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Noda Institute for Scientiﬁc Research GRANT
2010 Research Grant

Analysis of L-glucose metabolic pathway in Paracoccus sp.
43P1)
Akira NAKAMURA
Division of Integrative Environmental Sciences, Graduate School of Life and Environmental Sciences, University of Tsukuba
Research aims
Almost all the living organisms on the Earth can utilize
D-glucose as carbon and energy sources, and its metabolic

pathways are known in detail, such as the glycolysis and thepentose-phosphate pathway. On the other hand, L-glucose,
the enantiomer of D-glucose, is thought not to be present in
the natural environment, and any L-glucose-utilizing organisms are not known. Rather, since Rudney2) reported in1940 that some bacteria, yeast and mammals did not utilizeit, it has been generally accepted that there are no organisms that can utilize it. Therefore it can be assumed that
there is the homochirality of certain sugar molecules, especially for glucose, as in the case of the homochirality of Lamino acids in proteins.
To challenge this “homochirality in sugar metabolism”,
we have screened for microorganisms which could utilize Lglucose from natural environments, and succeeded in obtaining such a bacterium, termed Paracoccus sp. 43P. We
also revealed that the ﬁrst step for L-glucose utilization was
the reaction catalyzed by an NAD⫹-dependent L-glucose
dehydrogenase (L-GDH).
In this work, we have analyzed the enzymes and corresponding genes involved in L-glucose metabolism and succeeded in clarifying the metabolic pathway for L-glucose
utilization in this bacterium.
Methods
Sequence analysis of the region around lgdA, encoding LGDH, in strain 43P.
The DNA fragments of the region surrounding lgdA,which was already cloned and sequenced, were PCR-ampliﬁed by the primer-walking method from the genome ofstrain 43P, and their nucleotide sequences were determined.
Production and puriﬁcation of the enzymes involved in Lglucose metabolism.
The putative ORFs were PCR-ampliﬁed and cloned into
pET21a or pET28a, with the 6xHis-tag in the plasmid fusedin-frame with each ORF at its C- or N-terminus, respectively, and the protein products were produced in Escherichia coli BL21(DE3) harboring their respective plasmids. The enzymes were puriﬁed by an afﬁnity chromatography against the 6x His-tag.

Analyses of the enzyme activities.
The puriﬁed enzymes were used to detect their presumed
enzyme activities under the conditions described previously. The substrates used were commercially available
sugars and aldonates, or their derivatives made by chemical
and/or enzymatic reactions.
Identiﬁcation of the reaction products.
Some of the reaction products were puriﬁed by TLC, andidentiﬁed by HPLC analysis. GC/MS or 1H-NMR analysis
was also conducted to identify the products.
ResultsSequencing analysis of the region around lgdA.
The amino acid sequence of the cloned lgdA from strain
43P, encoding the L-GDH, showed 84% identity with thatof Pden_1680 from P. denitriﬁcans PD1222, and thePden_1680 gene was located in a cluster of genes annotatedas involved in inositol metabolism. Sequencing analysis ofthe region around lgdA in strain 43P showed the same geneorganization as that of strain PD1222, with amino acid
identities of 76–93%, suggesting that the LgdA might havea functional role in inositol metabolism (Fig. 1). In fact, thepuriﬁed L-GDH showed dehydrogenase activity against several inositols. Especially, the activity against scyllo-inositol(190 U/mg protein) was about six times-higher than thatagainst L-glucose (31 U/mg protein), suggesting that thesubstrate of this enzyme was scyllo-inositol under the physiological conditions.
The reaction product generated from L-glucose by the action of L-GDH was identiﬁed as L-glucono-1,5-lactone by
GC/MS analysis, indicating that L-GDH catalyses oxidationof L-glucose at C1 position. This compound is known to beunstable in an aqueous solution and is easily hydrolyzed toL-gluconate.
Cloning of L-GnDH gene and sequencing analysis of theregion around the gene.
Next, we searched for an enzyme activity against Lgluconate, which was synthesized from L-glucose by I2 oxidation, in the cell-free extract of strain 43P, and detected anNAD⫹-dependent L-gluconate dehydrogenase activity. Theenzyme was puriﬁed and its N-terminal amino acid sequence was determined. The sequence coincided with thatof Pden_4931 of strain PD1222, indicating that the LGnDH is encoded by the orthologous gene of Pden_4931 in

63Fig. 1. Gene organization of the region around lgdA gene in the genome of strain 43P. The ORFs are shown withboxes, and the amino acid sequence identity of each ORF with the corresponding gene from strain PD1222 is indicated. Annotations for PD1222 genes were cited from the KEGG database.

Fig. 2. Gene organization of the lgn cluster of strain 43P. The ORFs of the lgn cluster are shown with boxes, and theamino acid sequence identity of each ORF with the corresponding gene from strain PD1222 is indicated. Annotations for
PD1222 genes were cited from the KEGG database.

strain 43P. Using the N-terminal sequence and an internalamino acid sequence of Pden_4931, two degenerate primers
were synthesized and used for cloning of the L-GnDH geneby PCR-based method. We also analyzed the sequence ofthe region around this gene. We found that the L-GnDH
gene was located in a cluster of nine genes, and foundan ORF of a putative transcriptional regulator for this cluster in the upstream region. These ORFs showed again
high identities of 71–91% in the amino acid sequenceswith the respective ORFs in strain PD1222 (Pden_4923Pden_4932). Also, these ORFs showed certain similarities
to the genes related to aldonate metabolism. Therefore weassumed that the ORFs in this cluster might be related to ametabolic pathway downstream of L-gluconate, and designated lgnR and lgnA-I (Fig. 2). Of these ORFs, lgnE-I wereexpected to encode enzymes for L-gluconate metabolism,
therefore we analyzed the products of these ORFs further.

Analysis of a metabolic pathway downstream of Lgluconate.
To investigate whether LgnE-I proteins could function as
enzymes for L-gluconate metabolism, we overproduced theproteins in E. coli using the pET expression system and puriﬁed the recombinant proteins. The puriﬁed proteins were
examined for their enzymatic activity as follows.
LgnH (L-GnDH)
As expected, LgnH showed the same enzymatic activity
as the puriﬁed L-GnDH. Of the related compounds tested,L-galactonate, the C4 epimer of L-gluconate, could serve asa substrate, but L-mannonate and D-idonate, the C2 and C5
epimer of L-gluconate, respectively, could not. The reaction
product from L-gluconate was identiﬁed as 5-keto-Lgluconate by HPLC analysis with 5-keto-D-gluconate as astandard.
LgnI ( D -idonate dehydrogenase/5-keto- L -gluconate
reductase)
LgnI showed an NADP⫹-dependent dehydrogenase activity against commercial D-idonate, and at the same time, the64

Fig. 3. Model of L-glucose metabolic pathway in strain 43P. The compounds and the enzymes in this pathway areshown, and the atoms and bonds altered by each enzyme reaction are indicated by red color.

enzyme showed a reductase activity against the LgnH reaction product, 5-keto-L-gluconate, in an NADPH-dependent
manner. As this enzyme was more active in the reductase
reaction than in the dehydrogenase reaction, it is thought tofunction as the reductase under the physiological conditions. Therefore we concluded that, with LgnH, LgnI catalyses D/L conversion to produce D-idonate from L-gluconate
through the dehydrogenase/reductase reactions.
LgnE (D-idonate dehydratase)
LgnE catalyzed conversion of D-idonate to a compoundwith reducing activity in the presence of Fe2⫹ and DTT, asdetected by TLC. The puriﬁed reaction product was identiﬁed by HPLC and NMR as 2-keto-3-deoxy-D-galactonate(KDGal). Thus we concluded that LgnE was a D-idonate
dehydratase.
LgnF (KDGal kinase) and LgnG (6-phospho-KDGal
aldolase)
The orthologues of LgnF and LgnG in strain PD1222,
the gene products of Pden_4929 and Pden_4930, wereannotated as a KDGal kinase and a 6-phospho-KDGal(PKDGal) aldolase, respectively. Therefore we examined
LgnF and LgnG proteins for the respective enzymatic activity.
LgnF catalyzed the conversion of KDGal to anothercompound in the presence of ATP, as determined by TLC
analysis. Moreover, addition of LgnG to the reaction mixture resulted in the generation of pyruvate and D-glyceraldehyde-3-phosphate (G3P), which were detected using acommercially available lactate dehydrogenase and G3P dehydrogenase, respectively. Therefore we concluded that
LgnF functions as a kinase that phosphorylates KDGal at
C6 position, and that LgnG functions as an aldolase that
produces pyruvate and G3P from PKDGal.

Conclusion
A model for metabolic pathway of L-glucose in strain
43P is shown in Fig. 3. In general, the metabolic pathways
converting aldose sugars to aldonates by oxidation at C1
positions and subsequently converting them to TCA substrates by dehydratase and aldolase reactions are called theEntner-Doudoroff (ED) pathways. Of the ED pathways, thepathway identiﬁed here is classiﬁed in the semi-phosphorylative (semi-P) ED pathway, because phosphorylation ofthe substrate occurred after the dehydratase reaction. The
semi-P ED pathway is found only in the D-glucose metabolism of some hyperthermophilic archaea3). Compared withthe pathways found in those archeal strains, the pathway
identiﬁed in this study is distinct in the reaction steps of theD/L conversion by LgnH/LgnI. Also, the reaction steps
after L-gluconate formation in this pathway are similar to
those of the L-galactonate metabolic pathway in E. coli 4),
but the corresponding enzymes does not show any sequence similarities with each other. Therefore we concluded that, together with the uniqueness of the starting
substrate, L-glucose, the newly identiﬁed pathway differs inmany points from the known metabolic pathways.
References
1) Shimizu, T., Takaya, N., and Nakamura, A. (2012) An L-glucose
catabolic pathway in Paracoccus species 43P. J. Biol. Chem. 287:
40448–40456.
2) Rudney, H. (1940) The utilization of L-glucose by mammalian tissues and bacteria. Science 92: 112–113.
3) Ahmed, H., Ettema, T. J. G., Tjaden, B., Geerling, A. C. M., van derOost, J., and Siebers, B. (2005) The semi-phosphorylative EntnerDoudoroff pathway in hyperthermophilic archaea: a re-evaluation.
Biochem. J. 390: 529–540.
4) Cooper, R. A. (1979) The pathway for L-galactonate catabolism inEscherichia coli K-12. FEBS Lett. 103: 216–220.