Crystal structure of an archaeal homologue of multidrug resistance repressor protein, EmrR, from hyperthermophilic archaea Sulfolobus tokodaii strain 7.

MarR family proteins, MarR, MexR, and EmrR, are known as bacterial regulators for a phenotype resistant to multiple antibiotic drugs. Genomic data have indicated the presence of bacterial-type transcriptional regulators, including MarR family proteins in archaea, though the archaeal transcription system is close to that of eukaryote. To elucidate the structural basis of the transcriptional regulation mechanism of archaeal MarR family proteins, the crystal structure of the ST1710 protein, which was identified as an archaeal EmrR homologue, StEmrR, from hyperthermophilic archaeon Sulfolobus tokodaii strain 7 was determined at 1.45-A resolution. The protein was composed of two N- and C-terminal dimerization domains, and the DNA-binding domain consisted of a winged helix motif, as in the case of bacterial MarR family proteins. Despite the relatively low overall structural similarity between StEmrR and bacterial MarR family proteins, the structure of the DNA-binding domain displayed high structural similarity. A comparison with the crystal structures of bacterial MarR family proteins revealed that structural variation was mainly due to the different orientation of the two helices at the N- and C-termini. Our results indicated that the distance between the two DNA-binding domains of MarR family proteins would be changed by the rotation of the two terminal helices to interact with the target DNA.

Increasing in archaeal GlcNAc-1-P uridyltransferase activity by targeted mutagenesis while retaining its extreme thermostability.

UDP-GlcNAc, an activated and essential form of GlcNAc which is an important component in the polysaccharide structure of most organisms, is synthesized from GlcNAc-1-P and UTP by GlcNAc-1-P UTase. We previously reported the identification of the extremely thermostable ST0452 protein, which has dual sugar-1-P NTase activities (Glc-1-P TTase and GlcNAc-1-P UTase activities) from an acidothermophilic archaeon, Sulfolobus tokodaii strain 7. Detailed analyses of the protein indicated that the activity is slightly lower than that of bacteria. For industrial applications, activity needs to be increased without decreasing thermostability. Therefore, to enhance this activity, we introduced mutations into the amino acid residues located within the predicted reaction centre by targeted mutagenesis. All 12 mutant ST0452 proteins showed no decrease in thermostability. Among them, six mutant proteins were found to have increased GlcNAc-1-P UTase activity under optimal reaction conditions with sufficient substrates or an appropriate metal ion. Our results indicate that targeted mutagenesis is a powerful technique for in vitro production of a thermostable enzyme with enhanced activity. The results of this study also indicate that the space for the metal ion is important for selecting the type of metal ion and also affects the rate of the reaction.

Crystal structure of archaeal photolyase from Sulfolobus tokodaii with two FAD molecules: implication of a novel light-harvesting cofactor.

UV exposure of DNA molecules induces serious DNA lesions. The cyclobutane pyrimidine dimer (CPD) photolyase repairs CPD-type - lesions by using the energy of visible light. Two chromophores for different roles have been found in this enzyme family; one catalyzes the CPD repair reaction and the other works as an antenna pigment that harvests photon energy. The catalytic cofactor of all known photolyases is FAD, whereas several light-harvesting cofactors are found. Currently, 5,10-methenyltetrahydrofolate (MTHF), 8-hydroxy-5-deaza-riboflavin (8-HDF) and FMN are the known light-harvesting cofactors, and some photolyases lack the chromophore. Three crystal structures of photolyases from Escherichia coli (Ec-photolyase), Anacystis nidulans (An-photolyase), and Thermus thermophilus (Tt-photolyase) have been determined; however, no archaeal photolyase structure is available. A similarity search of archaeal genomic data indicated the presence of a homologous gene, ST0889, on Sulfolobus tokodaii strain7. An enzymatic assay reveals that ST0889 encodes photolyase from S. tokodaii (St-photolyase). We have determined the crystal structure of the St-photolyase protein to confirm its structural features and to investigate the mechanism of the archaeal DNA repair system with light energy. The crystal structure of the St-photolyase is superimposed very well on the three known photolyases including the catalytic cofactor FAD. Surprisingly, another FAD molecule is found at the position of the light-harvesting cofactor. This second FAD molecule is well accommodated in the crystal structure, suggesting that FAD works as a novel light-harvesting cofactor of photolyase. In addition, two of the four CPD recognition residues in the crystal structure of An-photolyase are not found in St-photolyase, which might utilize a different mechanism to recognize the CPD from that of An-photolyase.

Identification of sn-glycerol-1-phosphate dehydrogenase activity from genomic information on a hyperthermophilic archaeon, Sulfolobus tokodaii strain 7.

sn-Glycerol-1-phosphate dehydrogenase is responsible for the formation of sn-glycerol-1-phosphate, the backbone of membrane phospholipids of Archaea. This activity had never been detected in cell-free extract of Sulfolobus sp. Here we report the detection of this activity on the thermostable ST0344 protein of Sulfolobus tokodaii expressed in Escherichia coli, which was predicted from genomic information on S. tokodaii. This is another line of evidence for the general mechanism of sn-glycerol-1-phosphate formation by the enzyme.

Characterization of a thermostable enzyme with phosphomannomutase/phosphoglucomutase activities from the hyperthermophilic archaeon Pyrococcus horikoshii OT3.

The phosphomannomutase/phosphoglucomutase (PMM/PGM) enzyme catalyzes reversibly the intra-molecular phosphoryl interconverting reaction of mannose-6-phosphate and mannose-1-phosphate or glucose-6-phosphate and glucose-1-phosphate. Glucose-6-phosphate and glucose-1-phosphate are known to be utilized for energy metabolism and cell surface construction, respectively. PMM/PGM has been isolated from many microorganisms. By performing similarity searches using existing PMM/PGM sequences, the homologous ORFs PH0923 and PH1210 were identified from the genomic data of Pyrococcus horikoshii OT3. Since PH0923 appears to be part of an operon consisting of four carbohydrate metabolic enzymes, PH0923 was selected as the first target for the investigation of PMM/PGM activity in P. horikoshii OT3. The coding region of PH0923 was cloned and the purified recombinant protein was utilized for an examination of its biochemical properties. The enzyme retained half its initial activity after treatment at 95 degrees C for 90 min. Detailed analyses of activities showed that this protein is capable of utilizing a variety of metal ions that are not utilized by previously characterized PMM/PGM proteins. A mutated protein with an alanine residue replacing the active site serine residue indicated that this residue plays an important but non-essential role in PMM/PGM activity.

Identification of an extremely thermostable enzyme with dual sugar-1-phosphate nucleotidylyltransferase activities from an acidothermophilic archaeon, Sulfolobus tokodaii strain 7.

L-rhamnose is an essential component of the cell wall and plays roles in mediating virulence and adhesion to host tissues in many microorganisms. Glucose-1-phosphate thymidylyltransferase (RmlA, EC 2.7.7.24) catalyzes the first reaction of the four-step pathway of L-rhamnose biosynthesis, producing dTDP-D-glucose from dTTP and glucose-1-phosphate. Three RmlA homologues of varying size have been identified in the genome of a thermophilic archaeon, Sulfolobus tokodaii strain 7. In this study, we report the heterologous expression of the largest homologue (a 401 residue-long ST0452 protein) and characterization of its thermostable activity. RmlA enzymatic activity of this protein was detected from 65 to 100 degrees C, with a half-life of 60 min at 95 degrees C and 180 min at 80 degrees C. Analysis of a deletion mutant lacking the 170-residue C-terminal domain indicated that this region has an important role in the thermostability and activity of the protein. Analyses of substrate specificity indicated that the enzymatic activity of the full-length protein is capable of utilizing alpha-D-glucose-1-phosphate and N-acetyl-D-glucosamine-1-phosphate but not alpha-D-glucosamine-1-phosphate. However, the protein is capable of utilizing all four deoxyribonucleoside triphosphates and UTP. Thus, the ST0452 protein is an enzyme containing both glucose-1-phosphate thymidylyltransferase and N-acetyl-D-glucosamine-1-phosphate uridylyltransferase activities. This is the first report of a thermostable enzyme with dual sugar-1-phosphate nucleotidylyltransferase activities.

Motif CXCC in nitrile hydratase activator is critical for NHase biogenesis in vivo.

Nitrile hydratase (NHase) activator from Rhodococcus sp. N-771 is required for NHase functional expression. The motif 73CXCC76 in the NHase activator sequence was here revealed to be vital for its function by site-directed mutagenesis. All three substitutions of the cysteines by serines resulted in a much lower level of expression of active NHase. Furthermore, interaction between NHase activator and NHase was detected and the critical role of NHase activator was not exhibited in the cysteine oxidization process of NHase. These findings suggest NHase activator mainly participates in iron trafficking in NHase biogenesis as an iron type metallochaperone.

A novel inhibitor for Fe-type nitrile hydratase: 2-cyano-2-propyl hydroperoxide.

Nitrile hydratase (NHase) is a non-heme iron or non-corrin cobalt enzyme having two post-translationally modified ligand residues, cysteine-sulfinic acid (alphaCys112-SO(2)H) and -sulfenic acid (alphaCys114-SOH). We studied the interaction between Fe-type NHase and isobutyronitrile (iso-BN) which had been reported as a competitive inhibitor with a K(i) value of 5 microM. From detailed kinetic studies of the inhibitory effect of iso-BN on Fe-type NHase, we found that authentic iso-BN was hydrated normally and that the impurity present in commercially available iso-BN inhibited NHase activity strongly. The inhibitory compound induced significant changes in the UV-vis absorption spectrum of NHase, suggesting its interaction with the iron center. This compound was purified by using reversed-phase HPLC and identified as 2-cyano-2-propyl hydroperoxide (Cpx) by (1)H and PFG-HMBC NMR spectroscopy. Upon addition of a stoichiometric amount of Cpx, NHase was irreversibly inactivated, probably by the oxidation of alphaCys114-SOH to Cys-SO(2)H. This result suggests that the -SOH structure of alphaCys114 is essential for the catalytic activity. The oxygen atom in Cys-SO(2)H is confirmed to come from the solvent H(2)O. The oxidized NHase was found to induce the UV-vis absorption spectral changes by addition of Cpx, suggesting that Cpx strongly interacted with iron(III) in the oxidized NHase to form a stable complex. Thus, Cpx functions as a novel irreversible inhibitor for NHase.