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Function

Burkholderia species produce oxalate which helps maintain environmental pH.^[1] Two enzymes are required to produce oxalate, ObcA and ObcB. ObcA catalyzes first, resulting in a tetrahedral C6-CoA adduct from acetyl-CoA and oxaloacetate. ObcB then produces three products from the C6-CoA adduct which include oxalate, acetoacetate, and CoA. In Burkholderia thailandensis and Burkholderia pseudomallei carries out both steps of this reaction as it is a single bi-functional enzyme.

Disease

Burkholderia species have many pathogenic consequences for plants and humans. B. cepacia effects immunocompromised individuals that may have cystic fibrosis or chronic granulomatous disease. B. pseudomallei causes a lethal infection called melioidosis that results in formation of abscesses. In plants, B. glumae causes bacterial panicle blight in rice.

Relevance

Understanding the reaction that is catalyzed by ObcA and ObcB in two steps and Obc1 in one step has clinical relevance. Currently, the molecular basis of the Obc enzymes is unknown. By studying these enzymes, a treatment may be developed for disease control that is caused by Obc enzymes.

Structural highlights

Secondary structures of Obc1 include alpha helices, beta sheets, random coil, and turns. The most abundant is alpha helices which are shaded pink, followed by beta sheets (yellow) and random coil (gray), with turns being the least abundant (purple). Obc1's consists of two domains, an N-domain that is shaded red and C-domain that is shaded grey. The C-domain is composed of two regions: A cap region and an alpha/beta hydrolase fold. A space-filling view of a protein shows a 3D representation of how much space the atoms take up in the protein. The of Obc1 shows that the atoms in this protein consume a considerable amount of space as there are no gaps in the protein. With very little room it is difficult for other molecules to move through Obc1. Obc1 is composed of equal, dispersed amounts of hydrophobic and hydrophilic patches as seen in the . The areas shaded in gray represent hydrophobic areas and areas shaded in purple represent hydrophilic areas. The that interacts with Obc1 is glycerol. Important features of the ligand glycerol include that it is hydrophilic. This is demonstrated by the hydrogen bonds it creates in the active site. The helps the protein achieve its function by making Ser-935 act in a nucleophilic attack to generate a tetrahedral intermediate, which is the first step in producing oxalate. The catalytic triad consists of S935, D997, and H1069. Amino acids that make up the active site include S785, T786, P787, R856, H934, S936, F974, R999, D1061, D1067, S1070, and R1073 (). In Obc1, there are two subdomains that make up the C-domain. The first subdomain consists of Ser-740 to Gln 1106. The second subdomain consists of Arg-529 to Ala-739. The second subdomain forms a cap over a concave region formed by an alpha/beta hydrolase fold. In the image, the N-domain is shaded red, C-domain subdomain 1 is shaded teal, and the C-domain subdomain 2 is shaded green (). In the active site, there are two arginines that help stabilize the oxyanionic intermediate during the production of oxalate, Arg-856 and Arg-999 (). A loop consisting of Ser-785-Thr-786-Pro-787 connects beta27 and aplha27 in the area of Ser-935. In this loop, Thr-786 protrudes from the loop and hydrogen bonds with a water molecule ().

Kinetic Data

Formation of CoA from the adduct is dependent on an enzyme. This was proven in an Obc1 activity assay that showed C6-CoA adduct that was produced by ObcA was stable could not be converted into CoA without being in the presense of Obc1. Also, when mutants R856K, R999K, and H934 were tested, the mutants showed decreased Kcat and Kcat/Km. Mutant R999K and the Obc wildtype had very similiar Kms, where H934A had a much higher Km and R856K and the lowest Km of the four. These results show that the positive charge on R999 and R856 play a critical role for catalysis.