Abstract
The ydj gene cluster is annotated to catalyze the catabolism of an unknown carbohydrate. Previously, YdjI, a class II aldolase, was shown to catalyze the retro-aldol cleavage of l-glycero-l-galacto-octuluronate-1-phosphate into DHAP and l-arabinuronate. In this report, the functional characterization of YdjH is presented. YdjH catalyzes the phosphorylation of 2-keto-monosaccharides at the C1 hydroxyl group with a substrate profile significantly more stringent than that of YdjI. Similar to YdjI, YdjH shows a strong preference for higher-order monosaccharides (seven to nine carbons) with a carboxylate terminus. The best substrate was determined to be l-glycero-l-galacto-octuluronate, yielding l-glycero-l-galacto-octuluronate-1-phosphate with a k(cat) of 16 s(-1) and a k(cat)/K(m) of 2.1 × 10(4) M(-1) s(-1). This is apparently the first reported example of kinase activity with eight-carbon monosaccharides. Two crystal structures of YdjH were previously determined to 2.15 and 1.8 Å resolution (Protein Data Bank entries 3H49 and 3IN1 ). We present an analysis of the active site layout and use computational docking to identify potential key residues in the binding of l-glycero-l-galacto-octuluronate.