Eukaryotic Cell doi:10.1128/EC.00368-07
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.
The pyruvate formate-lyase (Pfl1) of Chlamydomonas reinhardtii – a biochemical and physiological characterization of a typically bacterial enzyme in a eukaryotic alga
Anja Hemschemeier*,
Jessica Jacobs,
and
Thomas Happe
Ruhr Universität Bochum, Fakultät für Biologie und Biotechnologie, Lehrstuhl für Biochemie der Pflanzen, AG Photobiotechnologie, 44780 Bochum, Germany
* To whom correspondence should be addressed. Email:
anja.hemschemeier{at}rub.de.
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Abstract |
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The unicellular green alga Chlamydomonas reinhardtii has a special type of anaerobic metabolism that is quite unusual for eukaryotes. It has two oxygen sensitive [FeFe]-hydrogenases (EC 1.12.7.2) that are coupled to photosynthesis and furthermore, a formate and ethanol producing fermentative metabolism, which was proposed to be initiated by pyruvate formate-lyase (Pfl, EC 2.3.1.54). Pfl enzymes are commonly found in prokaryotes, but only rarely in eukaryotes. Both the hydrogen and the formate/ethanol producing pathways are involved in a sustained anaerobic metabolism of the alga, which can be induced by sulphur-depletion in illuminated cultures. Until now, the presence of a Pfl protein in C. reinhardtii has been predicted from formate secretion and the homology of the deduced protein of the PFL1 gene model to known Pfl enzymes. In this study, we proved the formate producing activity of the putative Pfl1 enzyme by heterologous expression of the C. reinhardtii PFL1 cDNA in Escherichia coli and subsequent in vitro activity tests of the purified protein. Furthermore, a Pfl deficient E. coli strain secretes formate when expressing the PFL1 cDNA of C. reinhardtii. We also examined the Pfl1 fermentation pathway of C. reinhardtii under the physiological condition of sulphur-depletion. Genetic and biochemical analyses show that sulphur-depleted algae express genes encoding enzymes acting downstream of Pfl1 and further potentially ethanol producing enzymes such as pyruvate decarboxylase (Pdc, EC 4.1.1.1) or pyruvate ferredoxin oxidoreductase (Prf, EC 1.2.7.1). The latter enzymes might substitute for Pfl1 activity when Pfl1 is specifically inhibited by hypophosphite.
