Identification and Characterization of a Potential D-Lactate Oxidizing Enzyme from Rhodobacter sphaeroides

A cell like Rhodobacter sphaeroides can convert carbon sources into intermediates that \nare able to enter central carbon metabolism, which is the process of producing metabolic \nprecursors that are used to generate cellular biomass. D-Lactate is one of these potential carbon \nsources and the purpose of the study is to better understand how it is metabolized by R. \nsphaeroides and the genes involved in these processes. Wild-type R. sphaeroides underwent a \ntransposon mutagenesis procedure in which a random portion of the DNA of the organism was \ninterrupted, which inactivated the normal function of that gene. From this library, a mutant was \nisolated that was unable to grow using D-lactate but was able to utilize all other carbon substrates \ntested including L-lactate. The transposon insertion site was mapped to the gene rsp_1018 which \nis annotated to encode an iron-sulfur subunit of a glycolate oxidase. Two other nearby genes are \nlikely to encode two further subunits of this protein complex and based on nucleotide spacing it \nis likely that these three genes are co-transcribed along with another downstream gene of \nunknown function. In light of the fact that glycolate and D-lactate are structurally similar, we \npropose that rsp_1018, rsp_1019, and rsp_1020 encode for subunits of an enzyme that oxidizes \nD-lactate to pyruvate; pyruvate is an intermediate of central carbon metabolism. This conclusion \nis consistent with the D-lactate-negative phenotype of the mutant. Results indicate that growth of \nthe transposon mutant on D-lactate can be restored by introducing the genes rsp_1018 through \nrsp_1020 on a plasmid. There is also some evidence to indicate that the overexpression of the \nrsp_1018 gene product is detrimental to the cell. Our results suggest that rsp_1018 encodes a \nsubunit of a D-lactate oxidizing enzyme that is required by R. sphaeroides to metabolize D \nlactate.