51NISR Research GRANT
2007 Young Investigator Research Grant

Biochemical studies on unique enzymes in the biosynthesis ofbutirosin
Fumitaka KUDO
Department of Chemistry, Tokyo Institute of Technology

Research aims
The aminoglycosides are a diverse class of antibacterial
antibiotics including streptomycin and kanamycin as the effective anti-tuberculosis antibiotics. However, the appearance of resistant strains to the aminoglycosides interferedwith their therapeutic use in the history. To counter resistantorganisms, C1 position of the 2-deoxystreptamine (DOS)moiety of kanamycin A and dibekacin were semisynthetically modified with the (S)-4-amino-2-hydroxy butyryl(AHBA) side chain to afford amikacin and arbekacin, respectively. The AHBA was originally observed in the natural product butirosin, which inhibits a variety of aminoglycoside resistant pathogens due to the presence of the AHBA
substituent on the DOS moiety. The butirosin producer
Bacillus circulans should have the AHBA biosynthetic enzymes to introduce it onto ribostamycin. Thus, the AHBA
biosynthetic enzymes were expected to modify the otherDOS-containing aminoglycosides including kanamycin Aand dibekacin via biocatalytic route.
We have investigated the biosynthetic pathway for butirosin and neomysin for the last decade, and characterized
most of biosynthetic enzymes leading to ribostamycin fromglucose-6-phosphate. Comparison of biosynthetic genes forseveral DOS containing aminoglycosides showed that BtrF,G, H, I, J, K, N, O, and U are specifically encoded in thebutirosin biosynthetic gene cluster suggesting that these
could be involved in the specific enzymatic reactions in thebiosynthesis of butirosin including the AHBA biosynthesis
and transfer reactions. Among the enzymes, Spencer groupin UK reported that BtrI, J, K, O, and U are responsible forthe g -glutamyl AHBA acyl carrier protein (ACP) formation
prior to the acylation of ribostamycin leading to butirosin.1)
Thus, the other remaining enzyme BtrF, G, H, and N
seemed to be responsible for the AHBA transfer reaction
and other specific enzymatic steps in the biosynthesis ofbutirosin. In this research, we aimed to characterize BtrF,G, H, and N using the recombinant enzymes expressed inE. coli and also by gene inactivation experiments.
Methods
The btrF, G, H, and N genes were amplified by PCR withappropriate primers, and the amplified PCR products weredigested by restriction enzymes and sub-cloned into thecorresponding sites of LITMUS 28. After confirmation of
the sequence, the appropriate DNA fragments were inserted

into expression vectors such as pET30, pET28, and pColdI
to obtain expression plasmids, which were subsequently introduced into E. coli BL21(DE3). E. coli BL21(DE3) carrying each expression plasmid were grown in LB mediumcontaining appropriate antibiotics. The expression was induced by addition of isopropyl b -D-thiogalactopyranoside(IPTG) and the cultivation was continued at 1537°C for318 hr. The cells were harvested by centrifugation,
washed with buffer, and stored at 30°C until use. The wet
cells were suspended in buffer and disrupted by sonication
at 0°C to obtain cell free extracts containing recombinant
enzymes.
To investigate the function of BtrN, all experiments werecarried out under anaerobic conditions. The recombinant
BtrN protein was purified by a standard Ni affinity chromatography. The purified BtrN was treated with dithiothreitol (DTT) at room temperature. FeII(NH4)2(SO4)2 and Na2S
were then added and the mixture was incubated at roomtemperature. The BtrN sample was then desalted by passage through HiTrapTM desalting column (Amersham Pharmacia Biotech AB). The obtained reconstituted BtrN wasreduced by sodium dithionite for 1 hr at room temperature
prior to use. The reduced BtrN was incubated with DOIA,SAM in 50 mM HEPES-NaOH (pH 8.0) in the presence ofsodium dithionite. The reaction solution was analyzed for
3-amino-2,3-dideoxy-scyllo-inosose (amino-DOI) formation by HPLC after treatment with acetic anhydride and O(p-nitrobenzyl)-hydroxylamine. To investigate the speculated hydrogen abstraction mechanism, [3-2H]DOIA wasprepared from amino-DOI by reduction of the keto groupwith NaB2H4. For the EPR measurements during the catalytic cycle, BtrN was incubated with SAM and DOIA in
the presence of sodium dithionite at room temperature, andthe reaction was freeze-quenched in liquid nitrogen at specific periods of time. EPR spectra were recorded with aJEOL JES-FA300 ESR spectrometer (9.02 GHz).
Results
BtrF, BtrG, BtrH, and BtrN were successfully overexpressed in E. coli and the soluble recombinant proteins
were obtained. In addition, several co-expression systems
for BtrF, BtrG, BtrH, BtrI, BtrJ, BtrK, BtrO, and BtrU wereconstructed to study the AHBA transfer reaction in thebiosynthesis of butirosin. However, Spencer group at Cambridge University, UK has reported the g -glutamyl AHBA
(ACP): ribostamycin acyltransferase activity of BtrH and g -52glutamyl cyclotransferase activity of BtrG during our investigation.2,3)
On the other hand, inactivation of the btrN gene caused
the interruption of the biosynthetic pathway between 2deoxy-scyllo-inosamine (DOIA) and 2-deoxystreptamine(DOS) indicating its involvement in the oxidation ofDOIA.4) BtrN possesses a CXXXCXXC motif conserved
within the radical S-adenosyl methionine (SAM) superfamily and its involvement in the oxidation was hard to be presumed. Thus, we initiated to verify the function of thisunique radical SAM enzyme with its recombinant proteinexpressed in E. coli. BtrN was anaerobically purified by Ni
nitriloacetic acid (NTA) affinity chromatography, sinceother radical SAM enzymes were reported to be sensitive tooxygen. The anaerobically purified BtrN was further treatedwith FeII(NH4)2(SO4)2 and Na2S in the presence of dithiothreitol. The reconstituted BtrN was then reduced with 20mM sodium dithionite at room temperature. The reduced
BtrN was, then, incubated with DOIA and SAM. HPLC
analysis of the oxime derivative of the reaction product revealed that amino-DOI was efficiently formed. This resultclearly showed that BtrN itself catalyzes the oxidation ofDOIA into amino-DOI in the presence of SAM. Furtheranalysis of the other reaction products showed that 5-deoxyadenosine and methionine were formed by incubationof SAM with BtrN and DOIA.
From the mechanistic point of view, the 5-deoxyadenosyl radical generated through the reductive cleavage ofSAM by the [4Fe–4S] cluster was presumed to be involved in the abstraction of a substrate hydrogen atom toproduce a radical intermediate. To investigate this hypothesis, 2-deoxy-scyllo-[3-2H]inosamine ([3-2H]DOIA) wasprepared and subjected to the BtrN reaction. As a result, the2H NMR clearly showed incorporation of deuterium atom
into the methyl group of 5-deoxyadenosine. Furthermore,
the reaction was carried out in a deuterium oxide bufferusing non-labeled DOIA and no incorporation of the deuterium was observed in 5-deoxyadenosine. These suggestthat the BtrN reaction proceeds through direct hydrogen
atom abstraction from DOIA by the 5-deoxyadenosyl radical, not via the protein radical produced on a cysteine or atyrosine residue, since such a proton is exchangeable withsolvent.
Furthermore, the BtrN reaction was analyzed by EPR
spectroscopy. A sharp double triplet EPR signal was observed when the EPR spectrum of the enzyme reaction

mixture was recorded at 50 K. The spin coupling with protons partially disappeared by reaction with [2,2-2H2]DOIA,
which unambiguously proved the observed signal to be aradical on C-3 of DOIA. On the other hand, the EPR spectrum of the 4Fe–4S cluster of BtrN during the reaction
showed complex signal due to the presence of several
species. Comparison of signals derived from a 4Fe-4S center of BtrN incubated with various combinations of products (5-deoxyadenosine, L-methionine, and amino-DOI)
and substrates (SAM and DOIA) indicated that the EPR
signals observed during the reaction were derived from afree BtrN, a BtrN-SAM complex, and a BtrN-SAM-DOIAcomplex. Significant changes in the EPR signals uponbinding of SAM and DOIA suggest the close interaction ofboth substrates with the 4Fe–4S.
Conclusion
Among several specific enzymes encoded in the butirosinbiosynthetic gene cluster, we have successfully characterized BtrN as an unusual radical SAM dehydrogenase catalyzing the oxidation of the hydroxyl group by a radicalmechanism.4) We have also observed the DOIA radical intermediate by EPR measurements, and clearly showed theradical mechanism of BtrN.5) Our observations should help
understand the whole catalytic cycle of the BtrN and otherradical SAM dehydrogenases.
During our investigation, functional analysis of g -glutamyl AHBA acyltransferase BtrH and g -glutamyl cyclotransferase BtrG have been reported.2,3) These reports
demonstrated that my research proposal was reasonable andindicated potential of the enzymes for creation of modified
aminoglycosides. Two enzymes BtrE and F encoded in thebutirosin biosynthetic gene cluster now remain to be characterized. These enzymes seem to be involved in the unsolved epimerization step from butirosin B to A, which isalso significant reaction to modify aminoglycosides. In addition to these enzymes, other unique modification enzymes
such as methyltransferases and deoxygenation enzymes involved in other aminoglycoside biosynthesis will be further
investigated in future and applied for creation of diversestructural aminoglycosides with improved activity against
resistant strains.

53References
1)
2)

Y. Li, N. M. Llewellyn, R. Giri, F. Huang and J. B. Spencer, Chem.
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N. M. Llewellyn and J. B. Spencer, Chem. Commun. 2008,3786–3788.
K. Yokoyama, M. Numakura, F. Kudo, D. Ohmori and T. Eguchi, J.
Am. Chem. Soc. 2007, 129, 15147–15155.
K. Yokoyama, D. Ohmori, F. Kudo and T. Eguchi, Biochemistry
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