Structural and Biochemical Analysis of Oxalate-Biosynthetic Components, ObcA and Obc1

Abstract

In Burkholderia species, the quorum sensing-dependent production of oxalic acid is an indispensable process for bacterial growth during stationary phase. Oxalic acid produced plays a central role in maintaining the environmental pH, which counteracts inevitable population-collapsing alkaline toxicity in amino acid-based culture medium. In B. glumae, two enzymes are responsible for oxalic acid production. First, the oxalate biosynthetic component (Obc) A catalyzes the formation of a tetrahedral C6-CoA adduct from acetyl-CoA and oxaloacetate. Then the ObcB enzyme liberates three products from the C6-CoA adduct: oxalic acid, acetoacetate, and CoA. Interestingly, these two stepwise reactions are catalyzed by a single bifunctional enzyme, Obc1, from B. thailandensis and B. pseudomallei. Obc1 has an ObcA-like N-terminal domain and shows ObcB activity in its C-terminal domain, despite no sequence homology with ObcB. In this thesis, crystal structure and functional analysis of ObcA and Obc1 are reported, revealing structural and functional insights of oxalogenesis. Overall structure of ObcA and N-terminal domain of Obc1 exhibits (β/α)8-barrel fold, with a metal ion coordinated in its active site. In catalysis, substrate oxaloacetate serves as a nucleophile by forming an enolate intermediate mediated by Tyr residue as a general base, which then attacks the thioester carbonyl carbon of a second substrate acetyl-CoA to yield a tetrahedral adduct. In many reactions involving tetrahedral CoA intermediate, the presence of a negative charge in the intermediate leads to collapse of the intermediate, ejecting CoA moiety form the active site. However, the presence of the metal-coordination shell and absence of general acid(s) could produce an unusual tetrahedral CoA adduct as a stable product. Structure of C-terminal domain of Obc1 has an α/β hydrolase fold that contains a catalytic triad for oxalic acid production and a novel oxyanion hole distinct from the canonical HGGG motif in other α/β hydrolases. Functional analyses through mutagenesis studies suggest that His934 is an additional catalytic acid/base for its lyase activity and liberates two additional products, acetoacetate and CoA. These results provide structural and functional insights into bacterial oxalogenesis

an example of the functional diversity of an enzyme to survive and adapt in the environment and divergent evolution of the α/β hydrolase fold, which has both hydrolase and lyase activity.