oxlT

Transporter proteins change their conformations to carry their substrate across the cell membrane. The conformational dynamics is vital to understanding the transport function. We have studied the oxalate transporter (OxlT), an oxalate:formate antiporter from Oxalobacter formigenes, significant in avoiding kidney stone formation. The atomic structure of OxlT has been recently solved in the outward-open and occluded states. However, the inward-open conformation is still missing, hindering a complete understanding of the transporter. Here, we performed a Gaussian accelerated molecular dynamics simulation to sample the extensive conformational space of OxlT and successfully predicted the inward-open conformation where cytoplasmic substrate formate binding was preferred over oxalate binding. We also identified critical interactions for the inward-open conformation. The results were complemented by an AlphaFold2 structure prediction. Although AlphaFold2 solely predicted OxlT in the outward-open conformation, mutation of the identified critical residues made it partly predict the inward-open conformation, identifying possible state-shifting mutations.

An oxalate-degrading bacterium in the gut microbiota absorbs food-derived oxalate to use this as a carbon and energy source, thereby reducing the risk of kidney stone formation in host animals. The bacterial oxalate transporter OxlT selectively uptakes oxalate from the gut to bacterial cells with a strict discrimination from other nutrient carboxylates. Here, we present crystal structures of oxalate-bound and ligand-free OxlT in two distinct conformations, occluded and outward-facing states. The ligand-binding pocket contains basic residues that form salt bridges with oxalate while preventing the conformational switch to the occluded state without an acidic substrate. The occluded pocket can accommodate oxalate but not larger dicarboxylates, such as metabolic intermediates. The permeation pathways from the pocket are completely blocked by extensive interdomain interactions, which can be opened solely by a flip of a single side chain neighbouring the substrate. This study shows the structural basis underlying metabolic interactions enabling favourable symbiosis.

Oxalate serves various functions in the biological processes of plants, fungi, bacteria, and animals. It occurs naturally in the minerals weddellite and whewellite (calcium oxalates) or as oxalic acid. The environmental accumulation of oxalate is disproportionately low compared to the prevalence of highly productive oxalogens, namely plants. It is hypothesized that oxalotrophic microbes limit oxalate accumulation by degrading oxalate minerals to carbonates via an under-explored biogeochemical cycle known as the oxalate-carbonate pathway (OCP). Neither the diversity nor the ecology of oxalotrophic bacteria is fully understood. This research investigated the phylogenetic relationships of the bacterial genes oxc, frc, oxdC, and oxlT, which encode key enzymes for oxalotrophy, using bioinformatic approaches and publicly available omics datasets. Phylogenetic trees of oxc and oxdC genes demonstrated grouping by both source environment and taxonomy. All four trees included genes from metagenome-assembled genomes (MAGs) that contained novel lineages and environments for oxalotrophs. In particular, sequences of each gene were recovered from marine environments. These results were supported with marine transcriptome sequences and description of key amino acid residue conservation. Additionally, we investigated the theoretical energy yield from oxalotrophy across marine-relevant pressure and temperature conditions and found similar standard state Gibbs free energy to "low energy" marine sediment metabolisms, such as anaerobic oxidation of methane coupled to sulfate reduction. These findings suggest further need to understand the role of bacterial oxalotrophy in the OCP, particularly in marine environments, and its contribution to global carbon cycling.