DTDP-4-keto-6-deoxy-D-Glucose

The multi-enzyme system was recently utilized for the synthesis of dTDP-4-keto-6-deoxy-a-D-glucose 3 and dTDP-P-L-rhamnose 5 generating dlTP from dTMP by dTMP kinase and acetate kinase [66, 71]. Further combination with GerB, a dTDP-4-keto-6-deoxy-D-glucose aminotransferase, gave dTDP-4-amino-4,6-dideoxy-D-glucose [72]. 

The keto-sugar nucleotide dTDP-L-rhamnose is synthesized from dTDP-4-keto-6-deoxy-D-glucose by dTDP-i-rhamnose synthase [104, 105). The enzyme consists of two components, a cofactor independent epimerase and an NADH-dependent reductase. The epimerase component is inactive without the reductase component. The mechanism involves epimerization of two stereocenters flanking a carbonyl group, via sequential deprotonation/reprotonation, with two enol intermediates. Complete solvent isotope incorporation into both epimerized stereocenters was observed, and primary substrate-derived KIEs have been determined [104],... 

From the formation of dTDP-4-keto-6-deoxy-D-glucose to dTDP-L-mycarose, six proteins are involved, five of which have potential functions assigned 3,5-epimerization (EryVII), 2,3-dehydration and reduction (EryBVI and EryBII), 3-C-methylation (EryBIII), and 4-ketoreduction (EryBIV) [4]. 

Tylosin contains a 16-membered lactone to which three deoxysugar moieties are attached. A disaccharide D-mycaminosyl-D-mycarose is linked at C5 and a D-mycinose at C23. Several genes have been cloned that encode deoxysugar biosynthetic enzymes most of them are involved in dTDP-D-mycaminose biosynthesis [48,49]. TylAl and TylA2 correspond to the dTDP-o-glucose synthase and dTDP-4,6-dehydratase, respectively. A putative isomerase not yet identified acts on dTDP-4-keto-6-deoxy-D-glucose, and then aminotransferase (TyiB) and C-methyltransferase (TylMl) to generate the final dTDP-D-mycaminose. 

Some unresolved issues remain concerning the biosynthesis of the deoxysugar moieties of nogalamycin. After formation of the common pathway intermediate dTDP-4-keto-6-deoxy-D-glucose, nogalose biosynthesis requires C-3 methy-lation (Scheme 9, step 29) in addition to epimerization (Scheme 9, step 26)... 

Scheme 9 Possible biosynthetic pathway of dTDP-nogalose as proposed by Torkkell et al. [53]. The pathway deviates from deoxysugar biosynthetic routes after formation of the common intermediate dTDP-4-keto-6-deoxy-D-glucose...

Nelsestuen and Kirkwood obtained additional evidence in support of the oxidation-reduction mechanism by study of the reaction catalyzed by a highly purified epimerase containing NAD . When treated with sodium borohydride in the presence of UDP-D-glucose, the enzyme-bound NAD was reduced, with a concomitant loss of enzymic activity towards UDP-D-glucose. The reduced enzyme regained practically all of its activity when it was incubated with dTDP-6-deoxy-D-xyfo-hexos-4-ulose ( 4-keto-6-deoxy-D-glucose ), and NADH was simultaneously oxidized with a loss of tritium,... 

In order to provide dTDP-deoxy sugars by combinatorial biocatalysis we have utiHzed the enzymes for the dTDP- 3-L-rhamnose pathway. The successful combination of pathway enzymes with optimized enzyme productivities (amount of product per unit of enzyme) needs a concise kinetic and inhibition analysis. Scheme 5.1 depicts the biosynthetic pathway of dTDP- 3-L-rhamnose with important km and Ki constants. The enzymes RmlA and RmlB are highly controlled by the intermediate, dTDP-4-keto-6-deoxy-a-D-glucose 3, the product 5 or by... 

Scheme 6 Putative early common steps in the formation of deoxysugar and aminodeoxy-sugars in anthracydine biosynthesis. Glucose-3-phosphate is converted to dTDP-4-keto-2,6-deoxy-D-glucose and dTDP-3-amino-2,3,6-deoxy-4-keto-D-glucose via an instable 3,4-diketointermediate...

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