Abstract
BackgroundGentisate (2,5-dihydroxybenzoate) is a key ring-cleavage substrate involved in various aromatic compounds degradation. Corynebacterium glutamicum ATCC13032 is capable of growing on gentisate and genK was proposed to encode a transporter involved in this utilization by its disruption in the restriction-deficient mutant RES167. Its biochemical characterization by uptake assay using [14C]-labeled gentisate has not been previously reported.Methodology/Principal FindingsIn this study, biochemical characterization of GenK by uptake assays with [14C]-labeled substrates demonstrated that it specifically transported gentisate into the cells with V max and Km of 3.06±0.16 nmol/min/mg of dry weight and 10.71±0.11 µM respectively, and no activity was detected for either benzoate or 3-hydoxybenzoate. When GenK was absent in strain RES167 ΔgenK, it retained 85% of its original transport activity at pH 6.5 compared to that of strain RES167. However, it lost 79% and 88% activity at pH 7.5 and 8.0, respectively. A number of competing substrates, including 3-hydroxybenzoate, benzoate, protocatechuate and catechol, significantly inhibited gentisate uptake by more than 40%. Through site-directed mutagenesis, eight amino acid residues of GenK, Asp-54, Asp-57 and Arg-386 in the hydrophobic transmembrane regions and Arg-103, Trp-309, Asp-312, Arg-313 and Ile-317 in the hydrophilic cytoplasmic loops were shown to be important for gentisate transport. When conserved residues Asp-54 and Asp-57 respectively were changed to glutamate, both mutants retained approximately 50% activity and were able to partially complement the ability of strain RES167 ΔgenK to grow on gentisate.Conclusions/SignificanceOur results demonstrate that GenK is an active gentisate transporter in Corynebacterium glutamicum ATCC13032. The GenK-mediated gentisate transport was also shown to be a limiting step for the gentisate utilization by this strain. This enhances our understanding of gentisate transport in the microbial degradation of aromatic compounds.
Highlights
Transport of aromatic acids across the microbial cytoplasmic membrane is a step ahead of their catabolism
The catabolic pathways for a number of aromatic acids have been characterized genetically and biochemically, only three transporters in the aromatic acid/H+ symporter (AAHS) family have so far been functionally identified by uptake assays using their corresponding [14C]-labeled substrates: 4-hydroxybenzoate and protocatechuate transporter PcaK from Pseudomonas putida PRS2000 [2], benzoate transporter BenK from Acinetobacter sp. strain ADP1 [3] and Corynebacterium glutamicum [4], and 2,4-dichlorophenoxyacetate transporter TfdK from Ralstonia eutropha JMP134 [5]
GenK Actively Transports Gentisate To detect the capability of transporting gentisate by GenK, intracellular [14C]-labeled substrate accumulation through gentisate transport was measured for the resting cells of C. glutamicum RES167 and its variants, which were grown in lysogeny broth (LB) with gentisate and Isopropyl b-D-1thiogalactopyranoside (IPTG) induction
Summary
Transport of aromatic acids across the microbial cytoplasmic membrane is a step ahead of their catabolism. Most aromatic acids are in their dissociated form under neutral or basic conditions, and need to be actively transported across membranes. This procedure is assisted by members of the aromatic acid/H+ symporter (AAHS) family within the major facilitator superfamily (MFS) [1]. The catabolic pathways for a number of aromatic acids have been characterized genetically and biochemically, only three transporters in the AAHS family have so far been functionally identified by uptake assays using their corresponding [14C]-labeled substrates: 4-hydroxybenzoate and protocatechuate transporter PcaK from Pseudomonas putida PRS2000 [2], benzoate transporter BenK from Acinetobacter sp. Its biochemical characterization by uptake assay using [14C]-labeled gentisate has not been previously reported
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