Hydrogenases, which catalyze H2 to H ؉ conversion as part of the bioenergetic metabolism of many microorganisms, are among the metalloenzymes for which a gas-substrate tunnel has been described by using crystallography and molecular dynamics. However, the correlation between protein structure and gas-diffusion kinetics is unexplored. Here, we introduce two quantitative methods for probing the rates of diffusion within hydrogenases. One uses protein film voltammetry to resolve the kinetics of binding and release of the competitive inhibitor CO; the other is based on interpreting the yield in the isotope exchange assay. We study structurally characterized mutants of a NiFe hydrogenase, and we show that two mutations, which significantly narrow the tunnel near the entrance of the catalytic center, decrease the rates of diffusion of CO and H2 toward and from the active site by up to 2 orders of magnitude. This proves the existence of a functional channel, which matches the hydrophobic cavity found in the crystal. However, the changes in diffusion rates do not fully correlate with the obstruction induced by the mutation and deduced from the x-ray structures. Our results demonstrate the necessity of measuring diffusion rates and emphasize the role of side-chain dynamics in determining these.crystallography ͉ structure/function relationships ͉ substrate tunnel ͉ protein film voltammetry ͉ isotope exchange

Class 1 peptide release factors (RFs) in Escherichia coli are N 5 -methylated on the glutamine residue of the universally conserved GGQ motif. One other protein alone has been shown to contain N 5 -methylglutamine: E.coli ribosomal protein L3. We identify the L3 methyltransferase as YfcB and show that it methylates ribosomes from a yfcB strain in vitro, but not RF1 or RF2. HemK, a close orthologue of YfcB, is shown to methylate RF1 and RF2 in vitro. hemK is immediately downstream of and co-expressed with prfA. Its deletion in E.coli K12 leads to very poor growth on rich media and abolishes methylation of RF1. The activity of unmethylated RF2 from K12 strains is extremely low due to the cumulative effects of threonine at position 246, in place of alanine or serine present in all other bacterial RFs, and the lack of N 5 -methylation of Gln252. Fast-growing spontaneous revertants in hemK K12 strains contain the mutations Thr246Ala or Thr246Ser in RF2. HemK and YfcB are the ®rst identi®ed methyltransferases modifying glutamine, and are widely distributed in nature.

Hydrogenases catalyze the conversion between 2H(+) + 2e(-) and H(2)(1). Most of these enzymes are inhibited by O(2), which represents a major drawback for their use in biotechnological applications. Improving hydrogenase O(2) tolerance is therefore a major contemporary challenge to allow the implementation of a sustainable hydrogen economy. We succeeded in improving O(2) tolerance, which we define here as the ability of the enzyme to resist for several minutes to O(2) exposure, by substituting with methionines small hydrophobic residues strongly conserved in the gas channel. Remarkably, the mutated enzymes remained active in the presence of an O(2) concentration close to that found in aerobic solutions in equilibrium with air, while the wild type enzyme is inhibited in a few seconds. Crystallographic and spectroscopic studies showed that the structure and the chemistry at the active site are not affected by the mutations. Kinetic studies demonstrated that the inactivation is slower and reactivation faster in these mutants. We propose that in addition to restricting O(2) diffusion to the active site of the enzyme, methionine may also interact with bound peroxide and provide an assisted escape route for H(2)O(2) toward the gas channel. These results show for the first time that it is possible to improve O(2)-tolerance of [NiFe] hydrogenases, making possible the development of biohydrogen production systems.

Protein release factor eRF1 in Saccharomyces cerevisiae, in complex with eRF3 and GTP, is methylated on a functionally crucial Gln residue by the S-adenosylmethionine-dependent methyltransferase Ydr140w. Here we show that eRF1 methylation, in addition to these previously characterized components, requires a 15-kDa zinc-binding protein, Ynr046w. Co-expression in Escherichia coli of Ynr046w and Ydr140w allows the latter to be recovered in soluble form rather than as inclusion bodies, and the two proteins co-purify on nickel-nitrilotriacetic acid chromatography when Ydr140w alone carries a His tag. The crystal structure of Ynr046w has been determined to 1.7 Å resolution. It comprises a zinc-binding domain built from both the N-and C-terminal sequences and an inserted domain, absent from bacterial and archaeal orthologs of the protein, composed of three ␣-helices. The active methyltransferase is the heterodimer Ydr140w⅐Ynr046w, but when alone, both in solution and in crystals, Ynr046w appears to be a homodimer. The Ynr046w eRF1 methyltransferase subunit is shared by the tRNA methyltransferase Trm11p and probably by two other enzymes containing a Rossman fold.Termination codons in mRNA are recognized on the ribosome by class I protein termination factors (or release factors (RFs)) 5 in eubacteria, archaea, and eukaryotes (1-3). Three codons are used as stop signals in most organisms: UAA, UGA, and UAG. In bacteria, two class I RFs are required for termination: RF1, which recognizes UAA and UAG codons, and RF2, which recognizes UAA and UGA codons. In contrast, a single RF, eRF1 or aRF1, is sufficient for termination at all three stop codons in eukaryotes and archaea, respectively. eRF1 and aRF1 form closely related protein families but are evolutionarily distinct from the eubacterial RFs (4). Thus, despite a similar function, the only sequence element common to all RFs is a tripeptide sequence, GGQ. Structural and mutational analysis shows that this motif is essential for RF activity and is required to interact with the peptidyl transferase center of the large ribosomal subunit and trigger hydrolysis of the ester bond in peptidyl-tRNA (5, 6). The Gln residue of the GGQ motif is methylated in both bacteria (7) and Saccharomyces cerevisiae (8, 9), and probably in mammals. Bacterial RF methylation depends on the PrmC methyltransferase (MTase) (10, 11), the product of the gene prmC (previously named hemK) situated in Escherichia coli, and most other bacteria immediately downstream of the gene prfA encoding RF1. RF methylation in E. coli strongly stimulates the activity of the factors (7, 11). 6 It is remarkable that the modification of Gln in the GGQ motif is conserved from bacteria to eukaryotes despite the different evolutionary origin of the class I RFs themselves. The S. cerevisiae genome encodes two proteins, Ydr140w and Ynl063w, with significant similarity to bacterial PrmC that goes beyond the motifs known to be involved in AdoMet binding (7). Inactivation of Ydr140w was shown to lead to a loss of eRF1 methylation ...

Polypeptide release factors from eubacteria and eukaryotes, although similar in function, belong to different protein families. They share one sequence motif, a GGQ tripeptide that is vital to release factor (RF) activity in both kingdoms. In bacteria, the Gln residue of the motif in RF1 and RF2 is modified to N 5 -methylGln by the S-adenosyl L-methionine-dependent methyltransferase PrmC and the absence of Gln methylation decreases the release activity of Escherichia coli RF2 in vitro severalfold. We show here that the same modification is made to the GGQ motif of Saccharomyces cerevisiae release factor eRF1, the first time that N 5 -methyl-Gln has been found outside the bacterial kingdom. The product of the YDR140w gene is required for the methylation of eRF1 in vivo and for optimal yeast cell growth. YDR140w protein has significant homology to PrmC but lacks the N-terminal domain thought to be involved in the recognition of the bacterial release factors. Overproduced in S. cerevisiae, YDR140w can methylate eRF1 from yeast or man in vitro using S-adenosyl L-methionine as methyl donor provided that eRF3 and GTP are also present, suggesting that the natural substrate of the methyltransferase YDR140w is the ternary complex eRF1⅐eRF3⅐GTP.

Class I release factors bind to ribosomes in response to stop codons and trigger peptidyl-tRNA hydrolysis at the P site. Prokaryotic and eukaryotic RFs share one motif: a GGQ tripeptide positioned in a loop at the end of a stem region that interacts with the ribosomal peptidyl transferase center. The glutamine side chain of this motif is specifically methylated in both prokaryotes and eukaryotes. Methylation in E. coli is due to PrmC and results in strong stimulation of peptide chain release. We have solved the crystal structure of the complex between E. coli RF1 and PrmC bound to the methyl donor product AdoHCy. Both the GGQ domain (domain 3) and the central region (domains 2 and 4) of RF1 interact with PrmC. Structural and mutagenic data indicate a compact conformation of RF1 that is unlike its conformation when it is bound to the ribosome but is similar to the crystal structure of the protein alone.

SummaryRelease factors RF1 and RF2 are required in bacteria for the cleavage of peptidyl-tRNA. A single sequence motif, GGQ, is conserved in all eubacterial, archaebacterial and eukaryotic release factors and may mimic the CCA end of tRNA, although the position of the motif in the crystal structures of human eRF1 and Escherichia coli RF2 is strikingly different. Mutations have been introduced at each of the three conserved positions. Changing the Gln residue to Ala or Glu allowed the factors to retain about 22% of tetrapeptide release activity in vitro , but these mutants could not complement thermosensitive RF mutants in vivo . None of several mutants with altered Gly residues retained activity in vivo or in vitro . Many GGQ mutants were poorly expressed and are presumably unstable; many were also toxic to the cell. The toxic mutant factors or their degradation products may bind to ribosomes inhibiting the action of the normal factor. These data are consistent with a common role for the GGQ motif in bacterial and eukaryotic release factors, despite strong divergence in primary, secondary and tertiary structure, but are difficult to reconcile with the hypothesis that the amide nitrogen of the Gln plays a vital role in peptidyl-tRNA hydrolysis.

Clostridium cellulolyticum produces cellulolytic complexes (cellulosomes) made of 10-13 cell wall degrading enzymes tightly bound to a scaffolding protein (CipC) by means of their dockerin domain. It has previously been shown that the receptor domains in CipC are the cohesin domains and that the cohesin/dockerin interaction is calcium-dependent. In the present study, surface plasmon resonance was used to demonstrate that the free cohesin1 from CipC and dockerin from CelA have the same K(D) (2.5 x 10(-)(10) M) as that of the entire CelA and a larger fragment of CipC, the latter of which contains, in addition to cohesin1, a cellulose binding domain and a hydrophilic domain of unknown function. This demonstrates that neither the catalytic domain of CelA nor the noncohesin domains of CipC have any influence on the interaction. Dockerin domains are composed of two conserved segments of 22 residues: removal of the second segment abolishes the affinity for cohesin1, whereas modified dockerins having twice the first segment, twice the second, or both segments but in a reverse order have K(D) values for cohesin1 in the same range as that observed for wild-type dockerin. These data indicate that if two segments are required for the complexation with the cohesin, segments 1 and 2 are similar enough to replace each other. Calcium overlay experiments revealed that the dockerin domain has one calcium binding site per conserved segment. Circular dichroism performed on wild-type and mutant dockerins indicates that this domain is well structured and that removal of calcium only weakly affects the secondary structure, which remains 40-45% helical.