Characterization of Sugar Biosynthesis Enzymes

Sequence analysis of genes in sugar biosynthesis gene clusters has resulted in tentative assignments for putative functions for the encoded proteins in the biosynthesis of the corresponding deoxy sugars. However, a lack of biochemical 

The first two enzymatic steps are common to the biosynthesis of all 6DOHs and lead to the biosynthesis of the intermediate TDP-4-keto-6-deoxy-D-glucose. These enzymatic reactions have been extensively studied in different sugar biosynthesis pathways, such as tylosin or mithramycin [13]. 

Many DOHs, such as L-daunosamine, L-epivancosamine or L-ristosamine, contain an amino group at C3, which is introduced by an aminotransferase. The substrate for this reaction is the 3-keto sugar intermediate that arises as a consequence of the action of a 2,3-dehydratase. This transaminahon reaction has been biochemically characterized in the biosynthesis of L-epivancosamine [10]. Using a coupled reaction with EvaB (2,3-dehydratase) and EvaC (aminotransferase), with pyridoxal-5-phosphate (PEP) as a coenzyme and L-glutamate as a cosubstrate, they were able to show conversion of TDP-4-keto-2,6-dideoxyglucose into thymidine-5 -diphospho-3-amino-2,3,6-trideoxy-D-threo-hexopyranos-4-ulose. 

Many DOHs, such as L-mycarose, L-epivancosamine, L-noviose or L-dauno-samine, show an L configuration. Formation of L-DOH requires the action of a 5- or 3,5-epimerase. The epimerase EvaD from the biosynthesis pathway of L-epivancosamine was shown to act as a 5-epimerase on the intermediate thymidine-5 -diphospho-3-amino-2,3,6-trideoxy-D-tIireo-hexopyranos-4-ulose [10]. On the 

The flnal step in the biosynthesis of most DOHs is the reduchon of the C4 ketone in a stereospecific manner. Ketoreductases responsible for these reactions in the biosynthesis of L-epivancosamine and L-mycarose have been tested in in vitro assays, using NADPH as cosubstrate [10, 19]. 

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