Determining the Biochemical Properties of the Oxalate Biosynthetic Component (Obc)1 from < i > Burkholderia mallei < /i >

by Peter M. Lambert, Paul A. Nakata Oxalic acid is produced by a variety of organisms ranging from simple microbes to complex animals. This acid has been proposed to fulfill various physiological and pathological functions which vary between organisms. In bacteria from theBurkholderia genus, oxalate secretion has been shown to be quorum sensing dependent and to support pathogenicity and cell viability. In light of the critical roles of oxalate inBurkholderia as well as other organisms, it is surprising that our understanding of how this simple dicarboxylate is biosynthesized remains incomplete. Here we report the expression, purification, and partial characterization of the first intact bacterial oxalate biosynthetic enzyme, Obc1, fromB.mallei. An N-terminal His-taggedBmobc1 was cloned into pDUET, expressed inE.coli BLR (DE3), and the recombinant enzyme purified by affinity chromatography. Oxalate biosynthetic enzyme assays coupled with HPLC analysis revealed thatBmObc1 catalyzed the biosynthesis of oxalate, acetoacetate, and free CoA from oxaloacetate and a short chain acyl-CoA following Michaelis-Menten kinetics. Optimal enzyme activity was measured at pH 8.0 and a temperature around 44 °C. Kinetic analysis conducted under conditions of saturating acetyl-CoA and varying oxaloacetate concentrations resulted in a calculated Km value for oxaloacetate of 94.3± 9.2 μM (mean ± SE). Under conditions of saturating oxaloacetate concentration and varying acyl-CoA (acetyl- or pro...
Source: PLoS One - Category: Biomedical Science Authors: Source Type: research