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Enzymatic Activity of a Glycosyltransferase Kanm2 Encoded in the Kanamycin Biosynthetic Gene Cluster

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Enzymatic Activity of a Glycosyltransferase Kanm2 Encoded in the Kanamycin Biosynthetic Gene Cluster The Journal of Antibiotics (2009) 62, 707–710 & 2009 Japan Antibiotics Research Association All rights reserved 0021-8820/09 $32.00 www.nature.com/ja NOTE Enzymatic activity of a glycosyltransferase KanM2 encoded in the kanamycin biosynthetic gene cluster Fumitaka Kudo1, Hilda Sucipto2 and Tadashi Eguchi2 The Journal of Antibiotics (2009) 62, 707–710; doi:10.1038/ja.2009.107; published online 13 November 2009 Keywords: aminoglycoside antibiotics; biosynthesis; glycosyltransferase; kanamycin; paromamine Kanamycin, discovered by Umezawa in 1957, is the most well-known 2-deoxy-scyllo-inosamine dehydrogenase, UDP-GlcNAc:2DOS N-acetyl- 2-deoxystreptamine (2DOS)-containing aminoglycoside antibiotic.1 glucosaminyltransferase and N-acetylparomamine deacetylase Three structurally related kanamycins were identified from the (Figure 1).11 Among these, the kanC gene was expressed in Escherichia producer Streptomyces kanamyceticus (Figure 1). Extensive biosyn- coli and the recombinant KanC protein was confirmed to be 2DOI thetic studies of this class of aminoglycoside antibiotics, especially synthase.6 Recombinant KanM1 protein was also reported to have butirosin and neomycin, clarified that a unique aminocyclitol, 2DOS, weak glycosyltransferase activity with 2DOS as a glycosyl acceptor, and 12 is biosynthesized from D-glucose 6-phosphate by three crucial TDP-glucose or GDP-mannose as a glycosyl donor. However, this enzymes, 2-deoxy-scyllo-inosose (2DOI) synthase, L-glutamine KanM1 activity is contradictory to the presumed enzymatic function (Gln):2DOI aminotransferase and 2-deoxy-scyllo-inosamine dehydro- as suggested by the above-mentioned heterologous expression of genase.2,3 After the 2DOS formation, uridine 5¢-diphospho-N-acetyl- UDP-GlcNAc:2DOS N-acetylglucosaminyltransferase in S. lividans. glucosamine (UDP-GlcNAc):2DOS N-acetylglucosaminyltransferase Detailed enzymatic analysis is thus necessary to elucidate the substrate catalyzes the glycosylation of 2DOS to give N-acetylparomamine, specificity of KanM1 on using a pure enzyme. which is then deacetylated by N-acetylparomamine deacetylase In this study, we heterologously expressed another glycosyltransfer- to yield paromamine.4 Paromamine is believed to be a branch- ase gene, kanM2,inthekan gene cluster and investigated its enzymatic ing biosynthetic intermediate, which is converted to either activities, which were expected to be catalysis of the glycosylation of a 4,6-disubstituted 2DOS aminoglycosides, such as kanamycin and pseudo-disaccharide, paromamine, to yield a pseudo-trisaccharide. To gentamicin, or 4,5-disubstituted 2DOS aminoglycosides, such as obtain soluble KanM2 protein expressed in E. coli, a low expression neomycin and butirosin. Therefore, the regio-specificities and sub- temperature (15 1C) was critically important after addition of iso- strate specificities of glycosyltransferases that recognize paroma- propyl b-D-thiogalactoside (final concentration 0.5 mM). The cell-free mine as a glycosyl acceptor determine the core structures of amino- extract of the KanM2-expressing E. coli was treated with 2 mM of glycoside antibiotics. paromamine as a glycosyl acceptor, and 2 mM of UDP-Glc or UDP- The biosynthetic gene cluster for kanamycin, comprising 24 open GlcNAc as a glycosyl donor. After incubation at 28 1C for 18 h, the reading frames, was identified in 2004.5 Two other research groups enzymatic reactions were quenched by addition of ethanol. The also deposited independently identical kanamycin biosynthetic genes resulting mixture was treated with 2,4-dinitrofluorobenzene to by different symbols in the public DNA databases simultaneously.6,7 convert aminoglycosides into N-dinitrophenyl derivatives, which To prevent confusion, herein we use the btr gene-based names, which show UV–Vis absorption at 350 nm. Consequently, only a reaction were used systematically by the Piepersberg group and also in our mixture of paromamine and UDP-Glc gave a new peak with 40% recent review.8,9 The entire kanamycin biosynthetic gene cluster was conversion from the substrate paromamine in HPLC traces expressed in Streptomyces venezuelae and the resultant recombinant (Figures 2a and b). Liquid chromatography–electrospray ionization– strain was reported to produce kanamycin A, which indicates that the MS analysis (negative mode) of the sample showed the new peak with kan genes are responsible for the biosynthesis of kanamycins.10 The m/z 982.4, which corresponded to [M-H]À for the derivative of kanC, kanS1, kanE, kanM1 and kanN genes expressing Streptomyces glucosylated paromamine (Figure 2c). lividans reportedly produce paromamine, confirming that these genes, Next, we attempted to isolate the KanM2 reaction product from respectively, encode 2DOI synthase, Gln:2DOI aminotransferase, paromamine and UDP-Glc, and determine the chemical structure. 1Department of Chemistry, Tokyo Institute of Technology, Tokyo, Japan and 2Department of Chemistry and Materials Science, Tokyo Institute of Technology, Tokyo, Japan Correspondence: Professor T Eguchi, Department of Chemistry and Materials Science, Tokyo Institute of Technology, 2-12-2 O-okayama, Meguro-ku, Tokyo 151-8551, Japan. E-mail: [email protected] Received 17 August 2009; revised and accepted 20 October; published online 13 November 2009 A glycosyltransferase in kanamycin biosynthesis FKudoet al 708 NH2 KanM2 KanD2 HO O KanS2 HO kanamycin B H2N H2N O HO NH2 Alternative OH pathway neamine KanQ KanB OH KanC KanS1 OH OH HO O OPO 2- KanE HO 1' 3 H2N HO O HO O H2N KanM1 KanN KanM2 H2N 2 O HO HO HO O HO NH H2N H2N 4 OH HO 2 AcHN H2N HO NH2 HO HO O O 6 HO NH NH O UDP-GlcNAc HO 2 HO 2 UDP-Glc OH glucose 6-phosphate 2-deoxystreptamine OH OH 1" O (2DOS) HO OH N-acetylparomamine paromamine OH 3"-deamino-3"-hydroxykanamycin C OH NH2 NH2 HO O HO O HO O KanD2 HO KanQ HO KanJ HO H2N H2N H2N KanS2 H2N KanB H2N KanK HO O O O HO NH2 HO NH2 HO NH2 O O O OH OH OH O O O HO OH HO OH HO OH NH2 NH2 NH2 kanamycin C kanamycin B kanamycin A Figure 1 Proposed biosynthetic pathway for kanamycin. Ammonium sulfate fractionation between 10 and 40% saturation was A disruptant mutant of the kanQ gene homolog encoding a effective in recovering most of the enzymatic activity from the cell-free FAD-dependent dehydrogenase in the tobramycin and apramycin extract of KanM2-expressing E. coli, although its activity was signifi- producer Streptomyces tenebrarius accumulated 3¢-deoxy-carbam- cantly decreased after a standard dialysis probably because of the oylkanamycin C.13 This accumulation suggests that the KanM2 instability of the enzyme. We then used the 10–40% (NH4)2SO4 homologous protein, TobM2, in tobramycin biosynthesis recognizes fractionated solution just after the fractionation, paromamine paromamine as a substrate to produce 300-deamino-300-hydroxykana- (3 mM,10mg),andUDP-Glc(4.7mM, 29 mg) in 50 mM HEPES buffer mycin C, which would be dehydrogenated and transaminated to + + (pH 8.0) containing 1 mM MgCl2 and 10% glycerol (total 10 ml) at give kanamycin C. An NAD /NADP -dependent dehydrogenase, 28 1C for 24 h for a large-scale enzymatic reaction. To ensure max- KanD2, and an aminotransferase, KanS2, are presumably responsi- imum consumption of the starting material, the reaction mixture was ble for this transformation based on bioinformatic analysis of the further treated under identical enzymatic conditions using freshly gene clusters for 2DOS-containing aminoglycoside antibiotics.9 prepared enzyme solution (0.25 ml) and UDP-Glc (5–10 mg) nine Alternatively, this set of enzymes might be responsible for the forma- times. Consequently, 84% of paromamine was converted to the new tion of NDP-3-amino-3-deoxyglucose (kanosamine), which is glycosylated product, which was obtained as the sulfate form of another possible glycosyl donor in the KanM2 reaction, to afford aminoglycoside (7.9 mg) by ion exchange chromatography. HR-FAB- kanamycin C.11 KanS2 is a protein that is homologous to KanS1 MS (positive mode, glycerol) of the compound gave an m/z of (37% identity/51% positive), which is involved in the double trans- 486.2307, which corresponds to the calculated mass for amination in 2DOS biosynthesis. The difference in substrate specifi- + 1 C18H36O12N3 (486.2299), [M+H] of glucosylated paromamine. city of these aminotransferases is an interesting issue to be H-NMR analysis of the product showed two anomeric protons and resolved. a methylene of the 2DOS moiety, although the other signals were very A FAD-dependent dehydrogenase, KanQ, and an aminotransferase, complex for assignment (see Supplementary information). Spectra of KanB, remaining in the kan gene cluster are apparently responsible for the compound obtained by total correlation spectroscopy (TOCSY) the transamination at C-6. The substrate specificity of this set of indicated that the signals for H-1 and H-3 of 2DOS at 3.3 p.p.m., those enzymes is also interesting because the homologous enzymes were for H-6 at 3.6 p.p.m., and those for H-4 and H-5 at 3.7 p.p.m. are reported to recognize mainly the glucosamine moiety and are respon- correlated with the signals for H-2ax and H-2eq at 1.7 and 2.3 p.p.m., sible for the transaminations at C-6 in neomycin biosynthesis.14,15 respectively (data not shown). The use of 1H–1H-COSY, heteronuclear Thus, these enzymes might convert paromamine to neamine, which multiple quantum coherence and heteronuclear multiple bond coher- could be glycosylated by KanM2 to yield kanamycin B. Thus, the ence facilitated assignment of 13C chemical shifts for C100,C1¢,C-2¢, substrate specificity of KanM2 is a key determinant for the production C-1 or C-3, C-4, C-5 and C-6. Consequently, a glycosidic linkage of kanamycin analogs in the producer strain. between C100 and C-6 was confirmed in addition to the C1¢–O–C4 Finally, kanamycin B is converted to the end product kanamycin A. 14 bond of the paromamine moiety. Therefore, the KanM2 reaction A feeding experiment of D-[ C]glucosamine into the kanamycin product from paromamine and UDP-Glc appeared to be 300-deamino- producer revealed that the labeled glucosamine was incorporated 300-hydroxykanamycin C.
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