Identification and Characterization of a Potential D-Lactate Oxidizing Enzyme from Rhodobacter sphaeroides
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Date
2020-05
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The Ohio State University
Abstract
A cell like Rhodobacter sphaeroides can convert carbon sources into intermediates that
are able to enter central carbon metabolism, which is the process of producing metabolic
precursors that are used to generate cellular biomass. D-Lactate is one of these potential carbon
sources and the purpose of the study is to better understand how it is metabolized by R.
sphaeroides and the genes involved in these processes. Wild-type R. sphaeroides underwent a
transposon mutagenesis procedure in which a random portion of the DNA of the organism was
interrupted, which inactivated the normal function of that gene. From this library, a mutant was
isolated that was unable to grow using D-lactate but was able to utilize all other carbon substrates
tested including L-lactate. The transposon insertion site was mapped to the gene rsp_1018 which
is annotated to encode an iron-sulfur subunit of a glycolate oxidase. Two other nearby genes are
likely to encode two further subunits of this protein complex and based on nucleotide spacing it
is likely that these three genes are co-transcribed along with another downstream gene of
unknown function. In light of the fact that glycolate and D-lactate are structurally similar, we
propose that rsp_1018, rsp_1019, and rsp_1020 encode for subunits of an enzyme that oxidizes
D-lactate to pyruvate; pyruvate is an intermediate of central carbon metabolism. This conclusion
is consistent with the D-lactate-negative phenotype of the mutant. Results indicate that growth of
the transposon mutant on D-lactate can be restored by introducing the genes rsp_1018 through
rsp_1020 on a plasmid. There is also some evidence to indicate that the overexpression of the
rsp_1018 gene product is detrimental to the cell. Our results suggest that rsp_1018 encodes a
subunit of a D-lactate oxidizing enzyme that is required by R. sphaeroides to metabolize D
lactate.
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Keywords
Rhodobacter sphaeroides, carbon metabolism, lactate, oxidizing enzyme