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High‐affinity l‐malate transporter DcuE of Actinobacillus succinogenes catalyses reversible exchange of C4‐dicarboxylates

Rhie, Mi Na, Cho, Young Bin, Lee, Yeon Joo, Kim, Ok Bin
Environmental microbiology reports 2019 v.11 no.2 pp. 129-139
Actinobacillus succinogenes, Escherichia coli, antiporters, catalytic activity
Actinobacillus succinogenes is a natural succinate producer, which is the result of fumarate respiration. Succinate production from anaerobic growth with C₄‐dicarboxylates requires transporters catalysing uptake and efflux of C₄‐dicarboxylates. Transporter Asuc_1999 (DcuE) found in A. succinogenes belongs to the Dcu family and was considered the main transporter for fumarate respiration. However, deletion of dcuE affected l‐malate uptake of A. succinogenes rather than fumarate uptake. DcuE complemented anaerobic growth of Escherichia coli on l‐malate or fumarate; thus, the transporter was characterized in E. coli heterologously. Time‐dependent uptake and competitive inhibition assays demonstrated that l‐malate is the most preferred substrate for uptake by DcuE. The Vₘₐₓ of DcuE for l‐malate was 20.04 μmol/gDW·min with Kₘ of 57 μM. The Vₘₐₓ for l‐malate was comparable to that for fumarate, whereas the Kₘ for l‐malate was 8 times lower than that for fumarate. The catalytic efficiency of DcuE for l‐malate was 7.3‐fold higher than that for fumarate, showing high efficiency and high affinity for l‐malate. Furthermore, DcuE catalysed the reversible exchange of three C₄‐dicarboxylates – l‐malate, fumarate and succinate – but the preferred substrate for uptake was l‐malate. Under physiological conditions, the C₄‐dicarboxylates were reduced to succinate. Therefore, DcuE is proposed as the l‐malate/succinate antiporter in A. succinogenes.