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

Abstract

A new enzyme family: The glycosylation steps in the biosynthesis of neomycin were clarified through recombinant enzyme assays. NeoD catalyzes the first transglucosaminylation, and NeoK catalyzes the second, while NeoL catalyzes both deacetylations. We found that NeoK and its homologous proteins constitute a novel glycosyltransferase family.

Biosynthetic genes for aminoglycoside antibiotics

“…27 Then, 2DOS is glycosylated using glycosyltransferase with UDP-N-acetyl-D-glucosamine (UDP-GlcNAc) as a glycosyl donor to give N-acetylparomamine, whose N-acetyl group is removed using a deacetylase to give paromamine. 28 Paromamine is believed to be a branching intermediate to neomycin-related aminoglycosides, Table 1. Genes are classified according to the biosynthetic functions summarized in Table 1 (a)-(k).…”

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

“…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.…”

Enzymatic preparation of neomycin C from ribostamycin

“…4 Furthermore, two neomycin biosynthetic enzymes NeoF and NeoD were recently found to catalyze the glycosylation of ribostamycin using UDP-N-acetylglucosamine (UDP-GlcNAc) as a sugar donor and the subsequent deacetylation to afford 6¢¢¢-deamino-6¢¢¢-hydroxyneomycin C (Figure 1). 5 (As the biosynthetic gene cluster for neomycin was deposited with different symbols in DNA databases by multiple groups, we here use the butirosin biosynthetic btr gene-based code names, which are systematically utilized by the Piepersberg et al 6 and also in our recent review. 7 ) NeoB has also been reported to catalyze two transaminations: the conversion of neomycin C to 6¢¢¢-oxoneomycin C, which is a reverse reaction of the postulated in vivo biosynthetic reaction (Figure 1), and the conversion of 6¢-oxoparo- Figure 2 NMR spectra of neomycin C in D 2 O synthesized with biosynthetic enzymes.…”

“…At first, we prepared 6¢¢¢-deamino-6¢¢¢-hydroxyneomycin C from ribostamycin by incubation with NeoF and NeoD according to earlier methods with minor modifications. 5 Overexpressed NeoF protein was partially purified by anion exchange chromatography with DEAE Sepharose Fast Flow (GE Healthcare, Chalfont St Giles, UK) and the obtained crude enzyme (17 ml, 22 mg as crude enzyme) was directly used for the glycosylation of ribostamycin (56 mg) with UDP-GlcNAc 2Na (86 mg) at 28 1C for 45 h. Without purification of the glycosylated product, the next deacetylation reaction was carried out by the addition of NeoD (0.3 mg, 0.25 ml) purified by TALON (Clontech, Mountain View, CA, USA) chromatography. After 24 h at 28 1C, the resulting enzymatic solution was heat-treated (100 1C, 2 min) and then applied to a Dowex AG1-X8 (Bio-Rad, Richmond, CA, USA, [OH -] form, 12 ml) column.…”

“…A comparison of aminoglycoside biosynthetic gene clusters indicated the involvement of NeoN, a putative radical SAM enzyme that is also encoded in the paromomycin and lividomycin biosynthetic clusters. 5,7 As such an epimerization by a radical SAM enzyme is unprecedented 10 and would be an important tool to synthesize structurally diverse aminoglycosides, the functional characterization of NeoN is important and is currently underway.…”