SorF: a glycosyltransferase with promiscuous donor substrate specificity in vitro.

Glycosylations are well-established steps in numerous biosynthetic pathways, and the attached sugar moieties often influence the specificity or pharmacology of the modified compounds. The sorangicins belong to the polyketide family of natural products, and exhibit antibiotic activity through inhibition of bacterial RNA polymerase. We have identified the sorangicin biosynthetic gene cluster in the producing myxobacterium Sorangium cellulosum So ce12. Within the cluster, sorF encodes a putative glycosyltransferase. To determine its function in sorangicin biosynthesis, SorF was heterologously expressed as a fusion protein in Escherichia coli. After purification by affinity chromatography, SorF was found to glucosylate sorangicin A in vitro, utilizing UDP-alpha-D-glucose as the natural donor substrate. Additionally, SorF showed high flexibility towards further UDP- and dTDP-sugars and was able to transfer several other sugar moieties-alpha-D-galactose, alpha-D-xylose, beta-L-rhamnose, and 6-deoxy-4-keto-alpha-D-glucose-onto the aglycon. SorF is therefore one of the rare glycosyltransferases able to transfer both D- and L-sugars, and could thus be used to generate novel sorangiosides.

Nucleotide deoxysugars: essential tools for the glycosylation engineering of novel bioactive compounds.

The irreversible spread of new resistance mechanisms against existing therapeutical antibiotics has led to the development of technologies and strategies for the glycosylation engineering of novel antibiotics. Amino-, C-branched and O-methylated 6-deoxyhexoses play a favourite role in the biosynthesis of clinically important antibiotics like tylosin, erythromycin or oleandomycin and are essential for the antibiotic activity. They are transferred onto the aglycon by glycosyltransferases using dTDP-activated deoxyhexoses. The in vitro biochemical characterization of the biosynthetic enzymes and the glycosyltransferases are, however, hampered due to the poor synthetic access to dTDP-activated deoxysugars and their biosynthetic intermediates. The overcoming of the poor availability of dTDP-activated sugars was the target of several researchers to fulfil their distinct aims with these sugars which were mostly involved in the synthesis of different biological active compounds. Several completely different strategies were used in the past years to improve the availability of dTDP-activated deoxysugars, varying from complete enzymatic synthesis via syntheses using reaction technology for yield optimization to full organic synthesis or shortcuts like the decomposition of commercially available antibiotics and later chemical activation of the sugar moieties. This review gives a survey of the synthesis of dTDP-activated sugars by chemical and chemoenzymatic approaches and discusses the promiscuity of glycosyltransferases to evaluate the chances for applying them for the production of new bioactive compounds. It summarizes the most important enzymes in the field of synthesis using biosynthetic pathway enzymes and describes solutions for occurring challenges during application. Finally, this review will give a survey about the availability of dTDP-activated sugars in sufficient scale and will also point at important sugars which are still bottlenecks and difficult to synthesize and therefore should become a target for enhanced research efforts.

An enzyme module system for the synthesis of dTDP-activated deoxysugars from dTMP and sucrose.

A flexible enzyme module system is presented that allows preparative access to important dTDP-activated deoxyhexoses from dTMP and sucrose. The strategic combination of the recombinant enzymes dTMP-kinase and sucrose synthase (SuSy), and the enzymes RmlB (4,6-dehydratase), RmlC (3,5-epimerase) and RmlD (4-ketoreductase) from the biosynthetic pathway of dTDP-beta-L-rhamnose was optimized. The SuSy module (dTMP-kinase, SuSy, +/-RmlB) yielded the precursor dTDP-alpha-D-glucose (2) or the biosynthetic intermediate dTDP-6-deoxy-4-keto-alpha-D-glucose (3) on a 0.2-0.6 g scale with overall yields of 62 % and 72 %, respectively. A two-step strategy in which the SuSy module was followed by the deoxysugar module (RmlC and RmlD) resulted in the synthesis of dTDP-beta-L-rhamnose (4; 24.1 micromol, overall yield: 35.9 %). Substitution of RmlC by DnmU from the dTDP-beta-L-daunosamine pathway of Streptomyces peucetius in this module demonstrated that DnmU acts in vitro as a 3,5-epimerase with 3 as substrate to yield 4 (32.2 mumol, overall yield: 44.7 %). Chemical reduction of 3 with NaBH4 gave a mixture of the C-4 epimers dTDP-alpha-D-quinovose (6) and dTDP-alpha-D-fucose (7) in a ratio of 2:1. In summary, the modular character of the presented enzyme system provides valuable compounds for the biochemical characterization of deoxysugar pathways playing a major role in microbial producers of antibiotic and antitumour agents.