Biosynthesis of the unusual 5,5-gem-dimethyl-deoxysugar noviose: investigation of the C-methyltransferase gene cloU

Abstract

The aminocoumarin antibiotic clorobiocin contains an unusual branched deoxysugar with a 5,5-gem-dimethyl structure. Inactivation of the putative C-methyltransferase gene cloU was carried out, which led to the loss of the axial methyl group at C-5 of this deoxysugar moiety. This result establishes the function of cloU, and at the same time it proves that the biosynthesis of the deoxysugar moiety of clorobiocin proceeds via a 3,5-epimerization of the dTDP-4-keto-6-deoxyglucose intermediate. The inactivation was carried out on a cosmid which contained the entire clorobiocin biosynthetic gene cluster. Expression of the modified cluster in a heterologous host led to the formation of desmethyl-clorobiocin and a structural isomer thereof. Both compounds were isolated on a preparative scale, their structures were elucidated by 1 H-NMR and mass spectroscopy and their antibacterial activity was assayed.

Deciphering Pactamycin Biosynthesis and Engineered Production of New Pactamycin Analogues

“…The library was screened by using a number of heterologous probes, including the ketosynthase domain of rifB (a polyketide synthase (PKS) module from the rifamycin gene cluster), [12] the cetM (aminotransferase) gene from the cetoniacytone A biosynthetic gene cluster, [13] and the cloU (C-methyltransferase) gene from the clorobiocin biosynthetic gene cluster. [14] Initial screening with the PKS probe (rifB) resulted in 44 positive clones. Further screening of these 44 PKS-positive clones with either cetM or cloU resulted in ten and six positives, respectively.…”

Natural‐Product Sugar Biosynthesis and Enzymatic Glycodiversification

“…They are all believed to employ a mechanism similar to that of TylC3. [39,40,82,94] 3.1.5. Deoxygenation…”

“…Their biosynthesis from 21 involves either 3,5-or 3-epimerization catalyzed by RmlC homologues to form 31 or 36, respectively, and subsequent 5-C-methyltransfer to give TDP-4-keto-6-deoxy-5-C-methyl-l-mannose (35). Results obtained from coupled assays of the purified epimerase NovW and 5-C-methyltransferase NovU from the novobiocin pathway, [39] along with gene disruption studies of cloU from the clorobiocin biosynthesis, [40] suggested that the biosynthesis of 30 involves 3,5-epimerization rather than 3-epimerization. However, a recent in vitro study showed that the epimerase NovW is kinetically competent only as a 3-epimerase.…”

Pathway and Enzyme Engineering and Applications for Glycodiversification

“…The activities of a few other NDP-sugar C-3 and C-5 methyltransferases have also been verified in vitro ( Table 2, entries 2.2 and 2.3). They are all believed to employ a mechanism similar to that of TylC3 [18,19,52,75].…”