Pyruvate Formate-lyase and a Novel Route of Eukaryotic ATP Synthesis in Chlamydomonas Mitochondria

Abstract

Pyruvate formate-lyase (PFL) catalyzes the non-oxidative conversion of pyruvate to formate and acetyl-CoA. PFL and its activating enzyme (PFL-AE) are common among strict anaerobic and microaerophilic prokaryotes but are very rare among eukaryotes. In a proteome survey of isolated Chlamydomonas reinhardtii mitochondria, we found several PFL-specific peptides leading to the identification of cDNAs for PFL and PFL-AE, establishing the existence of a PFL system in this photosynthetic algae. Anaerobiosis and darkness led to increased PFL transcripts but had little effect on protein levels, as determined with antiserum raised against C. reinhardtii PFL. Protein blots revealed the occurrence of PFL in both chloroplast and mitochondria purified from aerobically grown cells. Mass spectrometry sequencing of C. reinhardtii mitochondrial proteins, furthermore, identified peptides for phosphotransacetylase and acetate kinase. The phosphotransacetylase-acetate kinase pathway is a common route of ATP synthesis or acetate assimilation among prokaryotes but is novel among eukaryotes. In addition to PFL and pyruvate dehydrogenase, the algae also expresses pyruvate:ferredoxin oxidoreductase and bifunctional aldehyde/alcohol dehydrogenase. Among eukaryotes, the oxygen producer C. reinhardtii has the broadest repertoire of pyruvate-, ethanol-, and acetate-metabolizing enzymes described to date, many of which were previously viewed as specific to anaerobic eukaryotic lineages.

Characterization of Nda2, a Plastoquinone-reducing Type II NAD(P)H Dehydrogenase in Chlamydomonas Chloroplasts

“…Respective distributions of PSAD and ATPase (Fig. 6, B and C) indicate that chloroplasts were efficiently separated from mitochondria, although some unavoidable cross contamination was observed (26,42). Interestingly, the signal detected by the Nda2 antibody (Fig.…”

A Mutant in theADH1Gene ofChlamydomonas reinhardtiiElicits Metabolic Restructuring during Anaerobiosis

“…In the Enterobacteriaceae, the AdhE enzyme represents the major route for recycling NADH during fermentation (Clark and Cronan, 1980;Leonardo et al, 1996), as highlighted by the inability of an E. coli adhE mutant to grow on minimal medium under anoxic conditions (Cunningham and Clark, 1986;Gupta and Clark, 1989). Interestingly, an AdhE/ADH1 homolog is not present in the majority of prokaryotes, and among the eukaryotes, it has only been identified in a few amitochondriate protists and some green algae (Atteia et al, 2003(Atteia et al, , 2006. In the alga Polytomella, the ADHE protein was localized to the mitochondrion (Atteia et al, 2003), while in Chlamydomonas, ADH1 was found to be present in chloroplasts (Terashima et al, 2010).…”

“…A number of these fermentation pathways are typical of those present in various prokaryotes and some eukaryotes (Mus et al, 2007). Some enzymes that function in these pathways include pyruvate:ferredoxin oxidoreductase (PFR), pyruvate decarboxylase (PDC), lactate dehydrogenase (LDH), pyruvate formate lyase (PFL), alcohol dehydrogenase (ADH), phosphate acetyltransferase (PAT), acetate kinase (ACK), and the two [FeFe] hydrogenases (HYDA1 and HYDA2) and their maturation proteins, HYDG and HYDEF (Posewitz et al, 2004;Atteia et al, 2006;Ghirardi et al, 2007;Mus et al, 2007;Hemschemeier et al, 2008;Grossman et al, 2011). The anaerobic activities of these and other enzymes result in the secretion of organic acids (formate, lactate, malate, acetate, and succinate) and alcohols (ethanol and glycerol) as well as the evolution of H 2 and CO 2 (Gfeller and Gibbs, 1984;Kreuzberg, 1984;Ohta et al, 1987;Tsygankov et al, 2002;Kosourov et al, 2003;Mus et al, 2007;Dubini et al, 2009).…”

Concerted Up-regulation of Aldehyde/Alcohol Dehydrogenase (ADHE) and Starch in Chlamydomonas reinhardtii Increases Survival under Dark Anoxia

“…It is known for some time that in absence of oxygen, green algae such as Chlamydomonas and Chlorella have the ability to produce organic acids (acetate, formate, lactate, and succinate), alcohols (ethanol), and gases (CO 2 and H 2 ) (6 -9). The genome sequences of Chlamydomonas reinhardtii (10) and Chlorella variabilis NC64 (11) have greatly helped to get a good grasp of the anaerobic pathways in the two algae (12)(13)(14). Since then, in C. reinhardtii, these pathways have been actively investigated through molecular and biochemical studies of the fermentative enzymes (15)(16)(17)(18)(19), as well as physiological studies of mutant strains (20 -25).…”