The bacterium Thermus thermophilus,like hyperthermophilic archaea,uses a two-step pathway for the synthesis of mannosylglycerate

The biosynthetic pathway for the synthesis of the compatible solute alpha-mannosylglycerate (MG) in the thermophilic bacterium Thermus thermophilus HB27 was identified based on the activities of recombinant mannosyl-3-phosphoglycerate synthase (MPGS) (EC 2.4.1.217) and mannosyl-3-phosphoglycerate phosphatase (MPGP) (EC 3.1.3.70). The sequences of homologous genes from the archaeon Pyrococcus horikoshii were used to identify MPGS and MPGP genes in T. thermophilus HB27 genome. Both genes were separately cloned and overexpressed in Escherichia coli, yielding 3 to 4 mg of pure recombinant protein per liter of culture. The molecular masses were 43.6 and 28.1 kDa for MPGS and MPGP, respectively. The recombinant MPGS catalyzed the synthesis of alpha-mannosyl-3-phosphoglycerate (MPG) from GDP-mannose and D-3-phosphoglycerate, while the recombinant MPGP catalyzed the dephosphorylation of MPG to MG. The recombinant MPGS had optimal activity at 80 to 90 degrees C and a pH optimum near 7.0; MPGP had maximal activity between 90 and 95 degrees C and at pH 6.0. The activities of both enzymes were strictly dependent on divalent cations; Mn(2+) was most effective for MPGS, while Mn(2+), Co(2+), Mg(2+), and to a lesser extent Ni(2+) activated MPGP. The organization of MG biosynthetic genes in T. thermophilus HB27 is different from the P. horikoshii operon-like structure, since the genes involved in the conversion of fructose-6-phosphate to GDP-mannose are not found immediately downstream of the contiguous MPGS and MPGP genes. The biosynthesis of MG in the thermophilic bacterium T. thermophilus HB27, proceeding through a phosphorylated intermediate, is similar to the system found in hyperthermophilic archaea.     

Characterization of the biosynthetic pathway of glucosylglycerate in the archaeon Methanococcoides burtonii

The pathway for the synthesis of the organic solute glucosylglycerate (GG) is proposed based on the activities of the recombinant glucosyl-3-phosphoglycerate synthase (GpgS) and glucosyl-3-phosphoglycerate phosphatase (GpgP) from Methanococcoides burtonii. A mannosyl-3-phosphoglycerate phosphatase gene homologue (mpgP) was found in the genome of M. burtonii (http://www.jgi.doe.gov), but an mpgS gene coding for mannosyl-3-phosphoglycerate synthase (MpgS) was absent. The gene upstream of the mpgP homologue encoded a putative glucosyltransferase that was expressed in Escherichia coli. The recombinant product had GpgS activity, catalyzing the synthesis of glucosyl-3-phosphoglycerate (GPG) from GDP-glucose and d-3-phosphoglycerate, with a high substrate specificity. The recombinant MpgP protein dephosphorylated GPG to GG and was also able to dephosphorylate mannosyl-3-phosphoglycerate (MPG) but no other substrate tested. Similar flexibilities in substrate specificity were confirmed in vitro for the MpgPs from Thermus thermophilus, Pyrococcus horikoshii, and "Dehalococcoides ethenogenes." GpgS had maximal activity at 50 degrees C. The maximal activity of GpgP was at 50 degrees C with GPG as the substrate and at 60 degrees C with MPG. Despite the similarity of the sugar donors GDP-glucose and GDP-mannose, the enzymes for the synthesis of GPG or MPG share no amino acid sequence identity, save for short motifs. However, the hydrolysis of GPG and MPG is carried out by phosphatases encoded by homologous genes and capable of using both substrates. To our knowledge, this is the first report of the elucidation of a biosynthetic pathway for glucosylglycerate.     

The open reading frame TTC1157 of Thermus thermophilus HB27 encodes the methyltransferase forming N²-methylguanosine at position 6 in tRNA

N(2)-methylguanosine (m(2)G) is found at position 6 in the acceptor stem of Thermus thermophilus tRNA(Phe). In this article, we describe the cloning, expression, and characterization of the T. thermophilus HB27 methyltransferase (MTase) encoded by the TTC1157 open reading frame that catalyzes the formation of this modified nucleoside. S-adenosyl-L-methionine is used as donor of the methyl group. The enzyme behaves as a monomer in solution. It contains an N-terminal THUMP domain predicted to bind RNA and contains a C-terminal Rossmann-fold methyltransferase (RFM) domain predicted to be responsible for catalysis. We propose to rename the TTC1157 gene trmN and the corresponding protein TrmN, according to the bacterial nomenclature of tRNA methyltransferases. Inactivation of the trmN gene in the T. thermophilus HB27 chromosome led to a total absence of m(2)G in tRNA but did not affect cell growth or the formation of other modified nucleosides in tRNA(Phe). Archaeal homologs of TrmN were identified and characterized. These proteins catalyze the same reaction as TrmN from T. thermophilus. Individual THUMP and RFM domains of PF1002 from Pyrococcus furiosus were produced. These separate domains were inactive and did not bind tRNA, reinforcing the idea that the THUMP domain acts in concert with the catalytic domain to target a particular position of the tRNA molecule.     

Functional and evolutionary relationship between arginine biosynthesis and prokaryotic lysine biosynthesis through alpha-aminoadipate

Our previous studies revealed that lysine is synthesized through alpha-aminoadipate in an extremely thermophilic bacterium, Thermus thermophilus HB27. Sequence analysis of a gene cluster involved in the lysine biosynthesis of this microorganism suggested that the conversion from alpha-aminoadipate to lysine proceeds in a way similar to that of arginine biosynthesis. In the present study, we cloned an argD homolog of T. thermophilus HB27 which was not included in the previously cloned lysine biosynthetic gene cluster and determined the nucleotide sequence. A knockout of the argD-like gene, now termed lysJ, in T. thermophilus HB27 showed that this gene is essential for lysine biosynthesis in this bacterium. The lysJ gene was cloned into a plasmid and overexpressed in Escherichia coli, and the LysJ protein was purified to homogeneity. When the catalytic activity of LysJ was analyzed in a reverse reaction in the putative pathway, LysJ was found to transfer the epsilon-amino group of N(2)-acetyllysine, a putative intermediate in lysine biosynthesis, to 2-oxoglutarate. When N(2)-acetylornithine, a substrate for arginine biosynthesis, was used as the substrate for the reaction, LysJ transferred the delta-amino group of N(2)-acetylornithine to 2-oxoglutarate 16 times more efficiently than when N(2)-acetyllysine was the amino donor. All these results suggest that lysine biosynthesis in T. thermophilus HB27 is functionally and evolutionarily related to arginine biosynthesis.     

The X-ray crystallographic structure and activity analysis of a Pseudomonas-specific subfamily of the HAD enzyme superfamily evidences a novel biochemical function

The haloacid dehalogenase (HAD) superfamily is a large family of proteins dominated by phosphotransferases. Thirty-three sequence families within the HAD superfamily (HADSF) have been identified to assist in function assignment. One such family includes the enzyme phosphoacetaldehyde hydrolase (phosphonatase). Phosphonatase possesses the conserved Rossmanniod core domain and a C1-type cap domain. Other members of this family do not possess a cap domain and because the cap domain of phosphonatase plays an important role in active site desolvation and catalysis, the function of the capless family members must be unique. A representative of the capless subfamily, PSPTO_2114, from the plant pathogen Pseudomonas syringae, was targeted for catalytic activity and structure analyses. The X-ray structure of PSPTO_2114 reveals a capless homodimer that conserves some but not all of the intersubunit contacts contributed by the core domains of the phosphonatase homodimer. The region of the PSPTO_2114 that corresponds to the catalytic scaffold of phosphonatase (and other HAD phosphotransfereases) positions amino acid residues that are ill suited for Mg+2 cofactor binding and mediation of phosphoryl group transfer between donor and acceptor substrates. The absence of phosphotransferase activity in PSPTO_2114 was confirmed by kinetic assays. To explore PSPTO_2114 function, the conservation of sequence motifs extending outside of the HADSF catalytic scaffold was examined. The stringently conserved residues among PSPTO_2114 homologs were mapped onto the PSPTO_2114 three-dimensional structure to identify a surface region unique to the family members that do not possess a cap domain. The hypothesis that this region is used in protein-protein recognition is explored to define, for the first time, HADSF proteins which have acquired a function other than that of a catalyst.     

Post-translational phosphorylation of serine 74 of human deoxycytidine kinase favors the enzyme adopting the open conformation making it competent for nucleoside binding and release

Deoxycytidine kinase (dCK) uses either ATP or UTP as a phosphoryl donor to catalyze the phosphorylation of nucleoside acceptors. The kinetic properties of human dCK are modulated in vivo by phosphorylation of serine 74. This residue is a part of the insert region and is distant from the active site. Replacing the serine with a glutamic acid (S74E variant) can mimic phosphorylation of Ser74. To understand how phosphorylation affects the catalytic properties of dCK, we examined the S74E variant of dCK both structurally and kinetically. We observe that the presence of a glutamic acid at position 74 favors the adoption by the enzyme of the open conformation. Glu74 stabilizes the open conformation by directly interacting with the indole side chain of Trp58, a residue that is in the proximity of the base of the nucleoside substrate. The open dCK conformation is competent for the binding of nucleoside but not for phosphoryl transfer. In contrast, the closed conformation is competent for phosphoryl transfer but not for product release. Thus, dCK must make the transition between the open and closed states during the catalytic cycle. We propose a reaction scheme for dCK that incorporates the transition between the open and closed states, and this serves to rationalize the observed kinetic differences between wild-type dCK and the S74E variant.     

Structural and functional analysis of the phosphoryl transfer reaction mediated by the human small C‐terminal domain phosphatase,Scp1

Human small C‐terminal domain phosphatase 1 (Scp1) modulates the phosphorylation state of the C‐terminal domain (CTD) of eukaryotic RNA polymerase II (RNAP II), with preference for phosphorylated Ser5 in the tandem heptad repeats of the CTD. Additionally, Scp1 was identified as a conserved regulator of neuronal stem cell development. Scp1 is a member of haloacid dehalogenase (HAD) superfamily, whose catalysis depends on a Mg²⁺ ion and a DXDX(T/V) motif. The first Asp of the motif is identified as the nucleophile that is subject to phosphorylation leading to a phosphoryl‐aspartate intermediate. This high‐energy mixed anhydride intermediate is subsequently hydrolyzed to regenerate the enzyme. In the present study, we successfully captured the phosphoryl‐aspartate intermediate in the crystal structure of a Scp1D206A mutant soaked with para‐nitrophenyl phosphate (pNPP), providing strong evidence for the proposed mechanism. Furthermore, steady‐state kinetic analysis of a variety of Scp1 mutants revealed the importance of Asp206 in Mg²⁺ coordination mediated by a water molecule. Overall, we captured the snapshots of the phosphoryl transfer reaction at each stage of Scp1‐mediated catalysis. Through structural‐based sequence alignment, we show that the spatial position of the D206 side chain is strictly conserved throughout HAD family. Our results strongly suggest that Asp206 and its equivalent residues in other HAD family members play important structural and possible mechanistic roles.     

Characterization of DNA transport in the thermophilic bacterium Thermus thermophilus HB27

Horizontal gene transfer has been a major force for genome plasticity over evolutionary history, and is largely responsible for fitness-enhancing traits, including antibiotic resistance and virulence factors. In particular, for adaptation of prokaryotes to extreme environments, lateral gene transfer seems to have played a crucial role. Recently, by performing a genome-wide mutagenesis approach with Thermus thermophilus HB27, we identified the first genes in a thermophilic bacterium for the uptake of free DNA, a process called natural transformation. Here, we present the first data on the biochemistry and bioenergetics of the DNA transport process in this thermophile. We report that linear and circular plasmid DNA are equally well taken up with a high maximal velocity of 1.5 microg DNA.(mg protein)(-1).min(-1), demonstrating an extremely efficient binding and uptake rate of 40 kb.s(-1).cell(-1). Uncouplers and ATPase inhibitors immediately inhibited DNA uptake, providing clear evidence that DNA translocation in HB27 is an energy-dependent process. DNA uptake studies with genomic DNA of Bacteria, Archaea and Eukarya revealed that Thermus thermophilus HB27 takes up DNA from members of all three domains of life. We propose that the extraordinary broad substrate specificity of the highly efficient Thermus thermophilus HB27 DNA uptake system may contribute significantly to thermoadaptation of Thermus thermophilus HB27 and to interdomain DNA transfer in hot environments.     

Catalytic cycling in beta-phosphoglucomutase: a kinetic and structural analysis

Lactococcus lactis beta-phosphoglucomutase (beta-PGM) catalyzes the interconversion of beta-d-glucose 1-phosphate (beta-G1P) and beta-d-glucose 6-phosphate (G6P), forming beta-d-glucose 1,6-(bis)phosphate (beta-G16P) as an intermediate. Beta-PGM conserves the core domain catalytic scaffold of the phosphatase branch of the HAD (haloalkanoic acid dehalogenase) enzyme superfamily, yet it has evolved to function as a mutase rather than as a phosphatase. This work was carried out to identify the structural basis underlying this diversification of function. In this paper, we examine beta-PGM activation by the Mg(2+) cofactor, beta-PGM activation by Asp8 phosphorylation, and the role of cap domain closure in substrate discrimination. First, the 1.90 A resolution X-ray crystal structure of the Mg(2+)-beta-PGM complex is examined in the context of previously reported structures of the Mg(2+)-alpha-d-galactose-1-phosphate-beta-PGM, Mg(2+)-phospho-beta-PGM, and Mg(2+)-beta-glucose-6-phosphate-1-phosphorane-beta-PGM complexes to identify conformational changes that occur during catalytic turnover. The essential role of Asp8 in nucleophilic catalysis was confirmed by demonstrating that the D8A and D8E mutants are devoid of catalytic activity. Comparison of the ligands to Mg(2+) in the different complexes shows that a single Mg(2+) coordination site must alternatively accommodate water, phosphate, and the phosphorane intermediate during catalytic turnover. Limited involvement of the HAD family metal-binding loop in Mg(2+) anchoring in beta-PGM is consistent with the relatively loose binding indicated by the large K(m) for Mg(2+) activation (270 +/- 20 microM) and with the retention of activity found in the E169A/D170A double loop mutant. Comparison of the relative positions of cap and core domains in the different complexes indicated that interaction of cap domain Arg49 with the "nontransferring" phosphoryl group of the substrate ligand might stabilize the cap-closed conformation, as required for active site desolvation and alignment of Asp10 for acid-base catalysis. Kinetic analyses of the specificity of beta-PGM toward phosphoryl group donors and the specificity of phospho-beta-PGM toward phosphoryl group acceptors were carried out. The results support a substrate induced-fit mechanism of beta-PGM catalysis, which allows phosphomutase activity to dominate over the intrinsic phosphatase activity. Last, we present evidence that the autophosphorylation of beta-PGM by the substrate beta-G1P accounts for the origin of phospho-beta-PGM in the cell.     

Molecular analyses of the natural transformation machinery and identification of pilus structures in the extremely thermophilic bacterium Thermus thermophilus strain HB27

Thermus thermophilus HB27, an extremely thermophilic bacterium, exhibits high competence for natural transformation. To identify genes of the natural transformation machinery of T. thermophilus HB27, we performed homology searches in the partially completed T. thermophilus genomic sequence for conserved competence genes. These analyses resulted in the detection of 28 open reading frames (ORFs) exhibiting significant similarities to known competence proteins of gram-negative and gram-positive bacteria. Disruption of 15 selected potential competence genes led to the identification of 8 noncompetent mutants and one transformation-deficient mutant with a 100-fold reduced transformation frequency. One competence protein is similar to DprA of Haemophilus influenzae, seven are similar to type IV pilus proteins of Pseudomonas aeruginosa or Neisseria gonorrhoeae (PilM, PilN, PilO, PilQ, PilF, PilC, PilD), and another deduced protein (PilW) is similar to a protein of unknown function in Deinococcus radiodurans R1. Analysis of the piliation phenotype of T. thermophilus HB27 revealed the presence of single pilus structures on the surface of the wild-type cells, whereas the noncompetent pil mutants of Thermus, with the exception of the pilF mutant, were devoid of pilus structures. These results suggest that pili and natural transformation in T. thermophilus HB27 are functionally linked.     

A high-transformation-efficiency cloning vector for Thermus thermophilus.

The cloning vector pMK18 was developed through the fusion of the minimal replicative region from an indigenous plasmid of Thermus sp. ATCC27737, a gene cassette encoding a thermostable resistance to kanamycin, and the replicative origin and multiple cloning site of pUC18. Plasmid pMK18 showed transformation efficiencies from 10(8) to 10(9) per microgram of plasmid in Thermus thermophilus HB8 and HB27, both by natural competence and by electroporation. We also show that T. thermophilus HB27 can take pMK18 modified by the Escherichia coli methylation system with the same efficiency as its own DNA. To demonstrate its usefulness as a cloning vector, a gene encoding the beta-subunit of a thermostable nitrate reductase was directly cloned in T. thermophilus HB27 from a gene library. Its further transfer to E. coli also proved its utility as a shuttle vector.     

Crystal structure of phosphoserine phosphatase from Methanococcus jannaschii, a hyperthermophile, at 1.8 A resolution

BACKGROUND: D-Serine is a co-agonist of the N-methyl-D-aspartate subtype of glutamate receptors, a major neurotransmitter receptor family in mammalian nervous systems. D-Serine is converted from L-serine, 90% of which is the product of the enzyme phosphoserine phosphatase (PSP). PSP from M. jannaschii (MJ) shares significant sequence homology with human PSP. PSPs and P-type ATPases are members of the haloacid dehalogenase (HAD)-like hydrolase family, and all members share three conserved sequence motifs. PSP and P-type ATPases utilize a common mechanism that involves Mg(2+)-dependent phosphorylation and autodephosphorylation at an aspartyl side chain in the active site. The strong resemblance in sequence and mechanism implies structural similarity among these enzymes. RESULTS: The PSP crystal structure resembles the NAD(P) binding Rossmann fold with a large insertion of a four-helix-bundle domain and a beta hairpin. Three known conserved sequence motifs are arranged next to each other in space and outline the active site. A phosphate and a magnesium ion are bound to the active site. The active site is within a closed environment between the core alpha/beta domain and the four-helix-bundle domain. CONCLUSIONS: The crystal structure of MJ PSP was determined at 1.8 A resolution. Critical residues were assigned based on the active site structure and ligand binding geometry. The PSP structure is in a closed conformation that may resemble the phosphoserine bound state or the state after autodephosphorylation. Compared to a P-type ATPase (Ca(2+)-ATPase) structure, which is in an open state, this PSP structure appears also to be a good model for the closed conformation of P-type ATPase.     

A gene from the mesophilic bacterium Dehalococcoides ethenogenes encodes a novel mannosylglycerate synthase

Mannosylglycerate (MG) is a common compatible solute found in thermophilic and hyperthermophilic prokaryotes. In this study we characterized a mesophilic and bifunctional mannosylglycerate synthase (MGSD) encoded in the genome of the bacterium Dehalococcoides ethenogenes. mgsD encodes two domains with extensive homology to mannosyl-3-phosphoglycerate synthase (MPGS, EC 2.4.1.217) and to mannosyl-3-phosphoglycerate phosphatase (MPGP, EC 3.1.3.70), which catalyze the consecutive synthesis and dephosphorylation of mannosyl-3-phosphoglycerate to yield MG in Pyrococcus horikoshii, Thermus thermophilus, and Rhodothermus marinus. The bifunctional MGSD was overproduced in Escherichia coli, and we confirmed the combined MPGS and MPGP activities of the recombinant enzyme. The optimum activity of the enzyme was at 50 degrees C. To examine the properties of each catalytic domain of MGSD, we expressed them separately in E. coli. The monofunctional MPGS was unstable, while the MPGP was stable and was characterized. Dehalococcoides ethenogenes cannot be grown sufficiently to identify intracellular compatible solutes, and E. coli harboring MGSD did not accumulate MG. However, Saccharomyces cerevisiae expressing mgsD accumulated MG, confirming that this gene product can synthesize this compatible solute and arguing for a role in osmotic adjustment in the natural host. We did not detect MGSD activity in cell extracts of S. cerevisiae. Here we describe the first gene and enzyme for the synthesis of MG from a mesophilic microorganism and discuss the possible evolution of this bifunctional MGSD by lateral gene transfer from thermophilic and hyperthermophilic organisms.     

Horizontal transference of S-layer genes within Thermus thermophilus.

The S-layers of Thermus thermophilus HB27 and T. thermophilus HB8 are composed of protein units of 95 kDa (P95) and 100 kDa (P100), respectively. We have selected S-layer deletion mutants from both strains by complete replacement of the slpA gene. Mutants of the two strains showed similar defects in growth and morphology and overproduced an external cell envelope inside of which cells remained after division. However, the nature of this external layer is strain specific, being easily stained and regular in the HB8 delta slpA derivative and amorphous and poorly stained in the HB27 delta slpA strain. The addition of chromosomic DNA from T. thermophilus HB8 to growing cultures of T. thermophilus HB27 delta slpA led to the selection of a new strain, HB27C8, which expressed a functional S-layer composed of the P100 protein. Conversely, the addition of chromosomic DNA from T. thermophilus HB27 to growing cultures of T. thermophilus HB8 delta slpA allowed the isolation of strain HB8C27, which expressed a functional S-layer composed of the P95 protein. The driving force which selected the transference of the S-layer genes in these experiments was the difference in growth rates, one of the main factors leading to selection in natural environments.     

Cloning and sequence analysis of tryptophan synthetase genes of an extreme thermophile, Thermus thermophilus HB27: plasmid transfer from replica-plated Escherichia coli recombinant colonies to competent T. thermophilus cells.

Tryptophan synthetase genes (trpBA) of the extreme thermophile Thermus thermophilus HB27 were cloned by a novel method of direct plasmid transfer from replica-plated Escherichia coli recombinant colonies to competent T. thermophilus HB27 trpB cells. The nucleotide sequences of the trpBA genes were determined. The amino acid sequences deduced from the nucleotide sequences of Thermus trpB and trpA were found to have identities of 54.8 and 28.7%, respectively, with those of E. coli trpB and trpA genes. Low cysteine content (one in trpB; zero in trpA) is a striking feature of these proteins, which may contribute to their thermostability.     

MDP-1 is a new and distinct member of the haloacid dehalogenase family of aspartate-dependent phosphohydrolases

MDP-1 is a eukaryotic magnesium-dependent acid phosphatase with little sequence homology to previously characterized phosphatases. The presence of a conserved motif (Asp-X-Asp-X-Thr) in the N terminus of MDP-1 suggested a relationship to the haloacid dehalogenase (HAD) superfamily, which contains a number of magnesium-dependent acid phosphatases. These phosphatases utilize an aspartate nucleophile and contain a number of conserved active-site residues and hydrophobic patches, which can be plausibly aligned with conserved residues in MDP-1. Seven site-specific point mutants of MDP-1 were produced by modifying the catalytic aspartate, serine, and lysine residues to asparagine or glutamate, alanine, and arginine, respectively. The activity of these mutants confirms the assignment of MDP-1 as a member of the HAD superfamily. Detailed comparison of the sequence of the 15 MDP-1 sequences from various organisms with other HAD superfamily sequences suggests that MDP-1 is not closely related to any particular member of the superfamily. The crystal structures of several HAD family enzymes identify a domain proximal to the active site responsible for important interactions with low molecular weight substrates. The absence of this domain or any other that might perform the same function in MDP-1 suggests an "open" active site capable of interactions with large substrates such as proteins. This suggestion was experimentally confirmed by demonstration that MDP-1 is competent to catalyze the dephosphorylation of tyrosine-phosphorylated proteins.     

Genetic transformation of the extreme thermophile Thermus thermophilus and of other Thermus spp.

Genetic transformation of auxotrophs of the extreme thermophile Thermus thermophilus HB27 to prototrophy was obtained at high frequencies of 10(-2) to 10(-1) when proliferating cell populations were exposed to chromosomal DNA from a nutritionally independent wild-type strain. The transformation frequency was proportional to the DNA concentration from 10 pg/ml to 100 ng/ml. T. thermophilus HB27 cells did not require chemical treatment to induce competence, although optimal transformation was obtained by the addition of a divalent cation (Ca2+ or Mg2+). Competence was maintained throughout the growth phase, with the highest transformation frequencies at pH 6 to 9 and at 70 degrees C. T. thermophilus HB27 and four other typical Thermus strains, T. thermophilus HB8, T. flavus AT62, T. caldophilus GK24, and T. aquaticus YT1, were also transformed to streptomycin resistance by DNA from their own spontaneous streptomycin-resistant mutants. A cryptic plasmid, pTT8, from T. thermophilus HB8 was introduced into T. thermophilus HB27 Pro- at a frequency of 10(-2).     

Bioenergetics at extreme temperature: Thermus thermophilus ba(3)- and caa(3)-type cytochrome c oxidases

Seven years into the completion of the genome sequencing projects of the thermophilic bacterium Thermus thermophilus strains HB8 and HB27, many questions remain on its bioenergetic mechanisms. A key fact that is occasionally overlooked is that oxygen has a very limited solubility in water at high temperatures. The HB8 strain is a facultative anaerobe whereas its relative HB27 is strictly aerobic. This has been attributed to the absence of nitrate respiration genes from the HB27 genome that are carried on a mobilizable but highly-unstable plasmid. In T. thermophilus, the nitrate respiration complements the primary aerobic respiration. It is widely known that many organisms encode multiple biochemically-redundant components of the respiratory complexes. In this minireview, the presence of the two cytochrome c oxidases (CcO) in T. thermophilus, the ba(3)- and caa(3)-types, is outlined along with functional considerations. We argue for the distinct evolutionary histories of these two CcO including their respective genetic and molecular organizations, with the caa(3)-oxidase subunits having been initially 'fused'. Coupled with sequence analysis, the ba(3)-oxidase crystal structure has provided evolutionary and functional information; for example, its subunit I is more closely related to archaeal sequences than bacterial and the substrate-enzyme interaction is hydrophobic as the elevated growth temperature weakens the electrostatic interactions common in mesophiles. Discussion on the role of cofactors in intra- and intermolecular electron transfer and proton pumping mechanism is also included.     

Evolutionary and Structural Analyses of Mammalian Haloacid Dehalogenase-type Phosphatases AUM and Chronophin Provide Insight into the Basis of Their Different Substrate Specificities

Mammalian haloacid dehalogenase (HAD)-type phosphatases are an emerging family of phosphatases with important functions in physiology and disease, yet little is known about the basis of their substrate specificity. Here, we characterize a previously unexplored HAD family member (gene annotation, phosphoglycolate phosphatase), which we termed AUM, for aspartate-based, ubiquitous, Mg²⁺-dependent phosphatase. AUM is a tyrosine-specific paralog of the serine/threonine-specific protein and pyridoxal 5′-phosphate-directed HAD phosphatase chronophin. Comparative evolutionary and biochemical analyses reveal that a single, differently conserved residue in the cap domain of either AUM or chronophin is crucial for phosphatase specificity. We have solved the x-ray crystal structure of the AUM cap fused to the catalytic core of chronophin to 2.65 Å resolution and present a detailed view of the catalytic clefts of AUM and chronophin that explains their substrate preferences. Our findings identify a small number of cap domain residues that encode the different substrate specificities of AUM and chronophin.