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Summary The pseudoglycosyltransferase (PsGT) VldE is a glycosyltransferase-like protein that is similar to trehalose 6-phosphate synthase (OtsA). However, in contrast to OtsA, which catalyzes condensation between UDP-glucose and glucose 6-phosphate, VldE couples two pseudosugars to give a product with an α,α- N -pseudoglycosidic linkage. Despite their unique catalytic activity and important role in the biosynthesis of natural products, little is known about the molecular basis governing their substrate specificity and catalysis. Here, we report comparative biochemical and kinetic studies using recombinant OtsA, VldE, and their chimeric proteins with a variety of sugar and pseudosugar substrates. We found that the chimeric enzymes can produce hybrid pseudo-(amino)disaccharides, and an amino group in the acceptor is necessary to facilitate a coupling reaction with a pseudosugar donor. Furthermore, we found that the N-terminal domains of the enzymes not only play a major role in selecting the acceptors, but also control the type of nucleotidyl diphosphate moiety of the donors. [ABSTRACT FROM AUTHOR]

Myxobacteria are efficient producers of natural products with various biological activities. Nevertheless, genetic systems for markerless mutagenesis are only available for Myxococcus xanthus DK1622, a strain that is not known to produce any secondary metabolites. We report here the development of such a technique for Stigmatella aurantiaca DW4/3-1, a multiproducer of natural products. The system is used to further characterize the combined polyketide synthase/peptide synthetase system responsible for myxothiazol biosynthesis. Analysis of a series of point mutations and in-frame deletions shows that the myxothiazol megasynthetase is a rather specialized and delicate system that is hardly capable of processing unnatural intermediates. An in-frame deletion of the MtaC oxidation domain results in the production of unchanged bis-thiazole myxothiazol instead of the expected thiazoline-thiazole derivative of the compound. This shows that this domain within a nonribosomal peptide synthetase is not necessary for product formation. [Copyright &y& Elsevier]

Summary: Pactamycin, one of the most densely functionalized aminocyclitol antibiotics, has pronounced antibacterial, antitumor, antiviral, and antiplasmodial activities, but its development as a clinical drug was hampered by its broad cytotoxicity. Efforts to modulate the biological activity by structural modifications using synthetic organic chemistry have been difficult because of the complexity of its chemical structure. However, through extensive biosynthetic studies and genetic engineering, we were able to produce analogs of pactamycin that show potent antimalarial activity, but lack significant antibacterial activity, and are about 10–30 times less toxic than pactamycin toward mammalian cells. The results suggest that distinct ribosomal binding selectivity or new mechanism(s) of action may be involved in their plasmodial growth inhibition, which may lead to the discovery of new antimalarial drugs and identification of new molecular targets within malarial parasites. [Copyright &y& Elsevier]

A DNA region of 41.847 base pairs from Melittangium lichenicola Me l46 is shown to be responsible for the biosynthesis of the potent electron transport inhibitor melithiazol. Melithiazol is formed by a combined polyketide synthase/peptide synthetase system resembling the myxothiazol megasynthetase from Stigmatella aurantiaca DW4/3-1. Both natural products share an almost identical core region but employ different starter molecules. Additionally, melithiazol contains a terminal methyl ester instead of the amide moiety found in myxothiazol. Similar to myxothiazol formation, the methyl ester is formed via an amide intermediate, which is converted by a hydrolase and an unusual type of SAM (S-adenosyl-L-methionine)-dependent methyltransferase into the methyl ester. When transferred into the myxothiazol A (amide) producer, these two genes lead to the formation of the methyl ester of myxothiazol. The methyl transferase described is a member of a protein subfamily of a previously unknown function lacking a typical SAM binding motif. [Copyright &y& Elsevier]

Summary: Gene valD, encodes a large vicinal oxygen chelate (VOC) superfamily protein, has been identified in the validamycin biosynthetic gene cluster. Inactivation of valD significantly reduced validamycin A production, which was fully restored with the full-length valD and partially restored with either N-terminal or C-terminal half by complementation. Heterologously expressed ValD catalyzed the epimerization of 2-epi-5-epi-valiolone to 5-epi-valiolone. This metalloenzyme is a homodimer with a metal ion-binding ratio of 0.73 mol/mole protein toward Fe2+, Mn2+, Ni2+, and Zn2+. Individual and combined site-directed mutations of eight putative active site residues revealed that the N-terminal H44/E107 and the C-terminal H315/E366 are more critical for the activity than the internal H130, E183, H229, and E291. Our data have established ValD as one of the largest proteins of the VOC superfamily, catalyzing an alternative epimerization for C7N-aminocyclitol biosynthesis. [Copyright &y& Elsevier]

Summary: A 45 kb DNA sequencing analysis from Streptomyces hygroscopicus 5008 involved in validamycin A (VAL-A) biosynthesis revealed 16 structural genes, 2 regulatory genes, 5 genes related transport, transposition/integration or tellurium resistance; another 4 genes had no obvious identity. The VAL-A biosynthetic pathway was proposed, with assignment of the required genetic functions confined to the sequenced region. A cluster of eight reassembled genes was found to support VAL-A synthesis in a heterologous host, S. lividans 1326. In vivo inactivation of the putative glycosyltransferase gene (valG) abolished the final attachment of glucose for VAL production and resulted in accumulation of the VAL-A precursor, validoxylamine, while the normal production of VAL-A could be restored by complementation with valG. The role of valG in the glycosylation of validoxylamine to VAL-A was demonstrated in vitro by enzymatic assay. [Copyright &y& Elsevier]