Thirty years of heme catalases structural biology.

About thirty years ago the crystal structures of the heme catalases from Penicillium vitale (PVC) and, a few months later, from bovine liver (BLC) were published. Both enzymes were compact tetrameric molecules with subunits that, despite their size differences and the large phylogenetic separation between the two organisms, presented a striking structural similarity for about 460 residues. The high conservation, confirmed in all the subsequent structures determined, suggested a strong pressure to preserve a functional catalase fold, which is almost exclusively found in these mono-functional heme catalases. However, even in the absence of the catalase fold an efficient catalase activity is also found in the heme containing catalase-peroxidase proteins. The structure of these broad substrate range enzymes, reported for the first time less than ten years ago from the halophilic archaebacterium Haloarcula marismortui (HmCPx) and from the bacterium Burkholderia pseudomallei (BpKatG), showed a heme pocket closely related to that of plant peroxidases, though with a number of unique modifications that enable the catalase reaction. Despite the wealth of structural information already available, for both monofunctional catalases and catalase-peroxidases, a number of unanswered major questions require continuing structural research with truly innovative approaches.

Characterization of alcohol dehydrogenase (ADH12) from Haloarcula marismortui, an extreme halophile from the Dead Sea.

Haloarchaeal alcohol dehydrogenases are of increasing interest as biocatalysts in the field of white biotechnology. In this study, the gene adh12 from the extreme halophile Haloarcula marismortui (HmADH12), encoding a 384 residue protein, was cloned into two vectors: pRV1 and pTA963. The resulting constructs were used to transform host strains Haloferax volcanii (DS70) and (H1209), respectively. Overexpressed His-tagged recombinant HmADH12 was purified by immobilized metal-affinity chromatography (IMAC). The His-tagged protein was visualized by SDS-PAGE, with a subunit molecular mass of 41.6 kDa, and its identity was confirmed by mass spectrometry. Purified HmADH12 catalyzed the interconversion between alcohols and aldehydes and ketones, being optimally active in the presence of 2 M KCl. It was thermoactive, with maximum activity registered at 60°C. The NADP(H) dependent enzyme was haloalkaliphilic for the oxidative reaction with optimum activity at pH 10.0. It favored a slightly acidic pH of 6.0 for catalysis of the reductive reaction. HmADH12 was significantly more tolerant than mesophilic ADHs to selected organic solvents, making it a much more suitable biocatalyst for industrial application.

A recurrent magnesium-binding motif provides a framework for the ribosomal peptidyl transferase center.

The ribosome is an ancient macromolecular machine responsible for the synthesis of all proteins in all living organisms. Here we demonstrate that the ribosomal peptidyl transferase center (PTC) is supported by a framework of magnesium microclusters (Mg(2+)-muc's). Common features of Mg(2+)-muc's include two paired Mg(2+) ions that are chelated by a common bridging phosphate group in the form Mg((a))(2+)-(O1P-P-O2P)-Mg((b))(2+). This bridging phosphate is part of a 10-membered chelation ring in the form Mg((a))(2+)-(OP-P-O5'-C5'-C4'-C3'-O3'-P-OP)-Mg((a))(2+). The two phosphate groups of this 10-membered ring are contributed by adjacent residues along the RNA backbone. Both Mg(2+) ions are octahedrally coordinated, but are substantially dehydrated by interactions with additional RNA phosphate groups. The Mg(2+)-muc's in the LSU (large subunit) appear to be highly conserved over evolution, since they are unchanged in bacteria (Thermus thermophilus, PDB entry 2J01) and archaea (Haloarcula marismortui, PDB entry 1JJ2). The 2D elements of the 23S rRNA that are linked by Mg(2+)-muc's are conserved between the rRNAs of bacteria, archaea and eukarya and in mitochondrial rRNA, and in a proposed minimal 23S-rRNA. We observe Mg(2+)-muc's in other rRNAs including the bacterial 16S rRNA, and the P4-P6 domain of the tetrahymena Group I intron ribozyme. It appears that Mg(2+)-muc's are a primeval motif, with pivotal roles in RNA folding, function and evolution.

First evidence for the salt-dependent folding and activity of an esterase from the halophilic archaea Haloarcula marismortui.

A gene encoding an esterase from Haloarcula marismortui, a halophilic archaea from the Dead Sea, was cloned, expressed in Escherichia coli, and the recombinant protein (Hm EST) was biochemically characterized. The enzymatic activity of Hm EST was shown to exhibit salt dependence through salt-dependent folding. Hm EST exhibits a preference for short chain fatty acids and monoesters. It is inhibited by phenylmethylsulfonyl fluoride, diethyl-p-nitrophenyl phosphate, and 5-methoxy-3-(4-phenoxyphenyl)-3H-[1,3,4]oxadiazol-2-one, confirming the conclusion from sequence alignments that Hm EST is a serine carboxylesterase belonging to the hormone-sensitive lipase family. The activity of Hm EST is optimum in the presence of 3 M KCl and no activity was detected in the absence of salts. Far-UV circular dichroism showed that Hm EST is totally unfolded in salt-free medium and secondary structure appears in the presence of 0.25-0.5 M KCl. After salt depletion, the protein was able to recover 60% of its initial activity when 2 M KCl was added. A 3D model of Hm EST was built and its surface properties were analyzed, pointing to an enrichment in acidic residues paralleled by a depletion in basic residues. This peculiar charge repartition at the protein surface supports a better stability of the protein in a high salt environment.

Wide distribution among halophilic archaea of a novel polyhydroxyalkanoate synthase subtype with homology to bacterial type III synthases.

Polyhydroxyalkanoates (PHAs) are accumulated as intracellular carbon and energy storage polymers by various bacteria and a few haloarchaea. In this study, 28 strains belonging to 15 genera in the family Halobacteriaceae were investigated with respect to their ability to synthesize PHAs and the types of their PHA synthases. Fermentation results showed that 18 strains from 12 genera could synthesize polyhydroxybutyrate (PHB) or poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). For most of these haloarchaea, selected regions of the phaE and phaC genes encoding PHA synthases (type III) were cloned via PCR with consensus-degenerate hybrid oligonucleotide primers (CODEHOPs) and were sequenced. The PHA synthases were also examined by Western blotting using haloarchaeal Haloarcula marismortui PhaC (PhaC(Hm)) antisera. Phylogenetic analysis showed that the type III PHA synthases from species of the Halobacteriaceae and the Bacteria domain clustered separately. Comparison of their amino acid sequences revealed that haloarchaeal PHA synthases differed greatly in both molecular weight and certain conserved motifs. The longer C terminus of haloarchaeal PhaC was found to be indispensable for its enzymatic activity, and two additional amino acid residues (C143 and C190) of PhaC(Hm) were proved to be important for its in vivo function. Thus, we conclude that a novel subtype (IIIA) of type III PHA synthase with unique features that distinguish it from the bacterial subtype (IIIB) is widely distributed in haloarchaea and appears to be involved in PHA biosynthesis.

Production and characterization of esterase and lipase from Haloarcula marismortui.

The present study was conducted to investigate the capability of Haloarcula marismortui to synthesize esterases and lipases, and the effect of physicochemical conditions on the growth and the production of esterases and lipases. Finally, the effect of NaCl concentration and temperature on esterase and lipase activities was studied using intracellular crude extracts. In order to confirm the genomic prediction about the esterase and lipase synthesis, H. marismortui was cultured on a rich medium and the crude extracts (intra- or extracellular) obtained were assayed for both activities using p-nitrophenyl esters and triacylglycerides as substrates. Studies on the kinetics of growth and production of esterase and lipase of H. marismortui were performed, reaching a maximum growth rate of 0.053 h(-1) and maximal productions of intracellular esterase and lipase of 2.094 and 0.722 U l(-1) using p-nitrophenyl valerate and p-nitrophenyl laurate, respectively. Both enzymes were produced as growth-associated metabolites. The effects of temperature, pH, and NaCl concentration on the growth rate and production of enzymes were studied by using a Box-Behnken response surface design. The three response variables were significantly influenced by the physicochemical factors and an interaction effect between temperature and NaCl concentration was also evidenced. The surface response method estimated the following maximal values for growth rate and productions of esterase and lipase: 0.086 h(-1) (at 42.5 degrees C, pH 7.4, and 3.6 mol l(-1) NaCl), 2.3 U l(-1) (at 50 degrees C, pH 7.5, and 4.3 mol l(-1) NaCl), and 0.58 U l(-1) (at 50 degrees C, pH 7.6, and 4.5 mol l(-1) NaCl), respectively. Esterases were active at different salt concentrations, showing two optimal activities (at 0.5 and 5 mol l(-1) NaCl), which suggested the presence of two different esterases. Interestingly, in the absence of salt, esterase retained 50% residual activity. Esterases and lipase activities were maximal at 45 degrees C and inactive at 75 degrees C. This study represents the first report evidencing the synthesis of esterase and lipase by H. marismortui.

Role of chirality of the sugar ring in the ribosomal peptide synthesis.

We present a theoretical analysis of the role of the natural chirality of the sugar ring ( D-enantiomeric form) in the peptide synthesis reaction in ribosome. The study is based on a model from the crystal structure of the ribosomal subunit of Haloarcula marismortui using hybrid quantum mechanical-molecular mechanical method. The result indicates that the natural heterochiral sugar-amino acid combination ( D: L) is most favorable for the formation of the peptide bond within the structure of peptidyl transferase center (PTC). Other possible combinations of unnatural chiral form of the sugar-amino acid pair are unfavorable to perform the reaction within the PTC. The presence of the sugar ring has favorable influence on the rotatory path. The chirality of the 2' carbon of the sugar ring is vital for the peptide synthesis. Alteration of the stereochemistry or removal of chirality at the 2' center makes the rate as several orders slower in magnitude. This is in agreement with the recent experimental result that the replacement of the 2' OH by H or F reduces the rate by several orders of magnitude. Two different mechanisms for the catalytic effect of the stereochemistry of 2' OH are investigated. In one mechanism, the 2' OH is involved in proton shuttle, and in the second mechanism, the OH group acts as an anchoring group. The transition state barriers of both mechanisms are found to be comparable. The natural chirality of the 2' center helps lowering the transition state barrier height of the reaction substantially compared with the cases where the 2' center is made achiral or with altered chirality. Thus, the stereochemistry of the 2' center has a major role in synthesis. Few surrounding residues like U2620, A2486, G2618, and C2487 have favorable influence on rotatory path, while the residues like U2541, C2104, C2105, A2485, C2542, C2608, U2619, and A2637 have little influence. The present study shows that the natural chirality of the sugar ring and amino acid makes a perfect heteropair within the PTC to carry out peptide synthesis with high efficiency.

Influence of an anion-binding site in the stabilization of halophilic malate dehydrogenase from Haloarcula marismortui.

Halophilic proteins have evolved to be soluble, stable and active in high salt concentration. Crystallographic studies have shown that surface enrichment by acidic amino acids is a common structural feature of halophilic proteins. In addition, ion-binding sites have also been observed in most of the cases. The role of chloride-binding sites in halophilic adaptation was addressed in a site-directed mutagenesis study of tetrameric malate dehydrogenase from Haloarcula marismortui. The mutation of K 205, which is involved in an inter-subunit chloride-binding site, drastically modified the enzyme stability in the presence of KCl, but not in the presence of KF. The oligomeric state of the [K205A] mutant changes with the nature of the anion. At high salt concentration, the [K205A] mutant is a dimer when the anion is a chloride ion, whereas it is a tetramer when the fluoride ion is used. The results highlight the role of anion-binding sites in protein adaptation to high salt conditions.

The Oligomeric states of Haloarcula marismortui malate dehydrogenase are modulated by solvent components as shown by crystallographic and biochemical studies.

The three-dimensional crystal structure of the (R207S, R292S) mutant of malate dehydrogenase from Haloarcula marismortui was solved at 1.95A resolution in order to determine the role of salt bridges and solvent ions in halophilic adaptation and quaternary structure stability. The mutations, located at the dimer-dimer interface, disrupt two inter-dimeric salt bridge clusters that are essential for wild-type tetramer stabilisation. Previous experiments in solution, performed on the double mutant, had shown a tetrameric structure in 4M NaCl, which dissociated into active dimers in 2M NaCl. In order to establish if the active dimeric form is a product of the mutation, or if it also exists in the wild-type protein, complementary studies were performed on the wild-type enzyme by analytical centrifugation and small angle neutron scattering experiments. They showed the existence of active dimers in NaF, KF, Na(2)SO(4), even in the absence of NADH, and in the presence of NADH at concentrations of NaCl below 0.3M. The crystal structure shows a tetramer that, in the absence of the salt bridge clusters, appears to be stabilized by a network of ordered water molecules and by Cl(-) binding at the dimer-dimer interface. The double mutant and wild-type dimer folds are essentially identical (the r.m.s. deviation between equivalent C(alpha) positions is 0.39A). Chloride ions are also observed at the monomer-monomer interfaces of the mutant, contributing to the stability of each dimer against low salt dissociation. Our results support the hypothesis that extensive binding of water and salt is an important feature of adaptation to a halophilic environment.

Sequence and electron paramagnetic resonance analyses of nitrate reductase NarGH from a denitrifying halophilic euryarchaeote Haloarcula marismortui.

Genes encoding the NarG and NarH subunits of the molybdo-iron-sulfur enzyme, a nitrate reductase from a denitrifying halophilic euryarchaeota Haloarcula marismortui, were cloned and sequenced. An incomplete cysteine motif reminiscent of that for a [4Fe-4S] cluster binding was found in the NarG subunit, and complete cysteine arrangements for binding one [3Fe-4S] cluster and three [4Fe-4S] clusters were found in the NarH subunit. In conjunction with chemical, electron paramagnetic resonance, and subcellular localization analyses, we firmly establish that the H. marismortui enzyme is a new archaeal member of the known membrane-bound nitrate reductases whose homologs are found in the bacterial domain.

Purification and characterization of dissimilatory nitrate reductase from a denitrifying halophilic archaeon, Haloarcula marismortui.

Dissimilatory nitrate reductase was purified from a denitrifying halophilic archaeon, Haloarcula marismortui, to an electrophoretically homogeneous state. The purified enzyme was inferred to be a homotetramer composed of a 63 kDa polypeptide. The electron paramagnetic resonance spectrum of the purified enzyme revealed typical rhombic signals which were ascribed to Mo(V) in the Mo-molybdopterin complex. Like the bacterial membrane-bound (Nar-) enzyme, the purified enzyme supported the catalysis of chlorate. The enzyme was activated in extreme saline conditions and the values of k(cat) and K(m) toward nitrate were 145 s(-1) and 79 microM, respectively, in the presence of 2.0 M NaCl.

The protein sequence of an archaeal catalase-peroxidase.

The gene encoding a catalase-peroxidase of archaeal origin, the halophilic catalase-peroxidase from Haloarcula marismortui, was sequenced. The primary structure proposed was confirmed by Edman degradation and mass spectrometry analyses of proteolytic fragments of the purified protein. The open reading frame in the gene corresponds to 731 amino acids and the calculated mass of the mature protein (deleted of the N-terminal methionine) is 81,253.65 Da, in reasonable agreement with the value of 81,292 +/- 9 Da previously measured by mass spectrometry. Southern and Northern blot analyses showed that the protein is encoded by a single gene as a monocistronic transcript. The protein sequence shows a high level of identity with bacterial catalase-peroxidases, with strongly conserved regions around the heme binding histidines. Similarly to other soluble halophilic proteins, it shows the excess of acidic residues that has been associated with solvation in halophilic adaptation.

A methylaspartate cycle in haloarchaea.

Access to novel ecological niches often requires adaptation of metabolic pathways to cope with new environments. For conversion to cellular building blocks, many substrates enter central carbon metabolism via acetyl-coenzyme A (acetyl-CoA). Until now, only two such pathways have been identified: the glyoxylate cycle and the ethylmalonyl-CoA pathway. Prokaryotes in the haloarchaea use a third pathway by which acetyl-CoA is oxidized to glyoxylate via the key intermediate methylaspartate. Glyoxylate condensation with another acetyl-CoA molecule yields malate, the final assimilation product. This cycle combines reactions that originally belonged to different metabolic processes in different groups of prokaryotes, which suggests lateral gene transfer and evolutionary tinkering of acetate assimilation. Moreover, it requires elevated intracellular glutamate concentrations, as well as coupling carbon assimilation with nitrogen metabolism.

Gradual adaptive changes of a protein facing high salt concentrations.

Several experimental techniques were applied to unravel fine molecular details of protein adaptation to high salinity. We compared four homologous enzymes, which suggested a new halo-adaptive state in the process of molecular adaptation to high-salt conditions. Together with comparative functional studies, the structure of malate dehydrogenase from the eubacterium Salinibacter ruber shows that the enzyme shares characteristics of a halo-adapted archaea-bacterial enzyme and of non-halo-adapted enzymes from other eubacterial species. The S. ruber enzyme is active at the high physiological concentrations of KCl but, unlike typical halo-adapted enzymes, remains folded and active at low salt concentrations. Structural aspects of the protein, including acidic residues at the surface, solvent-exposed hydrophobic surface, and buried hydrophobic surface, place it between the typical halo-adapted and non-halo-adapted proteins. The enzyme lacks inter-subunit ion-binding sites often seen in halo-adapted enzymes. These observations permit us to suggest an evolutionary pathway that is highlighted by subtle trade-offs to achieve an optimal compromise among solubility, stability, and catalytic activity.

Orphan SelD proteins and selenium-dependent molybdenum hydroxylases.

Bacterial and Archaeal cells use selenium structurally in selenouridine-modified tRNAs, in proteins translated with selenocysteine, and in the selenium-dependent molybdenum hydroxylases (SDMH). The first two uses both require the selenophosphate synthetase gene, selD. Examining over 500 complete prokaryotic genomes finds selD in exactly two species lacking both the selenocysteine and selenouridine systems, Enterococcus faecalis and Haloarcula marismortui. Surrounding these orphan selD genes, forming bidirectional best hits between species, and detectable by Partial Phylogenetic Profiling vs. selD, are several candidate molybdenum hydroxylase subunits and accessory proteins. We propose that certain accessory proteins, and orphan selD itself, are markers through which new selenium-dependent molybdenum hydroxylases can be found.

Molecular characterization of the phaECHm genes, required for biosynthesis of poly(3-hydroxybutyrate) in the extremely halophilic archaeon Haloarcula marismortui.

Although many haloarchaea produce biodegradable polyhydroxyalkanoates (PHAs), the genes involved in PHA synthesis in the domain of Archaea have not yet been experimentally investigated yet. In this study, we revealed that Haloarcula marismortui was able to accumulate poly(3-hydroxybutyrate) (PHB) up to 21% of cellular dry weight when cultured in a minimal medium with excessive glucose and identified the phaE(Hm) and phaC(Hm) genes, probably encoding two subunits of a class III PHA synthase. These two genes were adjacent and directed by a single promoter located 26 bp upstream of the transcriptional start site and were constitutively expressed under both nutrient-rich and -limited conditions. Interestingly, PhaC(Hm) was revealed to be strongly bound with the PHB granules, but PhaE(Hm) seemed not to be. Introduction of either the phaE(Hm) or phaC(Hm) gene into Haloarcula hispanica, which harbors highly homologous phaEC(Hh) genes, could enhance the PHB synthesis in the recombinant strains, while coexpression of the both genes always generated the highest PHB yield. Significantly, knockout of the phaEC(Hh) genes in H. hispanica led to a complete loss of the PHA synthase activity. Complementation with phaEC(Hm) genes, but not a single one, restored the capability of PHB accumulation as well as the PHA synthase activity in this phaEC-deleted haloarchaeon. These results indicated that the phaEC genes are required for biosynthesis of PHB and might encode an active PHA synthase in the Haloarcula species.

Haloarcula marismortui cytochrome b-561 is encoded by the narC gene in the dissimilatory nitrate reductase operon.

The composition of membrane-bound electron-transferring proteins from denitrifying cells of Haloarcula marismortui was compared with that from the aerobic cells. Accompanying nitrate reductase catalytic NarGH subcomplex, cytochrome b-561, cytochrome b-552, and halocyanin-like blue copper protein were induced under denitrifying conditions. Cytochrome b-561 was purified to homogeneity and was shown to be composed of a polypeptide with a molecular mass of 40 kDa. The cytochrome was autooxidizable and its redox potential was -27 mV. The N-terminal sequence of the cytochrome was identical to the deduced amino acid sequence of the narC gene product encoded in the third ORF of the nitrate reductase operon with a unique arrangement of ORFs. The sequence of the cytochrome was homologous with that of the cytochrome b subunit of respiratory cytochrome bc. A possibility that the cytochrome bc and the NarGH constructed a supercomplex was discussed.

AMP-forming acetyl-CoA synthetase from the extremely halophilic archaeon Haloarcula marismortui: purification, identification and expression of the encoding gene, and phylogenetic affiliation.

Halophilic archaea activate acetate via an (acetate)-inducible AMP-forming acetyl-CoA synthetase (ACS), (Acetate+ATP+CoA --> Acetyl-CoA+AMP+PP(i)). The enzyme from Haloarcula marismortui was purified to homogeneity. It constitutes a 72-kDa monomer and exhibited a temperature optimum of 41 degrees C and a pH optimum of 7.5. For optimal activity, concentrations between 1 M and 1.5 M KCl were required, whereas NaCl had no effect. The enzyme was specific for acetate (100%) additionally accepting only propionate (30%) as substrate. The kinetic constants were determined in both directions of the reaction at 37 degrees C. Using the N-terminal amino acid sequence an open reading frame - coding for a 74 kDa protein - was identified in the partially sequenced genome of H. marismortui. The function of the ORF as acs gene was proven by functional overexpression in Escherichia coli. The recombinant enzyme was reactivated from inclusion bodies, following solubilization in urea and refolding in the presence of salts, reduced and oxidized glutathione and substrates. Refolding was dependent on salt concentrations of at least 2 M KCl. The recombinant enzyme showed almost identical molecular and catalytic properties as the native enzyme. Sequence comparison of the Haloarcula ACS indicate high similarity to characterized ACSs from bacteria and eukarya and the archaeon Methanosaeta. Phylogenetic analysis of ACS sequences from all three domains revealed a distinct archaeal cluster suggesting monophyletic origin of archaeal ACS.

Crystallization and preliminary X-ray analysis of catalase-peroxidase from the halophilic archaeon Haloarcula marismortui.

Catalase-peroxidases are bifunctional enzymes found in many microorganisms. Crystals of catalase-peroxidase from the halophilic archaeon Haloarcula marismortui were obtained using the hanging-drop vapour-diffusion method. The rhombic plate-shaped crystals were grown from purified protein solution using (NH(4))(2)SO(4) as precipitant at 293 K. The crystal belongs to the monoclinic system, space group C2, and diffracted beyond 2.0 A resolution.