Identification of the Elusive Pyruvate Reductase of Chlamydomonas reinhardtii Chloroplasts.

Steven J Burgess,Justin A Yeoman,James W Murray,Peter J Nixon,Wojciech Bialek,Jacob G Bundy,Volker Behrends,Hussein Taha,Marko Boehm,Oksana Iamshanova,Kher Xing Chan

doi:10.1093/pcp/pcv167

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.

Highlights

The unicellular green alga Chlamydomonas reinhardtii, which is found in fresh water and soil, has a versatile metabolism allowing it to acclimate to a wide range of environments and stresses (Catalanotti et al 2013)
Understanding the fermentative pathways in C. reinhardtii has attracted interest because of the potential in using this and related algae for producing biohydrogen (Kruse et al 2005), and the possibility that knocking out competing fermentation pathways might be one route to improve the yields of hydrogen
Hydrogen is produced by the combined activity of two chloroplast-localized [Fe–Fe]-hydrogenases (HYDA1 and HYDA2) (Happe and Naber 1993, Forestier et al 2003, Meuser et al 2012) with electrons derived from reduced ferredoxin (PETF) (Winkler et al 2009) produced by pyruvate:ferredoxin oxidoreductase (PFOR) and by photosystem one (PSI) in the light (Noth et al 2013, van Lis et al 2013)

Summary

Introduction

The unicellular green alga Chlamydomonas reinhardtii, which is found in fresh water and soil, has a versatile metabolism allowing it to acclimate to a wide range of environments and stresses (Catalanotti et al 2013). When cells encounter anoxic conditions, the tricarboxylic acid (TCA) cycle shuts down, necessitating ATP generation by glycolysis, with NAD+ regenerated through a number of different fermentative pathways, leading to the excretion of formate, ethanol and acetate (Gfeller and Gibbs 1984), with hydrogen (Gfeller and Gibbs 1984, Kreuzberg 1984), glycerol (Klein and Betz 1978, Gfeller and Gibbs 1984, Meuser et al 2009), succinate (Dubini et al 2009), lactate (Gfeller and Gibbs 1984, Husic and Tolbert 1985) and malic acid (Mus et al 2007) produced, but at lower levels This is accompanied by a concomitant reorganization in nitrogen metabolism, and activation of glyoxylate and reductive pentose phosphate pathways (Subramanian et al 2014). Our data allow us to provide a refined description of pyruvate metabolism during anoxia

Results

Discussion

Materials and Methods