Functional Characterization of YdjH, a Sugar Kinase of Unknown Specificity in Escherichia coli K12

• Published in: Biochemistry (Easton) 2019-08, Vol.58 (31), p.3354-3364
• Main Authors: Huddleston, Jamison P, Raushel, Frank M
• Format: Article
• Language: English
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/acs.biochem.9b00327

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 × 104 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.