Search for: All records

The lanthanide elements (Ln3), those with atomic numbers57–63 (excluding promethium, Pm3), form a cofactor complexwith pyrroloquinoline quinone (PQQ) in bacterial XoxF meth-anol dehydrogenases (MDHs) and ExaF ethanol dehydroge-nases (EDHs), expanding the range of biological elements andopening novel areas of metabolism and ecology. Other MDHs,known as MxaFIs, are related in sequence and structure to theseproteins, yet they instead possess a Ca2-PQQ cofactor. Animportant missing piece of the Ln3puzzle is defining what fea-tures distinguish enzymes that use Ln3-PQQ cofactors fromthose that do not. Here, using XoxF1 MDH from the modelmethylotrophic bacteriumMethylorubrum extorquensAM1, weinvestigated the functional importance of a proposed lantha-nide-coordinating aspartate residue. We report two crystalstructures of XoxF1, one with and another without PQQ, bothwith La3bound in the active-site region and coordinated byAsp320. Using constructs to produce either recombinant XoxF1or its D320A variant, we show that Asp320is needed forin vivocatalytic function,in vitroactivity, and La3coordination.XoxF1 and XoxF1 D320A, when produced in the absence ofLa3, coordinated Ca2but exhibited little or no catalytic activ-ity. We also generated the parallel substitution in ExaF to pro-duce ExaF D319S and found that this variant loses the capacityfor efficient ethanol oxidation with La3. These results provideevidence that a Ln3-coordinating aspartate is essential for theenzymatic functions of XoxF MDHs and ExaF EDHs, supportingthe notion that sequences of these enzymes, and the genes thatencode them, are markers for Ln3metabolism. 

Isomerization reactions are fundamental in biology. Lactate racemase, which isomerizes L- and D-lactate, is composed of the LarA protein and a nickel-containing cofactor, the nickel-pincer nucleotide (NPN). In this study, we show that LarA is part of a superfamily containing many different enzymes. We overexpressed and purified 13 lactate racemase homologs, incorporated the NPN cofactor, and assayed the isomerization of different substrates guided by gene context analysis. We discovered two malate racemases, one phenyllactate racemase, one α-hydroxyglutarate racemase, two D-gluconate 2-epimerases, and one short-chain aliphatic α-hydroxyacid racemase among the tested enzymes. We solved the structure of a malate racemase apoprotein and used it, along with the previously described structures of lactate racemase holoprotein and D-gluconate epimerase apoprotein, to identify key residues involved in substrate binding. This study demonstrates that the NPN cofactor is used by a diverse superfamily of α-hydroxyacid racemases and epimerases, widely expanding the scope of NPN-dependent enzymes.