Tài liệu Báo cáo khoa học: Expression of two [Fe]-hydrogenases in Chlamydomonas reinhardtii under

Expression of two [Fe]-hydrogenases in Chlamydomonas reinhardtii

under anaerobic conditions

Marc Forestier1,*, Paul King1

, Liping Zhang1

, Matthew Posewitz1

, Sarah Schwarzer2

, Thomas Happe2

,

Maria L. Ghirardi1 and Michael Seibert1

1

National Renewable Energy Laboratory, Golden, CO USA; 2

Ruhr-Universitaet-Bochum, Lehrstuhl Biochemie der Pflanzen AG

Photobiotechnologie, Bochum, Germany

We have isolated and characterized a second [Fe]-hydro￾genase gene from the green alga, Chlamydomonas reinhardtii.

The HydA2 gene encodes a protein of 505 amino acids that

is 74% similar and 68% identical to the known HydA1

hydrogenase from C. reinhardtii. HydA2 contains all the

conserved residues and motifs found in the catalytic core of

the family of [Fe]-hydrogenases. We demonstrate that both

the HydA1 and the HydA2 transcripts are expressed upon

anaerobic induction, achieved either by neutral gas purging

or by sulfur deprivation of the cultures. Furthermore, the

expression levels of both transcripts are regulated (in some

cases differently) by incubation conditions, such as the length

of anaerobiosis, the readdition of O2, the presence of acetate,

and/or the absence of nutrients such as sulfate during

growth. Antibodies specific for HydA2 recognized a protein

of about 49 kDa in extracts from anaerobically induced

C. reinhardtii cells, strongly suggesting that HydA2 encodes

for an expressed protein. Homology-based 3D modeling of

the HydA2 hydrogenase shows that its catalytic site models

well to the known structure of Clostridium pasteurianum

CpI, including the H2-gas channel. The major differences

between HydA1, HydA2 and CpI are the absence of the

N-terminal Fe-S centers and the existence of extra sequences

in the algal enzymes. To our knowledge, this work represents

the first systematic study of expression of two algal [Fe]-

hydrogenases in the same organism.

Keywords: green algae; anaerobic induction; hydrogenase;

sulfur deprivation; gene expression.

Hydrogen metabolism, catalyzed by hydrogenases in green

algae, was first observed over 60 years ago in Scenedesmus

obliquus [1,2]. Since then, hydrogenase enzymes that either

uptake or evolve H2 have been found in many other green

algae [3,4], including Chlamydomonas reinhardtii. This

particular alga is capable of evolving H2 gas in the dark

[5,6] or in the light, using H2O [7] or starch [8,9] as the source

of reductant. The reaction is catalyzed by a monomeric,

49 kDa reversible [Fe]-hydrogenase enzyme, which has been

isolated to purity by Happe and Naber [10].

Other [Fe]-hydrogenases, identified in a small group of

nonphotosynthetic anaerobic microbes (bacteria and pro￾tists) also catalyze either H2 production or H2 uptake in vivo

[11,12]. They play an important role in the anaerobic energy

metabolism of these organisms, mainly by reoxidizing

accumulated reducing equivalents. All [Fe]-hydrogenases

whose X-ray structures have been analyzed to date incor￾porate a [2Fe-2S] center bridged by a cysteine residue to a

[4Fe-4S] center at the catalytic site (the H-cluster). It is also

known that most [Fe]-hydrogenases contain additional

iron-sulfur centers that act as electron relays between carrier

molecules and the H-cluster [13,14]. However, the addi￾tional centers are absent in the green algal enzymes [15,16].

In addition, [Fe]-hydrogenases usually exhibit high specific

activity but are easily inactivated by either O2 or CO.

Green algal [Fe]-hydrogenases have been cloned and

sequenced from S. obliquus [15], C. reinhardtii [16,17] and

Chlorella fusca [18]. Besides the physiologically and bio￾chemically characterized HydA1 [Fe]-hydrogenase [15],

S. obliquus possesses a gene sequence that encodes a second

polypeptide with all the essential attributes of an [Fe]-

hydrogenase protein [19]. The expression of the second

putative S. obliquus hydrogenase gene was shown to be

constitutive (in contrast to the inducible expression of its

HydA1), suggesting different physiological roles for the two

enzymes. However, the expression and activities of the two

hydrogenases have not been studied concomitantly in the

same organism, under the same physiological conditions.

Hydrogenase activity in C. reinhardtii is induced by

anaerobiosis. Anaerobic states can be achieved either

Correspondence to M. L. Ghirardi, National Renewable Energy

Laboratory, 1617Cole Blvd., Golden CO 80401, USA.

Fax: + 1 303 384 6150; Tel.: + 1 303 384 6312;

E-mail: [email protected]

Abbreviations: CpI, one of the cloned [Fe]-hydrogenases from

Clostridium pasteurianum; PSII, photosystem II; BS, basal salts;

EST, expressed sequence tag; ORF, open-reading frame; PAR,

photosynthetically active radiation; TAP, tris-acetate-phosphate.

Accession numbers: AY055756 (C. reinhardtii HydA2 cDNA

sequence); AY090770 (C. reinhardtii HydA2 promoter region and

genomic DNA sequence).

Note: Operated for the US Department of Energy by the Midwest

Research Institute, Batelle and Bechtel under contract number

DE-AC36–99G010337.

*Present address: Limnological Station, Plant Biology Department,

University of Zurich, Seestrasse 187, 8802 Kilchberg, Switzerland.

(Received 1 April 2003, revised 29 April 2003,

accepted 30 April 2003)

Eur. J. Biochem. 270, 2750–2758 (2003)
FEBS 2003 doi:10.1046/j.1432-1033.2003.03656.x