Identification and Characterization of Oxalate Oxidoreductase, a Novel Thiamine Pyrophosphate-dependent Enzyme that Enables Anaerobic Growth on Oxalate.

Abstract

Moorella thermoacetica is an anaerobic bacterium that uses the Wood-Ljungdahl or reductive acetyl-CoA pathway as a terminal electron accepting pathway for heterotrophic and autotrophic growth on many substrates. One electron donor to M. thermoacetica is oxalate. Oxalate is an important substrate for various microbes and is produced in soil, where M. thermoacetica lives. We identified a novel enzyme, oxalate oxidoreductase (OOR) that enables growth on oxalate. This is the first known enzyme that directly oxidizes oxalate in an anaerobic organism, reducing an electron carrier by two electrons, and producing carbon dioxide or bicarbonate. In conjunction with the Wood-Ljungdahl pathway, OOR constitutes a novel path for oxalate metabolism. Exposure to oxalate induces expression of the three subunits of OOR. Like other members of the 2-oxoacid:ferredoxin oxidoreductase family, OOR contains thiamine pyrophosphate and three 4Fe-4S clusters. However, unlike previously characterized members of this family, OOR does not use coenzyme A as a substrate. Oxalate is oxidized with kcat of 0.09 per second and Km of 60 micromolar at pH 8.0. The enzyme transfers its reducing equivalents to a broad range of electron acceptors, including ferredoxin and carbon monoxide dehydrogenase. OOR can oxidize pyruvate, also without any requirement for CoA. Pyruvate oxidation leads to inhibition of OOR, probably through formation of stable TPP-bound intermediates. One of these intermediates is a hydroxyethyl-TPP radical like the one formed as part of the pyruvate:ferredoxin oxidoreductase catalytic cycle. The formation of this radical on OOR indicates a common mechanism with other 2-oxoacid:ferredoxin oxidoreductases.

A second line of research explores the importance for catalysis of a sodium binding site on the key Wood-Ljungdahl pathway enzyme acetyl-CoA synthase. Mutation of a residue in the sodium binding site decreases the CO/acetyl-CoA exchange activity by approximately 25-fold. The activity of wild-type ACS is dependent on the sodium concentration with an approximately four-fold increase in the activity between 40 micromolar and 5 millimolar. The sodium concentration where the effect is seen is lower than the expected concentration in the cell.