Structural analysis of ColD: a unique PLP‐dependent sugar‐3‐dehydratase

Abstract

Colitose is a 3,6‐dideoxysugar found in the O‐antigen of some Gram‐negative bacteria. This unusual sugar is synthesized from mannose‐1‐phosphate via an intriguing four enzyme pathway. The focus of this investigation, GDP‐4‐keto‐6‐deoxymannose‐3‐dehydratase (ColD), is a unique pyridoxal‐5′‐phosphate (PLP)‐dependent enzyme that removes the hydroxyl from the sugar C‐3′ position. It is an interesting PLP‐dependent enzyme because it acts as both an aminotransferase and a dehydratase, but also because it lacks the conserved active site lysine residue typically observed in PLP‐dependent enzymes. We have solved wild‐type and mutant crystal structures of this enzyme in which various reaction intermediates have been trapped in the active site, including a PLP‐glutamate ketimine intermediate, a PLP‐glutamate geminal diamine, and a GDP‐linked sugar substrate analog. From these structures, it is clear that His 188 is the active site acid/base acting in both the aminotransferase and dehydratase reactions. Assays have revealed that H188K and H188N mutant ColD proteins are unable to catalyze the dehydration reaction. However, we demonstrate that the wild‐type ColD enzyme is capable of using GDP‐perosamine as a substrate to make its natural product. Taken together, these investigations have helped shed new light on the facinating manner in which ColD removes the hydroxyl group about the sugar C‐3′ position.