Stock solutions of AO, berberine, jatrorrhizine, and BBI alkaloids were prepared by dissolving them in distilled water.

Mutagenicity assay
------------------

*E. gracilis* cells diluted to concentration 8 × 10^5^ cells/ml CM medium were used in the experiments. Aliquots (0.2 ml) of the cell suspension were dispensed into test tubes, along with 2.3, 11.4 or 22.8 μM AO, indicated concentrations of berberine (0.03, 0.06, 0.15, 0.30, 0.45 μg/ml), jatrorrhizine (0.72, 1.44, 14.4, 28.8, 43.2 μg/ml), BBI alkaloids (0.72, 1.44, 14.4, 28.8,43.2 μg/ml) or the *M. aquifolium* extract (5.5, 11.0, 110,220,330 μg/ml), and at last the content of the incubation mixture was completed to a final volume (5 ml) by the addition of CM medium. Following a 24-h co-treatment, the cells were centrifuged at 3000 rpm for 20 min, the resultant pellet suspended in fresh CM medium and again centrifuged. After the pellet was resuspended in fresh CM medium, the cells were finally cultivated 14 days at 27°C under permanent illumination (16.4 W/m^2^). The experiments were repeated in three independent series. Just before the counting, the movement of *E. gracilis* was stopped by adding a drop of EtOH and the counting of green and white (mutant) colonies was carried out in a Bürker chamber under a microscope. The viability of the *Euglena* cells was estimated by counting the total white mutants in the presence of mutagen (positive control) compared to the number of the spontaneous white mutants in the absence of mutagen (negative control). We defined the relative decrease of the bleaching as the antimutagenic potency (AP) which was calculated by the formula

![](1472-6882-2-2-i1.gif)

where B~0~ is the AO-induced *E. gracilis* bleaching (%) and B~r~ is the AO-induced and antimutagen-reduced *E. gracilis* bleaching (%).

The statistical significance of all the calculated values were determined by paired Student\'s t-test. The values represent the means ± standard deviation (SD).

Results and discussion
======================

The mutagenic effect of AO on *E. gracilis* was tested in three concentrations, 2.3, 11.4 and 22.8 μM, which induced, respectively, 43 ± 2 %, 58 ± 2 % and 65 ± 3 % of white mutant cells. The AO concentrations were chosen so as to ensure no significant change in the viability of cells compared to the negative controls (*Euglena* cells in the absence of the mutagen). Similarly, no spontaneous white mutants were found in any sets of positive controls.

The dose-dependent inhibitory effect of the crude *Mahonia* extract, BBI alkaloids fraction, berberine and jatrorrhizine on the mutagenicity of AO applied at the above 3 concentrations is displayed in Figs. [2](#F2){ref-type="fig"},[3](#F3){ref-type="fig"},[4](#F4){ref-type="fig"},[5](#F5){ref-type="fig"}. As shown in Figs. [2](#F2){ref-type="fig"} and [3](#F3){ref-type="fig"}, the plot of the percentage of bleached mutants vs. concentration for the *Mahonia* extract and the BBI fraction respectively displays typical curving even though over different concentration ranges. The lowest concentration that causes statistically significant (p \< 0.05) reduction in the percentage of white colonies (as compared to the positive control) is 0.7 and 11.0 μg/ml for the *Mahonia* extract and the BBI alkaloids, respectively. For both samples, the decrease of the percent proportion of mutant colonies gradually continued with further increasing the concentration; however, the antimutagenic efficiency was much higher for the BBI alkaloids sample, for which the bleaching percentage (corresponding to the AO 2.3 μM curve) reaches \~5% level for the highest concentration tested.

![A plot of percentage of bleached colonies of *E. gracilis* vs. concentration of *M. aquifolium* extract at 3 concentrations of acridine orange (AO). Symbols and brackets denote means ± standard deviation of 3 independent determinations.](1472-6882-2-2-2){#F2}

![A plot of percentage of bleached colonies of *E. gracilis* vs. concentration of BBI alkaloid fraction at 3 concentrations of acridine orange (AO). Symbols and brackets have the same meaning as in Fig. [2](#F2){ref-type="fig"}.](1472-6882-2-2-3){#F3}

![A plot of percentage of bleached colonies of *E. gracilis* vs. concentration of berberine at 3 concentrations of acridine orange (AO). Symbols and brackets have the same meaning as in Fig. [2](#F2){ref-type="fig"}.](1472-6882-2-2-4){#F4}

![A plot of percentage of bleached colonies of *E. gracilis* vs. concentration of jatrorrhizine at 3 concentrations of acridine orange (AO). Symbols and brackets have the same meaning as in Fig. [2](#F2){ref-type="fig"}.](1472-6882-2-2-5){#F5}

As to the protoberberines berberine and jatrorrhizine, which are of prime interest here, the concentration dependence of the bleaching effect of jatrorrhizine (Fig. [5](#F5){ref-type="fig"}) is markedly similar to those observed for the above two samples. In contrast, the behaviour of berberine is completely different; as shown in Fig. [4](#F4){ref-type="fig"}, berberine chloride suddenly reduced the number of AO-induced mutant cells (to ca 5% for AO 2.3 μM) at concentration 0.03 μg/ml but further increase of the concentration did not cause progressive reduction of the bleaching activity or even, in the case of the AO 22.8 μM series, the number of white colonies increased slightly but significantly with respect to that observed for the most effective concentration (p \< 0.05). For berberine, the antimutagenic potency (AP) was also calculated using the formula given at the end of the Material and methods section and the results are summarised in Table [1](#T1){ref-type="table"}. As can be seen, berberine exhibited significant antimutagenic potency (\>80%) almost over the whole concentration range tested when the concentration of the mutagen (AO) was 2.3 and 11.4 μM while for the increased AO concentration (22.8 μM) the antimutagenic potential of berberine was much less effective (AP \< 55%).

::: {#T1 .table-wrap}
::: {.caption}
######

Antimutagenic potency (AP) \[%\] of berberine in reducing the acridine orange (AO)-induced bleaching of *Euglena gracilis* cells.
:::

  mutagen   concentration of berberine \[μg/ml\]
  --------- -------------------------------------- ---------- ---------- ---------- ----------
  2.3       89 ± 2.2                               88 ± 5.3   90 ± 6.2   86 ± 1.7   78 ± 6.5
  11.4      80 ± 3.0                               86 ± 4.2   84 ± 1.6   83 ± 1.1   82 ± 3.6
  22.8      54 ± 3.5                               54 ± 1.8   49 ± 4.1   38 ± 2.3   36 ± 1.7
:::

Although the BBI and protoberberine alkaloids studied in this laboratory exhibit a broad spectrum of biological activities, for most of them the molecular mechanism of action is still unclear. In the case of the BBI alkaloids, one possible mechanism of their antimutagenic activity could be based on the reports that tetrandrine and other BBI alkaloids are excellent scavangers of reactive oxygen species (ROS) such as singlet oxygen and/or superoxide anion radical \[[@B22]\] since it is well documented that that ROS play a central role in multistage mutagenesis and carcinogenesis \[[@B23],[@B24]\]. However, the potential mechanism based on the ability of BBIs to prevent oxidative damage of DNA by ROS is unlikely since the ability of AO to produce ROS in a redox system has never been published. Instead, the antimutagenic potency of the BBI fraction is more likely linked to modulation of DNA at the transcriptional level. Recently, the role of eukaryotic transcription factors, NF-κB and AP-1, has been highlighted in mutagenesis and carcinogenesis \[[@B25],[@B26]\] and tetrandrine is known to inhibit the activation of NF-κB in the alveolar macrophage \[[@B22]\]. Nevertheless, the identification of the ultimate mechanism of the antimutagenic effect of the BBI alkaloids awaits further study.

As described above, the significant antimutagenic potency against AO was observed for the two protoberberines studied herein. The potency of jatrorrhizine alone is comparable to that found for the overall BBI fraction and the potency of berberine is even by more than 2 orders of magnitude higher. To our knowledge, the ability of protoberberines to reduce the mutagenicity of environmental chemicals has not yet been reported so that the mechanism of their antigenotoxic action was not discussed. However, the cytotoxic activity of berberine and its analogues has been attributed to DNA topoisomerase I (TOPO-I) and II (TOPO-II) poisoning \[[@B11]-[@B13]\]; recently, a mechanistic model \[[@B12]\] for TOPO-I poisoning by protoberberines was developed according to which berberine binds to DNA and TOPO-I at the interface of the binary complex DNA -- TOPO-I in such a manner that rings C and D (Fig. [1](#F1){ref-type="fig"}) intercalate into the DNA helix, while rings A and B protrude out of the helix interior into the minor groove, where they are accessible to interact with specific functional groups on the enzyme. Thus, both drug-DNA and drug-enzyme interactions contribute to the overall affinity (potency) of the drug. In this light it is reasonable to suggest that the antimutagenicity of berberine against the genotoxic effect of AO on plastid DNA is exerted by a similar, if not identical, mechanism. A comparison of the antimutagenic potency of the two protoberberines tested here supports this notion. The only difference between berberine and jatrorrhizine (Fig. [1](#F1){ref-type="fig"}) is restricted to the substitution on the A ring (2,3-methylenedioxy in berberine vs. 2-methoxy-3-hydroxy substitution in jatrorrhizine), i.e. to the portion of the molecule which is involved in interactions with TOPO-I. Thus, insertion of the protoberberine molecule between the base pairs of DNA prevents the interaction of the AO mutagen at the same binding site as AO is known as a frameshift mutagen due to strong intercalative binding. A similar detrimental effect of the free hydroxy group on the TOPO-II poisoning activity has also been observed for berberrubine \[[@B13]\]. In this context it is important to note that camptothecin, a natural product currently in clinical use for the treatment of a variety of cancers \[[@B12],[@B25]\], has a pharmacological profile very similar to that of protoberberines; its antineoplastic and antimutagenic activity was demonstrated against many organic mutagens and also the DNA-TOPO-II complex was identified as its molecular target \[[@B26]\]. It is therefore reasonable to consider that the drug-induced poisoning of TOPO enzymes may be of general importance for anticancer and antimutagenic drugs.

Conclusions
===========

In summary, the data presented here showed that the BBI alkaloid fraction and especially the protoberberines, jatrorrhizine and berberine, inhibited the AO-induced mutagenicity in the *E. gracilis* assay. Berberine demonstrated significant antimutagenic activity against the chloroplast damaging effects of AO with a high potency. The protective effects of the latter compound against the chemical carcinogen suggest its potential chemopreventive ability, which is under investigation using other bacterial tests as well as animal tumour models.

Competing interests
===================

None declared.

Pre-publication history
=======================

The pre-publication history for this paper can be accessed here:

<>

Acknowledgements
================

This research was supported by the Grant Agency of the Slovak Republic, Projects No. 1/7359/20, 1/8216/01, 1/8215/01 and 2005.


Background
==========

The Nudix hydrolase family comprises enzymes that hydrolyse predominantly the diphosphate (pyrophosphate) linkage in a variety of nucleoside triphosphates, dinucleoside polyphosphates, nucleotide sugars and nucleotide cofactors having the general structure of a **nu**cleoside **di**phosphate linked to another moiety, **X**\[[@B1],[@B2]\]. They are found in archaea, eubacteria, animal, plants, and fungi and all possess the Nudix box sequence signature motif Gx~5~Ex~5~ \[UA\]xREx~2~EExGU (where U is an aliphatic hydrophobic amino acid) \[[@B1],[@B3]\]. The proposed functions of this family are to eliminate potentially toxic nucleotide metabolites from the cell and to regulate the concentrations of nucleotide cofactors and signalling molecules for optimal cell growth and survival.

The number of genes encoding Nudix hydrolases varies widely, from zero in *Mycoplasma genitalium*\[[@B4]\] to 22 in *Deinococcus radiodurans*\[[@B5],[@B6]\]. This variation presumably reflects the growth or environmental adaptability, stress tolerance and metabolic capacity of the different organisms. The Nudix hydrolases thus offer an ideal system with which to study the evolution of a largely inessential protein family and its contribution to the individual biology of an organism. Understanding such variation requires a combination of detailed biochemical, genetic and cellular studies to reveal the individual functions of family members within the set in any given system. In the case of multicellular eukaryotes, the nematode *Caenorhabditis elegans* offers a genetically amenable model system with which to carry out such studies. There are 11 members of the Nudix hydrolase family in *C. elegans*. So far only two of these have been characterized -- a diadenosine tetraphosphate pyrophosphohydrolase (the orthologue of human NUDT2) \[[@B7]\] and an NADH diphosphatase \[[@B8]\]. Sequence comparisons would predict the existence of an ADP-sugar diphosphatase (NUDT5 orthologue) \[[@B9]\], an ADP-ribose diphosphatase (NUDT9 orthologue) \[[@B10]\], a diphosphoinositol polyphosphate pyrophosphohydrolase (NUDT3/4 orthologue) \[[@B11]\], two probable coenzyme A diphosphatases, one of which is highly similar to the mouse Nudt7 CoA diphosphatase \[[@B12]\], and 4 proteins of unknown function, including one with a strong similarity to the *Saccharomyces cerevisiae* PSU1/DCP2 protein \[[@B13],[@B14]\] and another similar to the developmentally-regulated mouse RP2 protein \[[@B15]\].

Recent characterization of the *S. cerevisiae* NADH and CoA diphosphatases \[[@B16],[@B17]\] and the mouse Nudt7 CoA diphosphatase \[[@B12]\] has revealed that they are located in peroxisomes. The function of these peroxisomal enzymes may be to regulate the concentration of these essential nucleotide cofactors for peroxisomal metabolism or, by analogy with the *E. coli* MutT 8-oxo-dGTPase, to eliminate toxic modified cofactor metabolites from the highly oxidizing peroxisomal environment. In order to investigate these possibilities in the *C. elegans* model system, we have cloned and characterised the putative *C. elegans* Y87G2A.14 CoA diphosphatase and shown that it displays the expected enzymatic activities and that it appears to be targetted to peroxisomes by a C-terminal PTS1 targeting signal.

Results and discussion
======================

Cloning, expression and purification of Y87G2A.14
-------------------------------------------------

The *C. elegans* Y87G2A.14 gene encodes a 234 amino acid protein with an expected molecular weight of 26,601 Da. It was amplified by PCR from a *C. elegans* cDNA library. The PCR fragment was inserted into the pET-32b(+) expression vector and the nucleotide sequence of the insert was determined to be exactly the same as that submitted to GenBank under accession no. CAB54476. The recombinant plasmid pETY87G2A.14 was then used to transform *E. coli* BL21 (DE3) cells to generate a His-tagged thioredoxin fusion protein with an expected molecular mass of 43,731 Da. When the Trx-Y87G2A.14 fusion protein was expressed at 37°C, it was confined to inclusion bodies, so the induction temperature was decreased to 25°C to enhance protein solubility. As the expression level was low at this temperature, the induction time was increased to 8 h. These conditions markedly increased the solubility of Trx-Y87G2A.14 which was then purified from the soluble fraction (Fig [1](#F1){ref-type="fig"}, lane 2) to apparent homogeneity on NiCAM™-HC resin (Fig [1](#F1){ref-type="fig"}, lane 3). To determine the molecular weight of the Y87G2A.14 protein itself, the Trx-Y87G2A.14 fusion was cleaved with thrombin, which generated Y87G2A.14 with an apparent molecular weight of 27 kDa (expected molecular weight, 29,807 Da) and thioredoxin (15 kDa, Fig [1](#F1){ref-type="fig"}, lane 4).

![**Purification and cleavage of Trx-Y87G2A.14 fusion protein.** Samples were analysed by SDS-PAGE (15% polyacrylamide) and stained with Coomassie Brilliant blue R 250. Lane 1, 2 μg protein standards: bovine serum albumin (66 kDa), ovalbumin (45 kDa), glyceraldehyde 3-phosphate dehydrogenase (36 kDa), carbonic anhydrase (29 kDa), trypsinogen (24 kDa), soybean trypsin inhibitor (20 kDa) and α-lactalbumin (14.2 kDa); lane 2, soluble cell extract of BL21 (DE3) cells transformed with recombinant plasmid pETY87G2A.14 and induced with 1 mM IPTG for 8 hours at 25°C before applying to a column of NiCAM™-HC resin ; lane 3, 3 μg purified Trx-Y87G2A.14 fusion protein; lane 4, 3 μg purified Trx-Y87G2A.14 fusion protein after cleavage with thrombin.](1471-2091-3-5-1){#F1}

Substrate specificity and product analysis
------------------------------------------

Purified Trx-Y87G2A.14 was inactive towards the following nucleotides when assayed at a fixed concentration of 0.5 mM: NADH, NAD^+^, NDP-sugars, 5\'-(d)NTPs, 5\'-NDPs, 5\'-NMPs and diadenosine polyphosphates. High activity was found with CoA and its derivatives. HPLC analysis of CoA hydrolysis by Trx-Y87G2A.14 showed that the enzyme was a CoA diphosphatase, cleaving the diphosphate linkage in CoA to yield adenosine 3\',5\'-bisphosphate (3\',5\'-ADP) and 4\'-phosphopantetheine (Fig [2](#F2){ref-type="fig"}).

![**Identification of reaction products of CoA hydrolysis.** Reaction mixtures containing 0.5 mM CoA were incubated at 37°C for 20 min with or without 0.1 μg Trx-Y87G2A.14 fusion protein and the products separated by HPLC as described in Materials and methods. Without enzyme (\-\-\-\-\--), with enzyme ([       ]{.ul}), gradient (--- --- --- --- ---). Positions of authentic standards are indicated.](1471-2091-3-5-2){#F2}

Reaction requirements and kinetic parameters
--------------------------------------------

Trx-Y87G2A.14 displayed optimal activity with 0.5 mM CoA as a substrate at pH 9.5. A divalent metal ion was absolutely required for activity, with optimal activity at 5 mM MgCl~2~. In common with all other Nudix hydrolases tested, fluoride was a strong inhibitor with a *K*~i~ value of approximately 3 μM (results not shown). *K*~m~, and *k*~cat~ values for CoA, CoA esters and oxidized CoA were calculated by non-linear regression from data obtained by HPLC analysis (Table [1](#T1){ref-type="table"}). A graphical example of the data for CoA in the form of a hyperbolic plot (Fig [3a](#F3){ref-type="fig"}) and double reciprocal plot (Fig [3b](#F3){ref-type="fig"}) show that the enzyme obeys simple Michaelis-Menten kinetics. The *k*~cat~*/ K*~m~ ratios show that the enzyme prefers reduced forms of CoA to oxidized CoA (Table [1](#T1){ref-type="table"}) with CoA itself the best substrate of those tested.

![**Lineweaver-Burk and Michaelis-Menten (inset) plots for the hydrolysis of CoA.** Reaction mixtures containing various concentrations of CoA (0.05--0.7 mM) were incubated at 37°C for up to 20 min with 0.1 μg Trx-Y87G2A.14 fusion protein. Initial rates of hydrolysis were determined after separation of the products by HPLC as described in Materials and methods.](1471-2091-3-5-3){#F3}

::: {#T1 .table-wrap}
::: {.caption}
######

Kinetic parameters for the hydrolysis of CoA and CoA derivatives by Trx-Y87G2A.14 fusion protein
:::

  Substrate                     *K*~m~ (mM)   *k*~cat~ (s^-1^)   *k*~cat~ / *K*~m~ (10^3^M^-1^.s^-1^)
  ----------------------------- ------------- ------------------ --------------------------------------
  CoA                           0.22          13.8               64
  Acetyl-CoA                    0.23          11.8               51
  Succinyl-CoA                  0.24          12.7               53
  3-Hydroxymethylglutaryl-CoA   0.20          10.5               54
  CoASSCoA                      0.32          2.9                9.1

Data were collected using the HPLC assay described in Materials and methods.
:::

Subcellular localization
------------------------

Y87G2A.14 has the C-terminal tripeptide sequence SKI. This conforms to the pattern typical of PTS1 peroxisomal targeting signals found in many peroxisomal matrix proteins, suggesting that Y87G2A.14 may be targeted to these organelles \[[@B18],[@B19]\]. However, possession of a potential PTS1 sequence is not always sufficient on its own to result in peroxisomal targeting and other elements of the protein sequence may also be involved. Since targeting of animal peroxisomal proteins expressed in yeast has often been observed, yeast cells were transformed with expression plasmids encoding C-terminal or N-terminal fusions of Y87G2A.14 to yeast-enhanced green fluorescent protein (yEGFP) in order to determine the subcellular location of Y87G2A.14. The cells were then examined by confocal microscopy. Cells transformed with pY87G2A.14-yEGFP, in which the C-terminus of Y87G2A.14 is fused to the N-terminus of yEGFP, showed a diffuse, cytoplasmic fluorescence with no clear subcellular localization (Fig [4a](#F4){ref-type="fig"}). In contrast, cells transformed with pyEGFP-Y87G2A.14, in which the C-terminal tripeptide SKI is free to act as a targeting signal showed the clear punctate fluorescence that is indicative of yeast peroxisomes \[[@B16]\] (Fig [4b](#F4){ref-type="fig"}). The identity of SKI as the targeting signal was confirmed by transformation of cells with pyEGFP-Y87G2A.14Δ SKI, in which the C-terminal tripeptide was deleted during construction. This again showed a diffuse, cytoplasmic, fluorescence (Fig [4c](#F4){ref-type="fig"}). Together, these results strongly suggest that Y87G2A.14 is targeted to peroxisomes by its C-terminal tripeptide, SKI.

![**Subcellular localization of Y87G2A.14 by fluorescence confocal microscopy.** yEGFP fluorescence of yeast cells transformed with (a) pY87G2A.14-yEGFP; (b) pyEGFP-Y87G2A.14 and (c) pyEGF-Y87G2A.14ΔSKI](1471-2091-3-5-4){#F4}

Conclusions
===========

On the basis of its sequence, the *C. elegans* Y87G2A.14 gene product was predicted to be a peroxisomal coenzyme A diphosphatase. In addition to the Nudix motif, Y87G2A.14 possesses the PROSITE UPF0035 motif (Fig [5](#F5){ref-type="fig"}), which we have previously suggested confers a specificity for coenzyme A and its derivatives \[[@B12]\], and a C-terminal tripeptide, SKI, that conforms to the pattern typical of PTS1 peroxisomal targeting signals. The experiments described here confirm these predictions. Fig [5](#F5){ref-type="fig"} shows a multiple sequence alignment of the motif-containing region of Y87G2A.14 with related sequences from other organisms. Those marked with a tick have been experimentally shown to be coenzyme A diphosphatases \[[@B6],[@B12],[@B17]\]. In most cases, higher organisms possess two related sequences, e.g. mouse Nudt7 and Nudt8, one of which encodes a peroxisomal enzyme (e.g. Nudt7). However, *S. cerevisiae* has only one sequence containing the UPF0035 motif while *Arabidopsis thaliana* has three, and the second of the two *Drosophila melanogaster* sequences, RH61317, is currently only represented in GenBank by a single expressed sequence tag, so its status is still questionable. For the peroxisomal enzymes, either a putative C-terminal PTS1 or an N-terminal PTS2 targeting signal is present. Interestingly, in each case, the putative PTS2 signal is contained within or near a predicted mitochondrial targeting or chloroplast transit peptide sequence \[[@B20]-[@B22]\], suggesting a possible dual location for these proteins. Such a possibility has not yet been experimentally observed; however, mutation of a glutamate five residues to the C-terminal side of the PTS2 of rat peroxisomal 3-ketoacyl-CoA thiolase to a neutral or basic amino acid has been shown to result in partial mitochondrial targeting, suggesting that the negative charge on glutamate may normally block translocation to the mitochondria \[[@B23]\]. Whether or not a system exists *in vivo* to regulate dual targeting is clearly a topic requiring further investigation. The non-peroxisomal sequences provide no clear indication of possible subcellular location, hence they are likely to be cytoplasmic. Given the existence of mitochondrial, peroxisomal and cytoplasmic pools of CoA and CoA esters \[[@B24]\], it would not be surprising to find CoA diphosphatase activity in all these locations. However, the precise substrate specificities of the \"cytoplasmic\" activities remain to be determined.

![**Partial sequence alignment of Y87G2A.14 and related sequences.** The partial sequence of Y87G2A.14 containing the UPF0035 and Nudix motifs (arrowed) was aligned using the Clustal W program with related sequences from other organisms retrieved from a BLAST search. Organisms and database accession numbers are: *Caenorhabditis elegans* Y38A8.1, Q23236; *Homo sapiens* NUDT7, XP_058753; *H. sapiens* NUDT8, AI743601; *Mus musculus* Nudt7, Q99P30; *M. musculus* Nudt8, AK009700; *Drosophila melanogaster* CG11095, Q9VY79; *D. melanogaster* RH61317, BI631687; *Schizosaccharomyces pombe* YDH5, Q92350; *S. pombe* YDZA, 013717; *Ambidopsis thaliana At2g33980*, 022951; *A. thaliana At1g28960*, Q9SHQ7; *A. thaliana* At5g45940, BAB09322; *Saccharomyces cerevisiae* PCD1, Q 12524; *Escherichia coli* YeaB, P43337; *Deinococcus radiodurans* DR1184, Q9RV46. Sequences encoding experimentally confirmed CoA diphosphatases are marked with a tick. Columns on the right indicate whether the full sequence contains a putative peroxisomal targeting signal (PTS1 or PTS2) and/or a putative mitochondrial targeting peptide (mTP) or chloroplast transit peptide (cTP).](1471-2091-3-5-5){#F5}

Regarding the possible function of these enzymes in general, and the *C. elegans* peroxisomal enzyme in particular, a recent functional genomic analysis by RNA-mediated interference of *C. elegans* chromosome I, on which the Y87G2A.14 gene is located, revealed no phenotype in relation to growth, survival, fecundity or morphology when the expression of Y87G2A.14 was ablated \[[@B25]\]. This would indicate that, within the limitations of RNAi, the CoA diphosphatase activity of Y87G2A.14 is not essential. However, now that the biochemical properties of this protein have been established, a more detailed and targeted biochemical analysis can be undertaken that should reveal its cellular function and benefit to the organism.

Nudix hydrolases are believed to regulate the concentrations of nucleotides for optimal cell performance and also to eliminate potentially toxic nucleotide metabolites from the cell. With regard to regulation, CoA diphosphatase activity is associated with the 400 kDa CoA synthesizing protein complex from *S. cerevisiae*, in which it forms part of an alternative pathway for CoA biosynthesis that differs from the principal route of 3\'-dephospho-CoA and CoA synthesis by this complex \[[@B26]\]. This CoA/4\'-phosphopantetheine cycle involves hydrolysis of CoA to 3\',5\'-ADP and 4\'-phosphopantetheine, which then reacts with ATP to give 3\'-dephospho-CoA then CoA. Whether such a pathway operates in peroxisomes and whether the *C. elegans* Y87G2A.14 protein is involved remain to be established. With regard to the elimination of toxic nucleotide metabolites, the 13-fold higher *k*~cat~ / *K*~m~ ratio for oxidized CoA (CoASSCoA) compared to CoA for the *S. cerevisiae PCD1* CoA diphosphatase previously suggested to us that this enzyme might preferentially remove non-functional and potentially toxic oxidized CoA and CoA esters from within the oxidizing environment of the peroxisomes \[[@B17]\]. However, neither the mouse Nudt7 \[[@B12]\] nor the *C. elegans* Y87G2A.14 proteins show this preference. Nevertheless, the potential production of adenine ring-oxidized derivatives of CoA by reactive oxygen species generated in the peroxisomes analogous to the 2-oxo-dATP and 8-oxo-dATP substrates of the mammalian MTH1 Nudix hydrolase \[[@B27]\] suggests that such species could be more relevant substrates for peroxisomal CoA diphosphatases *in vivo*. The amenability of *C. elegans* to studies of cellular and molecular stress will allow the question of the biological function of these enzymes to be addressed.

Materials and methods
=====================

*S. cerevisiae* strain BY4741 (*MAT a; his3D1; leu2D0; met15D0; ura3D0*) was from Research Genetics. Calf intestinal alkaline phosphatase, yeast inorganic pyrophosphatase, *Eco*R1 and *Bam*H1 were from Roche while *Bsp*H1 (*Pag1*) was from Helena Biosciences. *Pfu* DNA polymerase was from Stratagene. All other chemicals and nucleotides were from BDH or Sigma. The *E. coli* expression vector pET-32b(+) was from Novagen and the yeast-enhanced green fluorescent protein (yEGFP) fusion vectors pUG35 and pUG36 were a gift from J.H. Hegemann, Institute of Microbiology, University of Düsseldorf, Germany. The *C. elegans* cDNA library was prepared from adult nematodes by H. M. Abdelghany, School of Biological Sciences, University of Liverpool, U.K.

Cloning of Y87G2A.14 from C.elegans
-----------------------------------

A cDNA corresponding to the *C. elegans* Y87G2A.14 gene on chromosome 1 (GenBank accession no. CAB54476) was amplified from a cDNA library by PCR using as forward and reverse primers 5\' GCAAATCATGAAGTGTGTGGTTAGCCGAGCTG 3\' and 5\' TAAATGAATTCACTAAATTTTGGATTTCGGTTC 3\' respectively. These primers provided a *Bsp*H1 restriction site at the start of the amplified gene and an *Eco*R1 site at the end. After amplification with *Pfu* DNA polymerase, the DNA was recovered by phenol/chloroform extraction and digested with *Bsp*H1 and *Eco*R1. The digest was gel-purified and the restriction fragment ligated into the *Nco1* and *Eco*R1 restriction sites of pET-32b(+) as both *Bsp*H1 and *Nco1* form compatible ends with each other. The resulting construct, pETY87G2A.14, yielded Y87G2A.14 downstream of the 109-amino acid thioredoxin (Trx) fusion and His-tag and S-tag sequences under the control of an IPTG-inducible promoter. The structure of the insert was confirmed by sequencing. The construct was propagated by transformation of *E. coli* XL1-Blue cells.

Expression of Y87G2A.14 in E. coli and protein purification
-----------------------------------------------------------

*E. coli* strain BL21(DE3) was transformed with pETY87G2A.14. A single colony was picked from an LB agar plate containing 50 μg/ml ampicillin and inoculated into 10 ml LB medium containing 50 μg/ml ampicillin and incubated at 37°C. When the cells reached an A~600~ of 0.5, they were transferred to 1 litre of fresh LB medium containing 50 μg/ml ampicillin and grown to an A~600~ of 0.3 at 37°C, then transferred to an incubator at 25°C. Isopropyl-1-thio-β-D-galactopyranoside (IPTG) was added to 1 mM at an A~600~ of 0.8, and the cells induced for 8 h. The induced cells (4 g) were harvested, washed and resuspended in 20 ml breakage buffer (50 mM Tris-HCl, pH 8.0, 50 mM NaCl, 1 mM DTT). The cell suspension was sonicated and the cell lysate was then cleared by centrifugation at 10,000 × *g* at 4°C for 15 min. The supernatant was applied to a 15 × 50 mm column of NiCAM™-HC resin (Sigma) equilibrated with 50 mM Tris-HCl, pH 8.0, 0.5 M NaCl, 1 mM 2-mercaptoethanol at a flow rate of 0.5 ml/min. After eluting the unbound proteins, a linear gradient of 0--40 mM histidine in the same equilibration buffer was applied at flow rate of 1 ml/min and 1 ml fractions collected and analysed by SDS-PAGE. Those containing pure Trx-Y87G2A.14 fusion protein were collected, dialysed overnight at 4°C against 1 litre of 20 mM Tris-HCl, pH 8.0, 50 mM NaCl, ImM DTT and then concentrated by ultraflltration (Amicon) and stored at -20°C in 50% glycerol.

Enzyme assays
-------------

Potential substrates were screened by measuring the Pi released after nucleotide hydrolysis in presence of inorganic pyrophosphatase or alkaline phosphatase \[[@B9]\]. The standard assay (200 μl) for phosphodiester substrates was incubated at 37°C for 30 min and contained 50 mM l,3-bis \[tris(hydroxymethyl)-methylamino\]propane-HCl (BisTrisPropane-HCl), pH 8.0, 5 mM MgCl~2~, 1 mM DTT, 0.5 mM substrate, 0.1 μg of Trx-Y87G2A.14 fusion protein and 0.5 μg (1 unit) alkaline phosphatase. Assays with phosphomonoester substrates were as above, except 0.5 μg (100 mU) inorganic pyrophosphatase was used instead of alkaline phosphatase. The Pi released in each case was measured colorimetrically.

Chromatographic analysis
------------------------

Kinetic parameters and reaction products generated from hydrolysis of CoA and its derivatives were measured by high performance anion-exchange chromatography. The reaction mixtures (100 μl) contained 50 mM BisTrisPropane-HCl, pH 9.5, 5 mM MgCl~2~, 1 mM DTT (in cases of substrates requiring reducing conditions), substrate in the range of 0.05--0.7 mM, (0.1--1 mM in the case of oxidized CoA) and were incubated at 37°C for up to 20 min (during which time the reaction rates remained linear) with 0.1 μg Trx-Y87G2A.14 fusion protein. A 90 μl sample of each reaction mixture was applied to a 1 ml Resource-Q column (Amersham Pharmacia Biotech) equilibrated with 0.045 M CH~3~COONH~4~ (pH 4.6, adjusted with H~3~PO~4~), and eluted with a linear gradient from 0 to 0.45 M NaH~2~PO~4~ (pH 2.7 adjusted with CH~3~COOH) for 10 min at a flow rate of 2 ml/min \[[@B28]\]. Elution was monitored at 259 nm and peaks identified with the aid of standards and quantified by area integration.

GFP fusion constructs and subcellular localization
--------------------------------------------------

Expression plasmids encoding C-terminal and N-terminal fusions of Y87G2A.14 to yeast-enhanced green fluorescent protein (yEGFP) \[[@B29]\] were constructed by amplification of the coding region of Y87G2A.14 from *C. elegans* cDNA by PCR using the same forward primer 5\' CGACGGATCCATGAAGTGTGT 3\' and one of the reverse primers 5\' TAAATGAATTCACTAAATTTTGGATTTCGGTTC 3\', 5\' CACTAAGAATTCTATTTCGGTTCAAATTTCCTACTTGC 3\', or 5\' GCTCGAATGAATTCAATTTTGGATTTCGGTTC 3\' to give PCR products \"C\", \"CΔSKI\" or \"N\" respectively. These primers provided a *Bam*H1 restriction site at the start of the amplified gene and *Eco*R1 sites at the end. PCR products \"C\" and \"CΔSKI\" were cloned as C-terminal fusion proteins to yEGFP, while PCR product \"N\" with a deletion of the Y87G2A.14 termination codon was cloned as an N-terminal fusion to yEGFP. After amplification with *Pfu* DNA polymerase, the DNA products were recovered by phenol/chloroform extraction and digested with *Bam*H1 and *Eco*R1. The digested PCR products \"C\", and \"CΔSKI\" were gel purified and the restriction fragments ligated between the *Bam*H1 and *Eco*R1 restriction sites of pUG36 (yEGFP-C-fusion) to give pyEGFP-Y87G2A.14 and pyEGFP-Y87G2A.14ΔSKI respectively. The digested PCR product \"N\" was gel purified and the restriction fragment ligated between the *Bam*H1 and *Eco*R1 restriction sites of pUG35 (yEGFP-N-fusion) to give pY87G2A.14-yEGFP. The structures of the inserts were confirmed by sequencing. The plasmids were propagated by transformation of *E. coli* XL 1-Blue cells. For microscopy, *S. cerevisiae* strain BY4741 was transformed with pyEGFP-Y87G2A.14, pyEGFP-Y87G2A.14ΔSKI or pY87G2A.14-yEGFP and grown on solid SC-Ura medium containing 2% glucose. Cells were viewed by conventional and confocal fluorescent microscopy on a Zeiss LSM510 confocal microscope with a 100 × 1.4 NA objective.

Other methods
-------------

Protein concentrations were estimated by the Coomassie blue binding dye-based colorimetric method using equal weights of bovine serum albumin, conalbumin, cytochrome c and myoglobin as standards \[[@B30]\].

Acknowledgements
================

S.R. AbdelRaheim is the recipient of a postgraduate scholarship from the Egyptian government. The authors are grateful to D.G. Spiller for assistance with the confocal microscopy. The authors thank J.H. Hegemann and H.M. Abdelghany for their kind gift of materials.


Background
==========

*Lathyrus sativus*, belonging to the family of leguminoceae, is a popular drought resistant legume plant whose grains serve as food in the drought prone areas of Asia and Africa. The 2,3 α-β-Diamino propionic acid (DAP) present in *L. sativus* seeds when consumed causes Lathyrism in humans, affecting either the nervous system (Neurolathyrism) or bone formation (Osteolathyrism), \[[@B1]\]. Medical scientists evince keen interest in understanding the causes of neurolathyrism as a model for neurodegenerative diseases striking the more affluent sections of the human population. The scourge of neurolathyrism, known since ancient times, mainly affects the poor rural classes in developing countries. Though different methods such as boiling in water and development of DAP free cultivars \[[@B2]\], had been tried, as yet no effective method has been developed for safe consumption by the famine hit, poverty ridden, malnourished hungry millions in the drought prone areas in central India and Africa.

Rajagopal et al. (1970) \[[@B3]\] have studied the metabolism of DAP in *Pseudomonas* and demonstrated that it is metabolized to pyruvic acid through Ammonia lyase reaction. Very scant information is available on the physicochemical and catalytic properties of diamino propionic acid ammonia lyase. In this communication we report the production of DAP ammonia lyase by *S. typhimurium* PU011 and describe a few of its properties.

Results and Discussion
======================

Virulence Assay
---------------

The results of virulence assay performed in mice employing *S. typhimurium* PU011 strain are shown in Table [1](#T1){ref-type="table"}. To be absolutely certain that enough number of virulent *S. typhimurium* and *S. typhimurium* PU011 are present in the mice after oral administration, to bring about mortality, each of the Salmonella strain at 2.5 × 10^6^ CFU per mouse, which is approximately twice the reported 50% Lethal Dose (LD~50~) \[[@B4],[@B5]\] was employed. In the light of observations reported by Leung et al (1991) that the administration of *S. typhimurium* in mice resulted in death within 28 days, the experiment was continued up to 28 days post inoculation. The mice administered with virulent *S. typhimurium* died on 8^th^ and 9^th^ day of post inoculation. However, the mice administered with *S. typhimurium* PU011 survived even after 28 days.

::: {#T1 .table-wrap}
::: {.caption}
######

Virulence of *S. typhimurium* in an orally infected typhoid mouse model
:::

  **Bacteria**                   **Percent Survival^\*^**   **Clinical Symptoms of liver and spleen**   **Conclusion**
  ------------------------------ -------------------------- ------------------------------------------- ----------------
  *S. typhimurium* PU011         100%                       No symptoms                                 Avirulent
  *S. typhimurium*               0%                         Enlarged, inflamed & edematous              Virulent
  Control (Mice with PBS only)   0%                         No symptoms                                 Avirulent

^\*^Average of six replicates
:::

Further, the mice injected with virulent *S. typhimurium* showed the clinical symptom of diarrohea prior to death, which was conspicuously absent in mice that were inoculated with *S. typhimurium* PU011. The clinical examination of sacrificed mice injected with virulent *S. typhimurium* revealed typical symptoms of enlarged, inflamed and edematous liver and spleen, which were absent in mice, injected with *S. typhimurium* PU011 strain. Conclusively this is evident enough to show that *S. typhimurium* PU011 strain employed in our studies is a non-virulent strain. To the best of our knowledge this is the first report about the production of DAP ammonia lyase by a non-virulent *S. typhimurium* with wider ramifications.

Biomass and Enzyme production
-----------------------------

Vijayalakshmi (1975) \[[@B6]\] and Nagasawa (1988) \[[@B7]\] have identified *Pseudomonad* and *Salmonella typhimurium* respectively capable of producing DAP ammonia lyase that degrades the neurotoxin present in *L. sativus* seeds. We have accidentally stumbled upon a non-virulent bacterium *S. typhimurium* PU011 which was capable of producing DAP ammonia lyase. The kinetics of production of biomass and DAP ammonia lyase activity by this organism is shown in Fig [1](#F1){ref-type="fig"}. A continuous increase in biomass and specific activity of enzyme with increase in incubation period was observed, the maximum reaching at 16 h post inoculation. Interestingly, a direct correlation between biomass yield and enzyme activity up to 20 h after inoculation was observed. Nagasawa et al. (1988)\[[@B7]\] have also observed a similar pattern of growth and enzyme activity in *S. typhimurium*.

![Biomass and DAP ammonia lyase production in Salmonella typhimurium PU 011. The culture was incubated on a rotary shaker at room temperature. The biomass was estimated gravimetrically and expressed as mg/ml. The crude enzyme activity of the culture drawn at different intervals of growth was measured spectrophotometrically and expressed as (U/mg) × 10^-3^.](1471-2180-2-5-1){#F1}

As to whether any carbon source other than DAP in the growth medium could support growth and enzyme production, it was observed that none other than DAP could substitute any of the carbon sources (data not shown). Since *S. typhimurium* PU011 employed in the present study was found to synthesize DAP ammonia lyase, it was thought fit to employ this enzyme for detoxification of DAP present in *L. sativus* seeds prior to consumption. Therefore, the characteristics of this enzyme were studied in detail.

Kinetic properties
------------------

The Km value for the substrate, DL-DAP, was found to be 0.685 mM based on the Line weaver Burk plot (Fig [2](#F2){ref-type="fig"}). However, for the same enzyme from *Pseudomonas sp.* and *S. typhimurium*, Vijiyalakhsmi et al (1975) \[[@B6]\] and Nagasawa et al (1988) \[[@B7]\] have reported Km values at 0.267 mM and 0.12 mM respectively. The differences in Km values were attributed to D or L or DL forms of DAP used as substrate in the estimation of enzyme activity.

![Determination of Km value for the enzyme Diamino propionic acid ammonia lyase.](1471-2180-2-5-2){#F2}

Effect of pH
------------

With regard to specific activity of DAP ammonia lyase in different buffer systems at various pH regimes, it was found that the enzyme had a sharp pH optimum of 8.0 in phosphate buffer and at any pH less than 6 or more than 11 a drastic reduction in activity was observed (Fig [3](#F3){ref-type="fig"}). Vijiyalakhsmi et al. (1975) \[[@B6]\] also have observed an optimum pH of 8.0 for maximum DAP ammonia lyase enzyme activity produced by *Pseudomonas*. However, in their studies Tris HCl buffer at pH 8.0 was found to inhibit 50% of enzyme activity.

![Effect of pH on the Stability of DAP ammonia lyase. The crude enzyme extract was incubated in 10 mM phosphate buffer at different pH regimes and the enzyme activity measured. The enzyme activity is expressed as (U/mg) × 10^-3^.](1471-2180-2-5-3){#F3}

Effect of temperature
---------------------

The enzyme activity was measured using spectrophotometer at temperatures ranging between 30°C -- 60°C at increment of 5°C. Up to 45°C there was no decrease in enzyme activity; however, thereafter a decline in enzyme activity with increase in temperature was observed (Fig [4](#F4){ref-type="fig"}). The loss in DAP ammonia lyase enzyme activity with increase in temperature indicates the level of stability of the enzyme *per se* to different temperature regimes and not temperature activity relationship since the enzyme was not incubated along with the substrate. While 45°C was found to support maximum enzyme activity in *Salmonella sp*. (Nagasawa et al., 1988) \[[@B7]\], 40°C was found to support maximum activity in *Pseudomonas* (Vijiyalakshmi et al., 1975)\[[@B6]\]. The fact that maximum activity was scored at above ambient temperature is a good augury for the use of this enzyme in the field scale applications for detoxification of DAP in *L. sativus* in drought prone areas of the world where elevated temperatures and desiccation pose threats for inactivation of enzyme activity.

![Effect of Temperature on the Stability of DAP ammonia lyase. The crude enzyme extract was incubated at various temperatures ranging between 20°C -- 60°C in 10 mM phosphate buffer and the enzyme activity measured. The enzyme activity is expressed as (U/mg) × 10^-3^.](1471-2180-2-5-4){#F4}

Purification of DAP ammonia lyase from *S. typhimurium* PU011
-------------------------------------------------------------

According to the procedure described in the text, the enzyme was purified 68 fold (Table [2](#T2){ref-type="table"}). When the purified enzyme was subjected to SDS-PAGE, a single band was observed corresponding to the molecular weight of 43 KDa (Fig [5](#F5){ref-type="fig"}). This is in conformity with the results of Vijiyalakshmi (1975) \[[@B6]\]. However, Nagasawa et al (1988) \[[@B7]\] reported that the relative molecular mass of DAP ammonia lyase produced by *Salmonella typhimurium*, estimated by ultra centrifugal equilibrium method, was 89,000 Da and the enzyme consisted of 2 subunits identical in Molecular mass.

![SDS-PAGE analysis of DAP ammonia lyase produced by *Salmonella typhimurium* PU 011. The crude enzyme was purified and run on SDS-PAGE. Molecular weight marker (Lane a), Crude Enzyme Extract (Lane b), Enzyme obtained after passing through Ammonium Sulfate precipitation (Lane c), Sephadex G-100 (Lane d), CM-Sephadex (Lane e) and CM-Sephacel (Lane f).](1471-2180-2-5-5){#F5}

::: {#T2 .table-wrap}
::: {.caption}
######

Purification Table of Diamino propionic acid ammonia lyase
:::

  Treatments                        Total Protein (mg)   Total Activity (U)   Specific Activity (U/mg)
  --------------------------------- -------------------- -------------------- --------------------------
  Crude Extract                     214.0                40.02                0.19
  Ammonium Sulphate precipitated    97.7                 39.17                0.40
  Passed through Sephadex -- G100   10.27                36.74                3.58
  Passed through CM -- Sephadex     3.8                  36.71                9.66
  Passed through DEAE -- Sephacel   1.6                  21.76                13.60

^\*^Average of six replicates
:::

The discovery of non-virulent *S. typhimurium* PU011 capable of producing DAP ammonia lyase has opened new vistas in the application of this enzyme in the tribal tracts of arid and semi-arid regions of Central India. For commercial exploitation of this enzyme, attempts are underway to clone and overexpress the gene coding for DAP ammonia lyase for applicative purposes in field.

Conclusion
==========

At 16 h post inoculation *S. typhimurium* PU011 produced maximum biomass yield and maximum enzyme activity in minimal medium containing DAP. Interestingly no carbon source other than DAP supported DAP ammonia lyase synthesis in the medium effectively. With respect to specific enzyme activity of DAP ammonia lyase at different pH regimes, the enzyme had a sharp activity at pH 8.0 in phosphate buffer and at any pH less than 6 or more than 11 a drastic decrease in enzyme activity was observed. Up to 45°C there was no decrease in enzyme activity; however, thereafter there was decline in enzyme activity with increase in temperature. Based on SDS-PAGE analysis of the DAP ammonia lyase enzyme, the molecular weight was determined to be 43 KDa having a Km value of 0.685 mM.

Materials and Methods
=====================

Culture and media
-----------------

*Salmonella typhimurium* PU011 employed in the present study was isolated in our lab and further characterized by standard microbiological, serological and biochemical tests \[[@B8]\]. Cells were grown at 30°C in minimal medium (0.05% Yeast Extract, 500 μl Mineral salt solution and 2.0% Agar) supplemented with DL -- DAP (0.3%) for production of DAP ammonia lyase by the bacterium.

Virulence Assay
---------------

Virulent *S. typhimurium* (obtained from the Department of Microbiology, Jawaharlal Institute of Post Graduate Medical Education and Research (JIPMER), Pondicherry, India) and *S. typhimurium* PU011 suspensions were grown at 37C in LB broth overnight, with shaking at 200 rpm. The next day, bacteria were diluted 1:100 into fresh LB broth and incubated with shaking. After 4 h, bacteria were washed once with PBS and resuspended in PBS. BALB/c female mice, aged 6--10 weeks, were inoculated orally with bacterial suspensions at 2.5 × 10^6^ CFU per mouse, which is approximately twice the reported 50% Lethal Dose (LD~50~) \[[@B4]\], after being deprived of water for 4 h. Control mice were given PBS only. The liver and spleen of experimental mice that died on different days after administration of *S. typhimurium* were harvested, homogenized and the resultant slurry was spread on Mac Conkey plates to obtain bacterial counts. After sacrifice, the mice were critically examined for the presence of typical symptoms of enlarged, inflamed and edematous liver and spleen. Mice surviving after 28 days post inoculation were indicative of the non-virulence nature of *S. typhimurium* employed.

Determination of Biomass and Enzyme activity
--------------------------------------------

Cells were grown in minimal medium supplemented with 0.3% DAP at 30°C on a rotary shaker (180 rpm) as per the method outlined by Premkumar \[[@B9]\] and Rupesh \[[@B10]\]. For estimation of biomass, at various intervals of time 10 ml of culture was withdrawn, centrifuged at 8000 g for 10 min, washed once with distilled water, dried at 105°C and weighed. However, for estimation of enzyme activity 25 ml of culture was withdrawn, centrifuged at 8000 g for 10 min at 4°C, suspended in 0.5 ml of extraction buffer (10 mM Potassium phosphate buffer pH 8.0, 1 mM EDTA, 1 mM β -- Mercaptoethanol, 100 μM Pyridoxal-5-Phosphate and 5% Glycerol) and sonicated for 10 min using Branson Sonifier 250 (10 min bursts at 40% duty cycle with intermittent cooling every 2 min in order to maintain the suspension below 5°C). After sonication, the lysate was centrifuged at 12,000 g for 10 min at 4°C and the supernatant served as the crude extract for the estimation of enzyme activity. The DAP ammonia lyase activity was measured as per the method of Vijiyalakhsmi et al \[[@B6]\]. Protein estimation was done as per the method of Lowry et al \[[@B11]\].

Enzyme Assay (Vijayalakshmi et al., 1975)
-----------------------------------------

DAP ammonia lyase was assayed spectrophotometrically, using a coupled assay system, utilizing the following reaction.

![](1471-2180-2-5-i1.gif)

Sum: DAP + NADH + H~2~O + H^+^ → Lactate + NAD + 2NH~3~

The reduction in absorbance at 340 nm was measured in Hitachi U-2000 spectrophotometer. The assay system contained in a total volume of 3.0 ml in a 1 cm light path cuvette: 1 mmol Potassium phosphate buffer pH 8.0, 20 mmol DL-DAP, 0.3 μmol PLP, 15 Units of LDH, 1 μmol NADH and appropriate amount of enzyme. The reaction was studied at 28°C, and was started by the addition of DAP. The decrease in absorbance was recorded at 30 s intervals for a period of 2 min. One Unit of enzyme was defined as the amount of enzyme required for the conversion of 1 μmol of NADH to NAD^+^ in one minute. Specific activity is defined as Units per mg of protein.

![](1471-2180-2-5-i2.gif)

Effect of pH
------------

Optimum pH at which a maximum specific activity of DAP ammonia lyase could be obtained was determined by employing different buffer systems at various pH as indicated in parenthesis. The buffer systems employed were: Glycine-HCl buffer (pH 3), Sodium acetate buffer (pH 5), Potassium phosphate buffer (pH 7), Potassium phosphate buffer (pH 8), Glycine-NaOH (pH 9), Glycine-NaOH (pH 11) and KCl-NaOH (pH 13). The crude enzyme extract was taken and incubated with the above buffers for 30 min. After incubation, the crude extract was precipitated with ammonium sulfate (80% saturation) and the precipitate was resuspended in minimal volume of 10 mM potassium phosphate buffer. The effect of pH was studied by measuring DAP ammonia lyase activity as per the procedure described earlier.

Effect of Temperature
---------------------

The crude enzyme extract in 10 mM Potassium phosphate buffer pH 8.0 was incubated at various temperatures ranging from 0°C -- 70°C. After 30 min of incubation, the crude extract was analyzed for DAP ammonia lyase activity.

Purification of DAP ammonia lyase
---------------------------------

### Preparation of Cell free crude extract

Cell free crude extract of *S. typhimurium* PU011 was prepared by sonicating the pellet in 0.5 ml extraction buffer for 10 min using Branson Sonifier 250. The sonicate was then centrifuged at 12,000 g for 10 min at 4°C and the supernatant served as the crude enzyme extract.

Ammonium sulfate fractionation
------------------------------

The enzyme was precipitated by addition of appropriate amounts of ammonium sulfate (35--65% saturation) as per the method of Nagasawa et al, (1988) \[[@B7]\]. The precipitate was collected by centrifugation and dissolved in 1 ml of extraction buffer and dialyzed against the same buffer for 24 h.

Chromatography on Sephadex G-100
--------------------------------

The crude enzyme preparation was subjected to gel filtration on sephadex G-100 (1.6 × 20 cm) column chromatography. The dialyzed enzyme solution was applied to sephadex G-100 column, which had been equilibrated with 10 mM phosphate buffer. The active fractions that showed absorption at 280 nm were collected separately and DAP ammonia lyase in each fraction was quantified. The fractions that showed maximum enzyme activity were pooled and passed through CM-Sephadex column.

Chromatography on CM-Sephadex
-----------------------------

Chromatography on CM-Sephadex column was performed on column (2 × 12.5 cm) of the resin equilibrated with 10 mM phosphate buffer pH 8.0, 1 mM β-mercaptoethanol and 1 mM pyridoxal-5-phosphate. The unbound fraction containing DAP ammonia lyase activity was reloaded onto DEAE-Sephacel column.

Chromatography on DEAE-Sephacel
-------------------------------

The active enzyme fraction was applied to a DEAE-Sephacel column (2 × 20 cm), which had been equilibrated with 10 mM phosphate buffer pH 8.0, 1 mM β-mercaptoethanol and 1 mM pyridoxal-5-phosphate. The enzyme fractions were eluted using 0 -- 1 M KCl gradient.

All the column chromatography purifications were carried out in BIORAD Econo system. The active fractions were precipitated with 80% ammonium sulfate. The precipitate was resuspended in a minimum amount of 10 mM phosphate buffer and dialyzed against the same buffer for 24 h with three changes under continuous stirring at 4°C. The progress of purification of DAP ammonia lyase was monitored both by enzyme activity measurement and on 12% SDS-PAGE.

SDS-Polyacrylamide Gel Electrophoresis (SDA-PAGE) (Laemmli, 1970) \[[@B12]\]
----------------------------------------------------------------------------

Routinely 0.75 mm thick gels were cast in a protein gel apparatus using a separating gel (12 %) and stacking gel (5%). The separating gel solution was first poured in between a sealed clean glass plate cassette leaving 3 to 4 cm space at the top. Water saturated n-butanol was layered gently on the top of the separating gel to make an even surface. After the separating gel got polymerized, the upper layer was removed completely and rinsed with water. Water was removed completely using a blotting paper. Stacking gel solution was poured onto the separating gel up to the top. A suitable comb was immediately inserted into the cassette and polymerization was allowed to take place. The slots were cleaned by rinsing with electrophoresis buffer. Protein samples were loaded into the slots and the slots were filled with 1 × electrophoresis buffer. The gel cassette was then fixed with the upper buffer tank and then fixed onto the lower buffer tank. Both the tanks were filled with 1 × electrophoresis buffer and electrophoresis was carried out at a constant current of 15 mA.

After electrophoresis was over, the gel cassette was dismantled and the gel was placed in a tray containing Coomassiae Brilliant Blue staining solution (0.1 % Coomassie Brilliant Blue (R 250), 45 % Methanol, 10 % Glacial Acetic Acid and 45 % Distilled water) and the gel was agitated slowly for 4 h to overnight. The staining solution was poured out and replaced with destaining solution (45 % Methanol, 10 % Glacial Acetic Acid and 45 % Distilled water). Destaining was carried out for a minimum of 2 h with gentle agitation.

Acknowledgements
================

The assistance rendered by M. K. Natarajan, JIPMER, Pondicherry, India in carrying out the microbiology work is gratefully acknowledged.