Biosynthesis of Nucleotide Diphospho 6-Deoxy Sugars

6-Deoxygenation involves three enzyme activities the 4,6-dehydratase, the 3,5-epimerase and the reductase. The 4,6-dehydratases work by a broadly similar mechanism to the 5 -adenosylhomocysteine hydrolase. They contain one tightly bound NAD(P) molecule per active site. This oxidises the 4-position to a ketone and an iilcB reaction drives off the 6-OH. The glucosenone is then reduced by the NAD(P)H to the 4-keto-6-deoxyhexose. The intermediacy of the glucosenone has been verified by rapid quench/soft ionisation mass spectrometry.  

By monitoring exchange of the C5 proton and the C6 oxygen with solvent by MALDI-TOF mass spectrometry in both reactant and product, it was possible to show that the reaction was concerted. With the acid catalytic Asp mutated to Asn, however, deprotonation at C5 could occur without loss of 06. 

The GDP-6-deoxy-4-keto-D-mannose then can either be reduced, giving GDP-D-rhamnose (the unusual stereoisomer of rhamnose), or be isomerised at positions 3 and 5 and reduced to L-fucose at the active site of an unusual bifunctional enzyme, which is both reductase and isomerase. The reduction occurs with proS (B-side) specificity of the NADPH coenzyme and the enzyme will catalyse epimerisation and exchange of H3 and H5 in the absence of NADPH.The available structures of the E. coli enzyme show NADPH bound in the expected syn conformation and counterparts to the His and Lys suggested to play a role in the action of the dTDP-6-deoxy-4-ketoglucose isomerase in the correct position. 

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