Presence and expression of hydrogenase specific C-terminal endopeptidases in cyanobacteria
© Wünschiers et al; licensee BioMed Central Ltd. 2003
Received: 6 March 2003
Accepted: 7 May 2003
Published: 7 May 2003
Hydrogenases catalyze the simplest of all chemical reactions: the reduction of protons to molecular hydrogen or vice versa. Cyanobacteria can express an uptake, a bidirectional or both NiFe-hydrogenases. Maturation of those depends on accessory proteins encoded by hyp-genes. The last maturation step involves the cleavage of a ca. 30 amino acid long peptide from the large subunit by a C-terminal endopeptidase. Until know, nothing is known about the maturation of cyanobacterial NiFe-hydrogenases. The availability of three complete cyanobacterial genome sequences from strains with either only the uptake (Nostoc punctiforme ATCC 29133/PCC 73102), only the bidirectional (Synechocystis PCC 6803) or both NiFe-hydrogenases (Anabaena PCC 7120) prompted us to mine these genomes for hydrogenase maturation related genes. In this communication we focus on the presence and the expression of the NiFe-hydrogenases and the corresponding C-terminal endopeptidases, in the three strains mentioned above.
We identified genes encoding putative cyanobacterial hydrogenase specific C-terminal endopeptidases in all analyzed cyanobacterial genomes. The genes are not part of any known hydrogenase related gene cluster. The derived amino acid sequences show only low similarity (28–41%) to the well-analyzed hydrogenase specific C-terminal endopeptidase HybD from Escherichia coli, the crystal structure of which is known. However, computational secondary and tertiary structure modeling revealed the presence of conserved structural patterns around the highly conserved active site. Gene expression analysis shows that the endopeptidase encoding genes are expressed under both nitrogen-fixing and non-nitrogen-fixing conditions.
Anabaena PCC 7120 possesses two NiFe-hydrogenases and two hydrogenase specific C-terminal endopeptidases but only one set of hyp-genes. Thus, in contrast to the Hyp-proteins, the C-terminal endopeptidases are the only known hydrogenase maturation factors that are specific. Therefore, in accordance with previous nomenclature, we propose the gene names hoxW and hupW for the bidirectional and uptake hydrogenase processing endopeptidases, respectively. Due to their constitutive expression we expect that, at least in cyanobacteria, the endopeptidases take over multiple functions.
Hydrogenases catalyze the simplest of all chemical reactions: the reduction of protons to molecular hydrogen or vice versa. Depending on the metal content of the active site hydrogenases are classified into Fe-, NiFe-, and metal-free hydrogenases . Independent from the metal content, the enzymes are characterized as hydrogen uptake, bidirectional and hydrogen evolving hydrogenases, indicating their actual in vivo activity. A prominent and evolutionary old group of organisms possessing NiFe-hydrogenases are phototrophic cyanobacteria (formerly blue-green algae) . All cyanobacteria investigated so far, express an uptake, a bidirectional or both NiFe-hydrogenases [2–6]. The uptake hydrogenase is a dimeric enzyme consisting of a large subunit (HupL) containing the active site and a small subunit (HupS) with several FeS-clusters. The physiological role of the uptake hydrogenase appears to be coupled to nitrogen fixation [7–9]: the hydrogen evolved as a by-product from nitrogenase activity can be recycled by the action of the uptake hydrogenase . Consequently, the uptake hydrogenase is found in nitrogen-fixing cyanobacteria only [2, 10]. The bidirectional hydrogenase consists of an electron transmitting and anchoring diaphorase part (HoxFU), an active site containing large subunit (HoxH) and a FeS-cluster harboring small subunit (HoxY) [3, 11]. The presence of a third diaphorase subunit (HoxE) has been demonstrated for Anacystis nidulans (Synechococcus PCC 6301) and Synechocystis PCC 6803 . Neither is the bidirectional hydrogenase universally distributed among cyanobacteria nor is its function clearly understood, yet .
The maturation of nickel-containing enzymes, e.g. hydrogenases, ureases, and carbonmonoxide dehydrogenases, is a complex process requiring accessory proteins [13–19]. For hydrogenases, the first experimental results were obtained from Escherichia coli. A number of mutations in the 58–59 min region of the E. coli chromosome (location 2848670–2852287 in E. coli strain K12 genome ) affect the biosynthesis of all NiFe-hydrogenases of this organism . Sequencing of this region revealed 5 ORFs, which were designated hypABCDE, indicating that these genes affect hy drogenases p leiotropically  and which were to be the first identified genes associated with hydrogenase maturation. Later on, hyp homologous genes were also identified in cyanobacteria (see  and references therein).
The C-terminal part of deduced cyanobacterial hydrogenase large subunit amino acid sequences. Cyanobacterial sequences in the focus of this study are shown in bold. The accession number and the C-terminal protein sequence follow the gene name. The putative cutting site is represented by a gap. As reference, the cutting sites of Escherichia coli hydrogenase large subunits are shown. Corresponding GenBank accession numbers are indicated. The consensus sequence D(P/S)CxxCxx(H/R) is shown in italic letters.
Synechocystis PCC 6803
Nostoc ATCC 29133
Anabaena PCC 7120
Synechococcus PCC 6301
Results and Discussion
Genome Search and Sequence Analysis
Initially, we investigated the C-terminal parts of all available cyanobacterial hydrogenase large subunit sequences for the presence of the characteristic cutting site motif: DPCxxCxx(H/R) [1, 19]. This motif was found in all deduced amino acid sequences (Table 1). In contrary to all other analyzed sequences, in the uptake hydrogenase (HupL) cutting site motif the neutral proline (P) at position 2 is exchanged by an uncharged polar serine (S) (Table 1).
Protein cross-comparison Deduced amino acid sequence relationship among the putative cyanobacterial and known Escherichia coli hydrogenase specific C-terminal endopeptidases. Similarities are shown in bold.
It should be noted that, according to the structural classification of proteins (SCOP; ), HybD (1CFZ) belongs to the superfamily of HybD-like structures in the class of alpha/beta proteins. The only other known structural member of this superfamily is a germination protease from Bacillus megaterium (1C8B, ), which contains a phosphorylase/hydrolase-like fold. Although sequence-wise very different, germination proteases match structurally very good to hydrogenase maturating peptidases. The only exception is the absence of helices 4 and 5 in the former enzymes (Fig. 3).
Summary of the gene expression studies Note that Synechocystis PCC 6803 does not survive under nitrogen deprivation, i.e. nitrogen-fixing conditions.
Synechocystis PCC 6803
ATCC 29133/PCC 7120
Anabaena PCC 7120
Hydrogenase specific C-terminal endopeptidases share only low sequence similarity. Thus, the present study gives an example for the necessity to apply secondary and tertiary protein sequence information in order to facilitate unambiguous gene identification. Anabaena PCC 7120 possesses two NiFe-hydrogenases and two hydrogenase specific C-terminal endopeptidases but only one set of hyp-genes. Thus, in contrast to the universal Hyp-proteins, the C-terminal endopeptidases are the only known hydrogenase maturation factors that are specific. In order to keep in line with previous nomenclature we name the bidirectional hydrogenase (encoded by hox-genes) processing endopeptidases HoxW and propose to name the hydrogenase uptake (encoded by hup-genes) processing endopeptidases HupW.
Phylogenetic analysis of NiFe-hydrogenases revealed a close relationship between cyanobacterial uptake hydrogenases (HupSL) and bacterial hydrogen sensing hydrogenases (HoxBC, HupUV) . These hydrogen sensors lack the C-terminal extension, which is cleaved off after nickel insertion, but nevertheless contain the bimetallic NiFe-center [49–51]. This indicates that the final endopeptidase mediated hydrogenase maturation step is not absolutely necessary in order to obtain the fully assembled active site. From a phylogenetic point of view it seems that the last common ancestor of cyanobacterial uptake hydrogenases and hydrogen sensing hydrogenases possessed the C-terminal extension and the cutting site motive. Thus, it will be interesting to elucidate the advantage or necessity to loose that extension in the case of hydrogen sensing hydrogenases. Furthermore, it raises the question why the C-terminal extension was kept in all other known NiFe-hydrogenase. The fact, that cyanobacterial hydrogenase specific endopeptidase are constitutively expressed might point to multiple functions of these enzymes.
Genome data analysis
The genomes of Nostoc punctiforme ATCC 29133/PCC 73102 http://spider.jgi-psf.org/JGI_microbial/html/nostoc/nostoc_homepage.html, Anabaena PCC 7120 http://www.kazusa.or.jp/cyanobase/Anabaena/index.html and Synechocystis PCC 6803 http://www.kazusa.or.jp/cyanobase/Synechocystis/index.html were analyzed using the online program BLAST (Basic Local Alignment Search Tool)  provided by the respective genome initiative. For Nostoc punctiforme ATCC 29133/PCC 73102, preliminary sequence data was obtained from "The DOE Joint Genome Institute (JGI)".
Computational structure modeling
The derived protein sequences from the putative cyanobacterial hydrogenase maturation peptidase genes were structurally aligned using the online program SWISS MODEL http://www.expasy.ch/swissmod/SWISS-MODEL.html[53–55]. As reference the crystal structure from the hydrogenase specific C-terminal endopeptidase HybD from E. coli  was chosen.
Secondary structure prediction
The secondary structure was predicted using NNPREDICT http://www.cmpharm.ucsf.edu/~nomi/nnpredict.html. No special parameters were set. The secondary structure from the crystal structure data file of the hydrogenase maturating endopeptidase HybD from E. coli  (ProteinDataBank accession number: 1CFZ.pdb) was extracted by DSSP http://www.cmbi.kun.nl/swift/dssp/. These data were used as a template for secondary structure identity calculations. The calculations were performed with a homemade PERL script employing a weight matrix. Identities were weighted one, all other combinations zero.
Synechocystis PCC 6803
Product Size [bp]
AAT CCC ACG CCC TAA GTT TT
CAC TGA CCA AGC AGA GTG GA
TCA TCG GTT ACG GCA ATA CC
ATG GTT CGT TTG CTG ATT CC
Nostoc punctiforme ATCC 29133/PCC 73102
CTG TTG GGC GGA CAA TGG CCT CA
CCA CTT TTC ATA ATC AT
CGC AGG GAT GGA AGT AAT GT
TCA CAT CAT CGG GAA AGT CA
Anabaena PCC 7120
TAT ATC ACC CGT CGG TAG AG
CCA TGA TGC AGA GGT TAA GT
CGC AGG GAT GGA AGT TAT GT
GCG GAA AAT CAT CTG GAA A
CCA CTA TGC TCG TTT AAT TGA AAT
GGA ACA ACT TAA ACA GGG GTC AAA
ATC TCT TGC AGT CCA TCA AC
CGT AGA TGG CTT TGG TTA AG
Cell growth and harvesting
Nitrogen-fixing cultures of Nostoc punctiforme ATCC 29133/PCC 73102 and Anabaena PCC 7120 were grown in BG110  as previously described . Non-nitrogen-fixing cultures of Nostoc punctiforme ATCC 29133/PCC 73102, Anabaena PCC 7120 and Synechocystis PCC 6803 were grown in BG110 supplemented with 5 mM ammonium chloride and 10 mM HEPES (pH 7.5) (Nostoc and Anabaena) or BG110 supplemented with 1.5 g/l (17.7 mM) sodium nitrate (Synechocystis). All cultures were sparked with air. Cells were harvested by centrifugation at 4°C for 5 min in 50 ml Falcon tubes (Falcon#2070). Subsequently, the supernatant was removed, the cell pellet immediately frozen in liquid nitrogen, and the cells stored at -20°C.
DNA and RNA purification
Genomic DNA from all investigated cyanobacterial strains was isolated as described earlier . Total RNA was isolated from all strains as described by Axelsson et al. . Nucleic acid quality was assessed by gel electrophoresis (1% agarose) and photospectroscopy.
Transcription analysis (RT-PCR)
Transcription analysis was carried out as previously described . Total RNA (0.5–1 μg) was used for reverse transcription with AMV reverse transcriptase (Promega Corporation) using antisense primers (Table 4). cDNA produced in reverse transcription reaction was used for PCR reactions with sense/antisense primer pairs given in Table 4. The following PCR program profile was applied: 1 min at 94°C followed by 40 repetitive cycles of 10 s denaturation at 94°C, 1 min annealing at 55.1°C, and 30 s elongation at 72°C and finishing with 7 min at 72°C. Products of the PCR reactions were analyzed using 1% agarose gels. Images were captured on film, scanned and edited into Photoshop 4.0. Negative controls included no reverse transcriptase in the RT reaction prior to PCR and dH2O in the PCR, both resulting in no amplificates. For positive controls genomic DNA was used in PCR.
The authors research was financially supported by Ångpanneföreningens Forskningsstiftelse (Sweden), the Swedish Energy Agency (Statens Energimyndighet), and the Swedish Research Council (NFR/VR). Preliminary sequence data was obtained from "The DOE Joint Genome Institute (JGI)".
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