Masanari Tsujimura

Fe-type nitrile hydratase

The characteristic features of Fe-type nitrile hydratase (NHase) from Rhodococcus sp. N-771 are described. Through the biochemical analyses, we have found that nitric oxide (NO) regulates the photoreactivity of this enzyme by association with the non-heme iron center and photoinduced dissociation from it. The regulation is realized by a unique structure of the catalytic non-heme iron center composed of post-translationally modified cysteine-sulfinic (Cys-SO2H) and -sulfenic acids (Cys-SOH). To understand the biogenic mechanism and the functional role of these modifications, we constructed an over-expression system of whole NHase and individual subunits in Escherichia coli. The results of the studies on several recombinant NHases have shown that the Cys-SO2H oxidation of alphaC112 is indispensable for the catalytic activity of Fe-type NHase.

Structure of the Photoreactive Iron Center of the Nitrile Hydratase from Rhodococcus sp. N-771 EVIDENCE OF A NOVEL POST-TRANSLATIONAL MODIFICATION IN THE CYSTEINE LIGAND

Nitrile hydratase (NHase) fromRhodococcus sp. N-771 is a photoreactive enzyme that is inactivated by nitrosylation of the non-heme iron center and activated by photodissociation of nitric oxide (NO). To obtain structural information on the iron center, we isolated peptide complexes containing the iron center by proteolysis. When the tryptic digest of the α subunit isolated from the inactive form was analyzed by reversed-phase high performance liquid chromatography, the absorbance characteristic of the nitrosylated iron center was observed in the peptide fragment, Asn105-Val-Ile-Val-Cys-Ser-Leu-Cys-Ser-Cys-Thr-Ala-Trp-Pro-Ile-Leu-Gly-Leu-Pro-Pro-Thr-Trp-Tyr-Lys128. The peptide contained 0.79 mol of iron/mol of molecule as well as endogenous NO. Subsequently, by digesting the peptide with thermolysin, carboxypeptidase Y, and leucine aminopeptidase M, we found that the minimum peptide segment required for the nitrosylated iron center is the 11 amino acid residues from αIle107 to αTrp117. Furthermore, by using mass spectrometry, protein sequence, and amino acid composition analyses, we have shown that the 112th Cys residue of the α subunit is post-translationally oxidized to a cysteine-sulfinic acid (Cys-SO2H) in the NHase. These results indicate that the NHase from Rhodococcus sp. N-771 has a novel non-heme iron enzyme containing a cysteine-sulfinic acid in the iron center. Possible ligand residues of the iron center are discussed.

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.

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‐Åresolution. 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. Proteins 2007.

Crystal structure of STS042, a stand-alone RAM module protein, from hyperthermophilic archaeon Sulfolobus tokodaii strain7

The leucine-responsive regulatory protein (Lrp) was identified as a regulatory protein that affects the expression of many genes and operons. This protein belongs to the Lrp/ AsnC family of transcriptional regulators. Members of this family are often involved in cellular metabolism in response to amino acid effectors. Search results among the DNA database indicate that the gene encoding Lrp/AsnC family proteins is widely distributed among many bacteria and most archaea, but not identified in eukarya.1 The recent microarray data showed that transcription levels of at least 10% of all Escherichia coli genes are affected by Lrp, depending on the amount of leucine.2 The Lrp/AsnC family proteins with an approximate molecular mass of 15 kDa consist of three domains, the N-terminal DNA-binding domain, the C-terminal amino acid effector binding [regulation of amino-acid metabolism (RAM)] domain, which is essential for multimerization, and a flexible hinge region connecting the two domains. Analyses of the oligomeric state of the E. coli Lrp protein by dynamic light scattering, chemical cross-linking, and analytical ultracentrifugation have indicated that the protein converts their oligomeric structures among dimeric, tetrameric, octameric, and hexadecameric forms.3,4 However, the three-dimensional structures of various Lrp/AsnC family proteins such as LrpA from Pyrococcus furiosus, AsnC, and Lrp from E. coli, and LrpC from Bacillus subtilis have been determined only as an octameric form.5–7 Interestingly, proteins that possess on the RAM domain are frequently observed in the genome of many organisms. These are classified as stand-alone RAMdomain (SARD) proteins.8,9 The functions of these proteins remain unclear, though some of these are crystallized and structure determined.10–12 The structure of TTHA0845 from Thermus thermophilus, which is one of the SARD proteins, was determined in a unique homodecameric form.10 In one of the SARD proteins, DM1, self-association to a hexadecamer and octamer is promoted by the addition of hydrophobic amino acids.13 The four genes encoding Lrp homologous protein, ST1022, ST1115, ST0489, and ST1473, and one gene encoding SARD protein, STS042, were identified from the entire genomic data of the hyperthermophilic archaeon Sulfolobus tokodaii strain7. Because the structure of the SARD protein from crenarchaeota remains unclear, the structure of STS042 was determined to elucidate the function of SARD. Using the expressed and purified protein, we determined the crystal structure of STS042 at 1.9 Å resolution by the multiwavelength anomalous dispersion (MAD) phasing method. The structure has revealed that this protein forms a