Location: Children's Nutrition Research CenterTitle: Determining the biochemical properties of the Oxalate Biosynthetic Component (Obc)1 from Burkholderia mallei
|LAMBERT, PETER - Children'S Nutrition Research Center (CNRC)|
Submitted to: PLOS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/5/2016
Publication Date: 9/19/2016
Publication URL: https://handle.nal.usda.gov/10113/5695345
Citation: Lambert, P.M., Nakata, P.A. 2016. Determining the biochemical properties of the Oxalate Biosynthetic Component (Obc)1 from Burkholderia mallei. PLoS One. 11(9):e0163294. doi:10.1371/journal.pone.0163294.
Interpretive Summary: A number of pathogenic microbes make oxalic acid. Oxalic acid has been shown to be important for the infection process and to maintain cell viability within dense populations. Although oxalic acid has been shown to be important for the survival of certain microbes we still do not understand how this acid is made. Once we understand how this acid is made we may be able to develop strategies to inhibit its production. Such a strategy may find use in combatting the infection of both plants (including many crop plants) and animals by oxalate-secreting microbial pathogens. To gain a better understanding of how oxalic acid is made we expressed, isolated, and partially characterized the enzyme responsible for making the acid from the animal pathogen, Burkholderia mallei. Enzyme assays revealed that B. mallei used the same enzymatic reaction as the plant pathogenic bacteria, B. glumae for making the acid, but this enzyme was encoded by a single gene rather than two genes. Overall, this study expands our understanding about how oxalic acid is made and may prove useful in future efforts to design strategies to combat oxalate-secreting microbial pathogens.
Technical Abstract: 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 the Burkholderia 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 in Burkholderia 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, from B. mallei. An N-terminal His-tagged Bmobc1 was cloned into pDUET, expressed in E. coli BLR (DE3), and the recombinant enzyme purified by affinity chromatography. Oxalate biosynthetic enzyme assays coupled with HPLC analysis revealed that BmObc1 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 deg 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 uM (mean +/- SE). Under conditions of saturating oxaloacetate concentration and varying acyl-CoA (acetyl- or propionyl-CoA) concentrations kinetic analysis generated a calculated Km value of 26.8 +/- 2.3 uM (mean +/- SE) for acetyl-CoA and 104.4 +/- 12.7 uM for propionyl-CoA. The significantly lower Km for acetyl-CoA suggests that it is strongly favored as a substrate over propionyl-CoA.