Structural Insights into an Oxalate-producing Serine Hydrolase with an Unusual Oxyanion Hole and Additional Lyase Activity

Abstract

In Burkholderia species, the production of oxalate, an acidic molecule, is a key event for bacterial growth in the stationary phase. Oxalate plays a central role in maintaining environmental pH, which counteracts inevitable population-collapsing alkaline toxicity in amino acid-based culture medium. In the phytopathogen Burkholderia glumae, two enzymes are responsible for oxalate production. First, the enzyme oxalate biosynthetic component A (ObcA) catalyzes the formation of a tetrahedral C6-CoA adduct from the substrates acetyl-CoA and oxaloacetate. Then the ObcB enzyme liberates three products from the C6-CoA adduct: oxalate, acetoacetate, and CoA. Interestingly, these two stepwise reactions are catalyzed by a single bifunctional enzyme, Obc1, from Burkholderia thailandensis and Burkholderia pseudomallei. Obc1 has an ObcA-like N-terminal domain and shows ObcB activity in its C-terminal domain despite no sequence homology with ObcB. We report the crystal structure of Obc1 in its apo and glycerol-bound form at 2.5 Å and 2.8 Å resolution, respectively. The Obc1 N-terminal domain is essentially identical both in structure and function to that of ObcA. Its C-terminal domain has an ␣/␤ hydrolase fold that has a catalytic triad for oxalate production and a novel oxyanion hole distinct from the canonical HGGG motif in other ␣/␤ hydrolases. Functional analyses through mutagenesis studies suggested that His-934 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 and an example of divergent evolution of the ␣/␤ hydrolase fold, which has both hydrolase and lyase activity.

Proteomic Profiling of Burkholderia thailandensis During Host Infection Using Bio-Orthogonal Noncanonical Amino Acid Tagging (BONCAT)

“…While it is beyond the scope of this study to reannotate all proteins differentially expressed during B. thailandensis infection, we reviewed the annotation of the proteins showing the largest changes in expression during infection, which provided some intriguing insights. For example, the protein showing the highest expression level in host-associated bacteria (Q2T6D1, encoded by BTH_II1071) has been annotated as belonging to a “Domain of Unknown Function” protein family DUF849 generally associated with beta-keto acid cleavage enzymes (Bastard et al, 2014 ); however, recent structural and functional studies have concluded that the protein is Obc1, a bifunctional enzyme that catalyzes quorum sensing-dependent oxalogenesis, which is indispensable for Burkholderia survival in stationary phase (Oh et al, 2016 ). Similarly, the protein showing the highest fold change in overexpression in host-associated bacteria (Q2T3Y0, encoded by BTH_II1925) has been annotated as a “chitin binding domain protein” belonging to a family of lytic polysaccharide monooxygenases that mediate biomass degradation; but in recent years, several members of this family have been shown to contribute to virulence in a variety of bacterial pathogens, including B. mallei (A0A0H2WBG2, encoded by BMAA1785) (Frederiksen et al, 2013 ).…”

YqfB protein from Escherichia coli: an atypical amidohydrolase active towards N4-acylcytosine derivatives

“…The latter functions as an atypical oxyanion hole, which, in the case of hydrolases, is usually formed by the hydrogen bond donors from two or more residues, one of which is typically a Gly. Notably, only the Obc1 protein from Burkholderia thailandensis and O-acetyltransferase PatB1 from Bacillus cereus possess an oxyanion hole formed by arginines 23,24 . The role of YqfB His70 and Tyr89 is to provide the framework for substrate recognition.…”

Mechanism and Structural Insights Into a Novel Esterase, E53, Isolated From Erythrobacter longus

“…The family IV esterase contains two conserved motifs, GXSAG and HGGG motif. The HGGG motif (H-G-G-G) is involved in forming the oxyanion hole and plays a vital role in stabilizing the tetrahedral intermediate of the reaction ( Oh et al, 2016 ; Santiago et al, 2018 ). The GXSAG motif contains the serine, one of the catalytic triad of the enzyme.…”