Elaboration of Neosamine Rings in the Biosynthesis of Neomycin and Butirosin

Huang, Fanglu; Spiteller, Dieter; Koorbanally, Neil A.; Li, Yanyan; Llewellyn, Nicholas M.; Spencer, Jonathan B.

doi:10.1002/cbic.200600371

ChemBioChem

2007

DOI: 10.1002/cbic.200600371

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Elaboration of Neosamine Rings in the Biosynthesis of Neomycin and Butirosin

Fanglu Huang

Dieter Spiteller

Neil A. Koorbanally

et al.

Abstract: The proteins Neo-11 and Neo-18 encoded in the neomycin gene cluster (neo) of Streptomyces fradiae NCIMB 8233 have been characterized as glucosaminyl-6'-oxidase and 6'-oxoglucosaminyl:L-glutamate aminotransferase, respectively. The joint activity of Neo-11 and Neo-18 is responsible for the conversion of paromamine to neamine in the biosynthetic pathway of neomycin through a mechanism of FAD-dependent dehydrogenation followed by a pyridoxal-5'-phosphate-mediated transamination. Neo-18 is also shown to catalyze d… Show more

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Cited by 37 publications

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“…The substrate specificity of this set of enzymes is also interesting because the homologous enzymes were reported to recognize mainly the glucosamine moiety and are responsible for the transaminations at C-6 in neomycin biosynthesis. 14,15 Thus, these enzymes might convert paromamine to neamine, which could be glycosylated by KanM2 to yield kanamycin B. Thus, the substrate specificity of KanM2 is a key determinant for the production of kanamycin analogs in the producer strain.…”

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Enzymatic activity of a glycosyltransferase KanM2 encoded in the kanamycin biosynthetic gene cluster

2009

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Kanamycin, discovered by Umezawa in 1957, is the most well-known 2-deoxystreptamine (2DOS)-containing aminoglycoside antibiotic. 1 Three structurally related kanamycins were identified from the producer Streptomyces kanamyceticus (Figure 1). Extensive biosynthetic studies of this class of aminoglycoside antibiotics, especially butirosin and neomycin, clarified that a unique aminocyclitol, 2DOS, is biosynthesized from D-glucose 6-phosphate by three crucial enzymes, 2-deoxy-scyllo-inosose (2DOI) synthase, L-glutamine (Gln):2DOI aminotransferase and 2-deoxy-scyllo-inosamine dehydrogenase. 2,3 After the 2DOS formation, uridine 5¢-diphospho-N-acetylglucosamine (UDP-GlcNAc):2DOS N-acetylglucosaminyltransferase catalyzes the glycosylation of 2DOS to give N-acetylparomamine, which is then deacetylated by N-acetylparomamine deacetylase to yield paromamine. 4 Paromamine is believed to be a branching biosynthetic intermediate, which is converted to either 4,6-disubstituted 2DOS aminoglycosides, such as kanamycin and gentamicin, or 4,5-disubstituted 2DOS aminoglycosides, such as neomycin and butirosin. Therefore, the regio-specificities and substrate specificities of glycosyltransferases that recognize paromamine as a glycosyl acceptor determine the core structures of aminoglycoside antibiotics.The biosynthetic gene cluster for kanamycin, comprising 24 open reading frames, was identified in 2004. 5 Two other research groups also deposited independently identical kanamycin biosynthetic genes by different symbols in the public DNA databases simultaneously. 6,7 To prevent confusion, herein we use the btr gene-based names, which were used systematically by the Piepersberg group and also in our recent review. 8,9 The entire kanamycin biosynthetic gene cluster was expressed in Streptomyces venezuelae and the resultant recombinant strain was reported to produce kanamycin A, which indicates that the kan genes are responsible for the biosynthesis of kanamycins. 10 The kanC, kanS1, kanE, kanM1 and kanN genes expressing Streptomyces lividans reportedly produce paromamine, confirming that these genes, respectively, encode 2DOI synthase, Gln:2DOI aminotransferase, 2-deoxy-scyllo-inosamine dehydrogenase, UDP-GlcNAc:2DOS N-acetylglucosaminyltransferase and N-acetylparomamine deacetylase (Figure 1). 11 Among these, the kanC gene was expressed in Escherichia coli and the recombinant KanC protein was confirmed to be 2DOI synthase. 6 Recombinant KanM1 protein was also reported to have weak glycosyltransferase activity with 2DOS as a glycosyl acceptor, and TDP-glucose or GDP-mannose as a glycosyl donor. 12 However, this KanM1 activity is contradictory to the presumed enzymatic function as suggested by the above-mentioned heterologous expression of UDP-GlcNAc:2DOS N-acetylglucosaminyltransferase in S. lividans. Detailed enzymatic analysis is thus necessary to elucidate the substrate specificity of KanM1 on using a pure enzyme.In this study, we heterologously expressed another glycosyltransferase gene, kanM2, in the kan gene cluster and investig...

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Enzymatic activity of a glycosyltransferase KanM2 encoded in the kanamycin biosynthetic gene cluster

2009

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“…2D-NMRs including 1 H-1 H COSY and TOCSY spectra (data not shown) were also recorded to determine the structure of neomycin C. The signals were assigned by comparison with those of 6¢¢¢-deamino-6¢¢¢-hydroxyneomycin C 5 and neomycin B. Enzymatic preparation of neomycin C F Kudo et al mamine to neamine using L-glutamate or L-glutamine as an amino donor. 8 NeoQ was characterized as a flavin adenine dinucleotidedependent dehydrogenase of paromamine to give 6¢-oxoparomamine during neamine formation. 8 No dehydrogenase for the oxidation of 6¢¢¢-deamino-6¢¢¢-hydroxyneomycin C has been identified as yet.…”

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“…8 NeoQ was characterized as a flavin adenine dinucleotidedependent dehydrogenase of paromamine to give 6¢-oxoparomamine during neamine formation. 8 No dehydrogenase for the oxidation of 6¢¢¢-deamino-6¢¢¢-hydroxyneomycin C has been identified as yet.…”

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“…8 Characterization of the NeoQ function and reconstitution of the biosynthetic reactions between ribostamycin and neomycin C is important to elucidate the potential repetitive use of multiple enzymes in the neomycin biosynthetic pathway. At first, we prepared 6¢¢¢-deamino-6¢¢¢-hydroxyneomycin C from ribostamycin by incubation with NeoF and NeoD according to earlier methods with minor modifications.…”

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“…Despite the use of newly prepared enzymes for both reactions, 10-20% of the starting material remained after the reaction, probably because of the reversibility of the transamination. 8 However, the conversion of 6¢¢¢-deamino-6¢¢¢-hydroxyneomycin C to neomycin C using purified NeoQ and NeoB strongly suggests a repetitive use of NeoQ and NeoB in neomycin biosynthesis.…”

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Enzymatic preparation of neomycin C from ribostamycin

et al. 2009

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The high regio-and stereospecificities of enzymes and their ability to catalyze reactions in the absence of organic solvents make enzymatic synthesis of antibiotics an attractive way to prepare complex natural products. Enzymatic total syntheses of several complex natural products have been recently achieved, which illustrates the power of this synthetic methodology. 1,2 Combinations of biosynthetic enzymes and substrates, including unnatural types, can afford structurally diverse natural product-like compounds. 3 In this study, we report the enzymatic synthesis of the aminoglycoside antibiotic neomycin C from ribostamycin by the use of four neomycin biosynthetic enzymes. So far, all nine enzymes for ribostamycin biosynthesis from D-glucose 6-phosphate have been characterized using the enzymes derived from the butirosin producer Bacillus circulans and the neomycin producer Streptomyces fradiae. 4 Furthermore, two neomycin biosynthetic enzymes NeoF and NeoD were recently found to catalyze the glycosylation of ribostamycin using Figure 1 Biosynthesis of neomycin C from ribostamycin.

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Involvement of Two Distinct N‐Acetylglucosaminyltransferases and a Dual‐Function Deacetylase in Neomycin Biosynthesis

et al. 2008

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Elaboration of Neosamine Rings in the Biosynthesis of Neomycin and Butirosin

Cited by 37 publications

References 23 publications

Enzymatic activity of a glycosyltransferase KanM2 encoded in the kanamycin biosynthetic gene cluster

Enzymatic activity of a glycosyltransferase KanM2 encoded in the kanamycin biosynthetic gene cluster

Enzymatic preparation of neomycin C from ribostamycin

Involvement of Two Distinct N‐Acetylglucosaminyltransferases and a Dual‐Function Deacetylase in Neomycin Biosynthesis

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