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

Neomycin is one of the clinically important 2-deoxystreptamine (DOS)-containing aminoglycoside antibiotics, which specifically interact with bacterial rRNA and inhibit protein synthesis. These compounds have attracted attention through their potential as anti-HIV and antiplasmid agents owing to their unique nucleotide recognition ability. Structural diversification of aminoglycosides is therefore a promising approach for the generation of novel bioactive compounds. Glycoside diversification by the use of glycosyltransferases is an attractive way to carry this out. 5] However, in the biosynthetic pathway of aminoglycosides, only a phosphoribosyltransferase has been characterized, even though many putative glycosyltransferase genes have been identified in the biosynthetic gene clusters. The only readily apparent glycosyltransferase gene in the neomycin biosynthetic gene cluster is neoD (neo8). 9] The deduced product (NeoD) belongs to the GT4 family, members of which catalyze retaining glycosyl transfer reactions with NDP-sugars. Comparative genetics has revealed that neoD-homologous genes are conserved among all the reported gene clusters of DOScontaining aminoglycosides, and are thus proposed to be involved in the formation of a common biosynthetic intermediate, paromamine (4, Scheme 1). Recently, 2’-N-acetylparomamine (3) was proposed to be a biosynthetic intermediate ACHTUNGTRENNUNGaccording to the deacetylase activity toward 3 of BtrD, an enzyme encoded in the butirosin biosynthetic gene cluster. Thus, NeoD was presumed to catalyze N-acetylglucosaminylation of DOS. To confirm the function of NeoD, the enzymatic activity of the recombinant NeoD protein was investigated. NeoD was co-expressed with molecular chaperone GroES and GroEL in E. coli to increase the amount of soluble protein (Figure 1A). Because NeoD showed low binding affinity for any kind of resin, even Ni affinity resin when expressed as a His-tagged protein, the cell-free extract of E. coli expressing NeoD was used for enzymatic assays. After incubating recombinant NeoD with UDP-GlcNAc and DOS, enzyme reaction products were treated with 2,4-dinitrofluorobenzene (DNFB), and the derivatives were analyzed by HPLC. As a result, about 80% of DOS was consumed, and a new product was observed at a retention time of 18 min (Figure 2A). This peak was not observed in a control reaction with the cell-free extract of E. coli harboring an empty plasmid (Figure 2B). The new peak showed m/z 696.4 by LC–ESIMS analysis, indicating that bis(2,4-dinitrophenyl)-3 ([M H] 696.3) was produced. The NeoD reaction product was further isolated from a large-scale enzyme reaction (4 mL) and its structure was confirmed by NMR and FABMS to be 3 (Supporting Information). Another possible glycosyl donor, UDP-glucose, and other possible gly[a] Prof. Dr. T. Eguchi Department of Chemistry and Materials Science Tokyo Institute of Technology O-okayama, Meguro-ku, Tokyo 152-8551 (Japan) Fax: (+81)3-5734-2631 E-mail : eguchi@cms.titech. ac.jp [b] K. Yokoyama, Y. Yamamoto, Dr. F. Kudo Department of Chemistry, Tokyo Institute of Technology O-okayama, Meguro-ku, Tokyo 152-8551 (Japan) Supporting information for this article is available on the WWW under http://www.chembiochem. org or from the author. Scheme 1. Sequential glycosylation and deacetylation steps in neomycin biosynthesis.