Structural and Biochemical Analysis of Oxalate-Biosynthetic Components, ObcA and Obc1 : 옥살산 생합성에 관여하는 효소 ObcA 와 Obc1의 구조와 생화학적 기능 분석

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Juntaek Oh

농업생명과학대학 농생명공학부
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서울대학교 대학원
Quorum sensingPublic goodsOxalate biosynthetic componentOxalic acidAcetyl-CoACrystal structureBurkholderia species
학위논문 (박사)-- 서울대학교 대학원 : 농생명공학부, 2017. 2. 이상기.
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.
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College of Agriculture and Life Sciences (농업생명과학대학)Dept. of Agricultural Biotechnology (농생명공학부)Theses (Ph.D. / Sc.D._농생명공학부)
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