Structure of a Bacterial Ketoisomerase/N‐acetyltransferase

Abstract

3‐acetamido‐3,6‐dideoxy‐a‐D‐galactose, Fuc3NAc, is an unusual dideoxysugar found in the O‐antigens of various Gram‐negative bacteria and in the S‐layer glycans of some Gram‐positive bacteria. The biosynthetic pathway for its production in Aneurinibacillus thermoaerophilus L420‐91T was elucidated in 2003. In the case of A. thermoaerophilus, all of the enzymes required for the production of dTDP‐Fuc3NAc exist on separate polypeptide chains. However, this is not the case for other organisms. In Shewanella denitrificans, a Gram‐negative bacterium, a gene (fdtD) has been identified that putatively encodes for a bifunctional protein with both 3,4‐ketoisomerase and N‐acetyltransferase activities (the third and fifth steps in the pathway). The quaternary structure of this hypothetical protein is intriguing since the sugar isomerases and the N‐acetyltransferases are typically dimers and trimers, respectively. Curious as to its molecular architecture, we initiated a biochemical and structural analysis on this protein. From our investigation, it is now known that the quaternary structure of the enzyme is hexameric with 322‐symmetry. Each polypeptide chain of the hexamer folds into two specific domains connected by a flexible loop. The N‐terminal ß‐helix motif harbors the active site responsible for the N‐acetylation reaction. The C‐terminal region folds into an antiparallel flattened ß‐barrel and contains the active site responsible for isomerization. Biochemical assays verified the two proposed catalytic activities of the enzyme and revealed that the 3,4‐keto isomerization event leads to inversion of configuration about the hexose C‐4' carbon. This work represents the first molecular characterization of a bifunctional protein involved in the biosynthesis of Fuc3NAc.