Identification of the elusive pyruvate reductase of Chlamydomonas reinhardtii chloroplasts

Abstract

Under anoxic conditions the green alga Chlamydomonas reinhardtii activates various fermentation pathways leading to the creation of formate, acetate, ethanol and small amounts of other metabolites including D-lactate and hydrogen. Progress has been made in identifying the enzymes involved in these pathways and their subcellular locations; however, the identity of the enzyme involved in reducing pyruvate to D-lactate has remained unclear. Based on sequence comparisons, enzyme activity measurements, X-ray crystallography, biochemical fractionation and analysis of knock-down mutants, we conclude that pyruvate reduction in the chloroplast is catalyzed by a tetrameric NAD+-dependent D-lactate dehydrogenase encoded by Cre07.g324550. Its expression during aerobic growth supports a possible function as a 'lactate valve' for the export of lactate to the mitochondrion for oxidation by cytochrome- dependent D-lactate dehydrogenases and by glycolate dehydrogenase. We also present a revised spatial model of fermentation based on our immunochemical detection of the likely pyruvate decarboxylase, PDC3, in the cytoplasm.