Comparative proteomic analysis of glomerular proteins in IgA nephropathy and IgA vasculitis with nephritis.

BACKGROUND: IgA nephropathy (IgAN) and IgA vasculitis with nephritis (IgAVN) are related glomerular diseases characterized by marked similarities in immunological and histological findings. We herein performed a comparative proteomic analysis of glomerular proteins in IgAN and IgAVN. METHODS: We used renal biopsy specimens from 6 IgAN patients without nephrotic syndrome (NS) (IgAN-I subgroup), 6 IgAN patients with NS (IgAN-II subgroup), 6 IgAVN patients with 0-8.0% of glomeruli with crescent formation (IgAVN-I subgroup), 6 IgAVN patients with 21.2-44.8% of glomeruli with crescent formation (IgAVN-II subgroup), 9 IgAVN patients without NS (IgAVN-III subgroup), 3 IgAVN patients with NS (IgAN-IV subgroup), and 5 control cases. Proteins were extracted from laser microdissected glomeruli and analyzed using mass spectrometry. The relative abundance of proteins was compared between groups. An immunohistochemical validation study was also performed. RESULTS: More than 850 proteins with high confidence were identified. A principal component analysis revealed a clear separation between IgAN and IgAVN patients and control cases. In further analyses, 546 proteins that were matched with ≥ 2 peptides were selected. The levels of immunoglobulins (IgA, IgG, and IgM), complements (C3, C4A, C5, and C9), complement factor H-related proteins (CFHR) 1 and 5, vitronectin, fibrinogen chains, and transforming growth factor-ß inducible gene-h3 were higher (> 2.6 fold) in the IgAN and IgAVN subgroups than in the control group, whereas hornerin levels were lower (< 0.3 fold). Furthermore, C9 and CFHR1 levels were significantly higher in the IgAN group than in the IgAVN group. The abundance of some podocyte-associated proteins and glomerular basement membrane (GBM) proteins was significantly less in the IgAN-II subgroup than in the IgAN-I subgroup as well as in the IgAVN-IV subgroup than in the IgAVN-III subgroup. Among the IgAN and IgAVN subgroups, talin 1 was not detected in the IgAN-II subgroup. This result was supported by immunohistochemical findings. CONCLUSIONS: The present results suggest shared molecular mechanisms for glomerular injury in IgAN and IgAVN, except for enhanced glomerular complement activation in IgAN. Differences in the protein abundance of podocyte-associated and GBM proteins between IgAN and IgAVN patients with and without NS may be associated with the severity of proteinuria.

Comparative proteomic analysis of glomerular proteins in primary and bucillamine-induced membranous nephropathy.

BACKGROUND: Anti-phospholipase A2 receptor autoantibody (PLA2R Ab)-associated membranous nephropathy (MN) is the most common form of primary MN (pMN). On the other hand, bucillamine (BCL), an antirheumatic drug developed in Japan, was reported to cause a rare form of secondary MN (sMN). Between these MN forms, comparative proteomic analysis of glomerular proteins has not been performed. METHODS: We used renal biopsy specimens from 6 patients with PLA2R Ab (+) pMN, 6 patients with PLA2R Ab (‒) pMN, 6 patients with BCL-induced sMN, and 5 control cases (time 0 transplant biopsies). Proteins were extracted from laser-microdissected glomeruli and analyzed using mass spectrometry. The quantification values of protein abundance in each MN group were compared with those in the control group. RESULTS: More than 800 proteins with high confidence were identified. Principal component analysis revealed a different distribution between the pMN and sMN groups. For further analysis, 441 proteins matched with ≥ 3 peptides were selected. Among the pMN and sMN groups, we compared the profiles of several protein groups based on the structural and functional characteristics, such as immunoglobulins, complements, complement-regulating proteins, podocyte-associated proteins, glomerular basement membrane proteins, and several proteins that are known to be associated with kidney diseases, including MN. In all MN groups, increased levels of immunoglobulins (IgG, IgA, and IgM), complements (C3, C4, and C9), complement factor H-related protein 5, type XVIII collagen, calmodulin, polyubiquitin, and ubiquitin ligase were observed. For some proteins, such as type VII collagen and nestin, the fold-change values were significantly different between the pMN and sMN groups. CONCLUSIONS: Between the pMN and BCL-induced sMN groups, we observed common and different alterations in protein levels such as known disease-associated proteins and potential disease marker proteins.

Comparative proteomic analysis of renal proteins from IgA nephropathy model mice and control mice.

BACKGROUND: High-IgA ddY (HIGA) mice, an animal model of human IgA nephropathy (IgAN), spontaneously develop nephropathy with glomerular IgA deposition and markedly elevated serum IgA levels from 25 weeks of age. METHODS: We performed a comparative proteomic analysis of the renal proteins collected from HIGA mice and control C57BL/6 mice at 5 or 38 weeks of age (the H5, H38, C5, and C38 groups) (n = 4 in each group). Proteins were extracted from the left whole kidney of each mouse and analyzed using nano-liquid chromatography-tandem mass spectrometry. The right kidneys were used for histopathological examinations. RESULTS: Immunohistochemical examinations showed glomerular deposition of IgA and the immunoglobulin joining (J) chain, and increased numbers of interstitial IgA- and J-chain-positive plasma cells in the H38 group. In the proteomic analysis, > 5000 proteins were identified, and 33 proteins with H38/H5 ratios of > 5.0, H38/C38 ratios of > 5.0, and C38/C5 ratios of < 1.5 were selected. Among them, there were various proteins that are known to be involved in human IgAN and/or animal IgAN models. Immunohistochemical examinations validated the proteomic results for some proteins. Furthermore, two proteins that are known to be associated with kidney disease displayed downregulated expression (H38/H5 ratio: 0.01) in the H38 group. CONCLUSIONS: The results of comparative proteomic analysis of renal proteins were consistent with previous histopathological and serological findings obtained in ddY and HIGA mice. Various proteins that are known to be involved in kidney disease, including IgAN, and potential disease marker proteins exhibited markedly altered levels in HIGA mice.

Comparison of measurements of anti-PLA2R antibodies in Japanese patients with membranous nephropathy using in-house and commercial ELISA.

BACKGROUND: The prevalence of antibodies against M-type anti-phospholipase A2 receptor (PLA2R) was reported to be ~ 70-80% in early studies on idiopathic membranous nephropathy (iMN) cohorts from Western countries, China, and Korea, and ~ 50% in recent studies on two Japanese iMN cohorts. METHODS: We developed an in-house enzyme-linked immunosorbent assay (ELISA) for the detection of anti-PLA2R antibodies, and examined sera from 217 patients with iMN, 22 patients with secondary MN (sMN), and 50 healthy individuals. All patients and healthy individuals were Japanese. The relationships between levels of anti-PLA2R antibodies and clinical parameters were analyzed. Serum samples were also tested using a standardized commercial ELISA (Euroimmun, Germany). RESULTS: In our ELISA, OD values greater than the mean + 3 standard deviation of healthy subjects were considered to be positive for anti-PLA2R antibodies. Of the patients with iMN, 33.6% (73/217) were positive, but all sMN patients were negative. Our ELISA and the Euroimmun ELISA had a high concordance (93.5%). The proportion of patients with nephrotic syndrome was significantly higher in anti-PLA2R antibody-positive patients than in antibody-negative patients (65.8 vs. 37.5%, P < 0.001). Levels of anti-PLA2R antibodies were significantly correlated with levels of urinary protein and serum albumin (P = 0.004 and P < 0.001, respectively). CONCLUSIONS: The prevalence of anti-PLA2R antibodies in our Japanese iMN cohort was lower than that in the previous studies from other countries and other Japanese institutes. The low prevalence of antibodies may be related with the characteristics of enrolled patients with mild proteinuria and undetectable antibody levels.

Structural and functional characterization of aspartate racemase from the acidothermophilic archaeon Picrophilus torridus.

Functional and structural characterizations of pyridoxal 5'-phosphate-independent aspartate racemase of the acidothermophilic archaeon Picrophilus torridus were performed. Picrophilus aspartate racemase exhibited high substrate specificity to aspartic acid. The optimal reaction temperature was 60 °C, which is almost the same as the optimal growth temperature. Reflecting the low pH in the cytosol, the optimal reaction pH of Picrophilus aspartate racemase was approximately 5.5. However, the activity at the putative cytosolic pH of 4.6 was approximately 6 times lower than that at the optimal pH of 5.5. The crystal structure of Picrophilus aspartate racemase was almost the same as that of other pyridoxal 5'-phosphate -independent aspartate racemases. In two molecules of the dimer, one molecule contained a tartaric acid molecule in the catalytic site; the structure of the other molecule was relatively flexible. Finally, we examined the intracellular existence of D-amino acids. Unexpectedly, the proportion of D-aspartate to total aspartate was not very high. In contrast, both D-proline and D-alanine were observed. Because Picrophilus aspartate racemase is highly specific to aspartate, other amino acid racemases might exist in Picrophilus torridus.

Crystal structures of halohydrin hydrogen-halide-lyases from Corynebacterium sp. N-1074.

Halohydrin hydrogen-halide-lyase (H-Lyase) is a bacterial enzyme that is involved in the degradation of halohydrins. This enzyme catalyzes the intramolecular nucleophilic displacement of a halogen by a vicinal hydroxyl group in halohydrins to produce the corresponding epoxides. The epoxide products are subsequently hydrolyzed by an epoxide hydrolase, yielding the corresponding 1, 2-diol. Until now, six different H-Lyases have been studied. These H-Lyases are grouped into three subtypes (A, B, and C) based on amino acid sequence similarities and exhibit different enantioselectivity. Corynebacterium sp. strain N-1074 has two different isozymes of H-Lyase, HheA (A-type) and HheB (B-type). We have determined their crystal structures to elucidate the differences in enantioselectivity among them. All three groups share a similar structure, including catalytic sites. The lack of enantioselectivity of HheA seems to be due to the relatively wide size of the substrate tunnel compared to that of other H-Lyases. Among the B-type H-Lyases, HheB shows relatively high enantioselectivity compared to that of HheBGP1 . This difference seems to be due to amino acid replacements at the active site tunnel. The binding mode of 1, 3-dicyano-2-propanol at the catalytic site in the crystal structure of the HheB-DiCN complex suggests that the product should be (R)-epichlorohydrin, which agrees with the enantioselectivity of HheB. Comparison with the structure of HheC provides a clue for the difference in their enantioselectivity.

Packaging guest proteins into the encapsulin nanocompartment from Rhodococcus erythropolis N771.

The encapsulin nanocompartment from Rhodococcus erythropolis N771 (Reencapsulin) was expressed and purified in wild-type and C-terminally His-tagged forms. Negative-stained transmission electron microscopy, field-flow fractionation combined with multi-angle light scattering and dynamic light scattering analyses showed that 60 Reencapsulin monomers were assembled as a spherical particle with a diameter of 28 nm. Heterogeneous guest proteins such as EGFP and firefly luciferase were packaged into the internal cavity of the Reencapsulin nanocompartment by fusing the C-terminal 37-amino-acid sequence of the R. erythropolis N771 DypB peroxidase to the C-terminus. Reencapsulin has the potential to package target proteins in its internal cavity and/or display them on its external surface, making it a feasible carrier for nanotechnology applications.

Time-Resolved Crystallography of the Reaction Intermediate of Nitrile Hydratase: Revealing a Role for the Cysteinesulfenic Acid Ligand as a Catalytic Nucleophile.

The reaction mechanism of nitrile hydratase (NHase) was investigated using time-resolved crystallography of the mutant NHase, in which ßArg56, strictly conserved and hydrogen bonded to the two post-translationally oxidized cysteine ligands, was replaced by lysine, and pivalonitrile was the substrate. The crystal structures of the reaction intermediates were determined at high resolution (1.2-1.3 Å). In combination with FTIR analyses of NHase following hydration in H2 (18) O, we propose that the metal-coordinated substrate is nucleophilically attacked by the O(SO(-) ) atom of αCys114-SO(-) , followed by nucleophilic attack of the S(SO(-) ) atom by a ßArg56-activated water molecule to release the product amide and regenerate αCys114-SO(-) .

Carbonyl sulfide hydrolase from Thiobacillus thioparus strain THI115 is one of the ß-carbonic anhydrase family enzymes.

Carbonyl sulfide (COS) is an atmospheric trace gas leading to sulfate aerosol formation, thereby participating in the global radiation balance and ozone chemistry, but its biological sinks are not well understood. Thiobacillus thioparus strain THI115 can grow on thiocyanate (SCN(-)) as its sole energy source. Previously, we showed that SCN(-) is first converted to COS by thiocyanate hydrolase in T. thioparus strain THI115. In the present work, we purified, characterized, and determined the crystal structure of carbonyl sulfide hydrolase (COSase), which is responsible for the degradation of COS to H2S and CO2, the second step of SCN(-) assimilation. COSase is a homotetramer composed of a 23.4 kDa subunit containing a zinc ion in its catalytic site. The amino acid sequence of COSase is homologous to the ß-class carbonic anhydrases (ß-CAs). Although the crystal structure including the catalytic site resembles those of the ß-CAs, CO2 hydration activity of COSase is negligible compared to those of the ß-CAs. The α5 helix and the extra loop (Gly150-Pro158) near the N-terminus of the α6 helix narrow the substrate pathway, which could be responsible for the substrate specificity. The k(cat)/K(m) value, 9.6 × 10(5) s(-1) M(-1), is comparable to those of the ß-CAs. COSase hydrolyzes COS over a wide concentration range, including the ambient level, in vitro and in vivo. COSase and its structurally related enzymes are distributed in the clade D in the phylogenetic tree of ß-CAs, suggesting that COSase and its related enzymes are one of the catalysts responsible for the global sink of COS.

Expression, purification, crystallization and preliminary X-ray crystallographic studies of hepatitis B virus core fusion protein corresponding to octahedral particles.

Recombinant hepatitis B virus core proteins dimerize to form building blocks that are capable of self-assembly into a capsid. A core capsid protein dimer (CPD) linked to a green fluorescent protein variant, EGFP, at the C-terminus has been designed. The recombinant fusion CPD was expressed in Escherichia coli, assembled into virus-like particles (VLPs), purified and crystallized. The single crystal diffracted to 2.15 Å resolution and belonged to the cubic space group F432, with unit-cell parameters a = b = c = 219.7 Å. The fusion proteins assembled into icosahedral VLPs in aqueous solution, but were rearranged into octahedral symmetry through the crystal-packing process under the crystallization conditions.

Expression, purification, crystallization and preliminary crystallographic analysis of hepatitis B virus core protein dimerized via a peptide linker containing an EGFP insertion.

Virus-like particles (VLPs) have many potentially useful applications. The core proteins of human hepatitis B virus self-assemble into icosahedral VLPs. As previously reported, core protein dimers (CPDs), produced by connecting two core proteins via a peptide linker, can also assemble into VLPs. CPDs in which heterologous proteins were connected to the C-terminus (CPD1) were found to rearrange into symmetrical octahedra during crystallization. In this study, a heterologous protein was inserted into the peptide linker of the CPD (CPD2). CPD2 was expressed in Escherichia coli, assembled into VLPs, purified and crystallized. A single crystal diffracted to 2.8 Å resolution and belonged to the cubic space group F432, with unit-cell parameters a = b = c = 218.6 Å. Single-crystal analysis showed that CPD1 and CPD2 rearranged into the same octahedral organization in a crystallization solution.

Crystal structures of the lumazine protein from Photobacterium kishitanii in complexes with the authentic chromophore, 6,7-dimethyl- 8-(1'-D-ribityl) lumazine, and its analogues, riboflavin and flavin mononucleotide, at high resolution.

Lumazine protein (LumP) is a fluorescent accessory protein having 6,7-dimethyl-8-(1'-d-ribityl) lumazine (DMRL) as its authentic chromophore. It modulates the emission of bacterial luciferase to shorter wavelengths with increasing luminous strength. To obtain structural information on the native structure as well as the interaction with bacterial luciferase, we have determined the crystal structures of LumP from Photobacterium kishitanii in complexes with DMRL and its analogues, riboflavin (RBF) and flavin mononucleotide (FMN), at resolutions of 2.00, 1.42, and 2.00 A. LumP consists of two beta barrels that have nearly identical folds, the N-terminal and C-terminal barrels. The structures of LumP in complex with all of the chromophores studied are all essentially identical, except around the chromophores. In all of the structures, the chromophore is tethered to the narrow cavity via many hydrogen bonds in the N-terminal domain. These are absent in the C-terminal domain. Hydrogen bonding in LumP-FMN is decreased in comparison with that in LumP-RBF because the phosphate moiety of FMN protrudes out of the narrow cavity. In LumP-DMRL, the side chain of Gln65 is close to the ring system, and a new water molecule that stabilizes the ligand is observed near Ser48. Therefore, DMRL packs more tightly in the ligand-binding site than RBF or FMN. A docking simulation of bacterial luciferase and LumP suggests that the chromophore is located close enough for direct energy transfer to occur. Moreover, the surface potentials around the ligand-binding sites of LumP and bacterial luciferase exhibit complementary charge distributions, which would have a significant effect on the interaction between LumP and luciferase.

Kinetic and structural studies on roles of the serine ligand and a strictly conserved tyrosine residue in nitrile hydratase.

Nitrile hydratases (NHase), which catalyze the hydration of nitriles to amides, have an unusual Fe(3+) or Co(3+) center with two modified Cys ligands: cysteine sulfininate (Cys-SO(2) (-)) and either cysteine sulfenic acid or cysteine sulfenate [Cys-SO(H)]. Two catalytic mechanisms have been proposed. One is that the sulfenyl oxygen activates a water molecule, enabling nucleophilic attack on the nitrile carbon. The other is that the Ser ligand ionizes the strictly conserved Tyr, activating a water molecule. Here, we characterized mutants of Fe-type NHase from Rhodococcus erythropolis N771, replacing the Ser and Tyr residues, alphaS113A and betaY72F. The alphaS113A mutation partially affected catalytic activity and did not change the pH profiles of the kinetic parameters. UV-vis absorption spectra indicated that the electronic state of the Fe center was altered by the alphaS113A mutation, but the changes could be prevented by a competitive inhibitor, n-butyric acid. The overall structure of the alphaS113A mutant was similar to that of the wild type, but significant changes were observed around the catalytic cavity. Like the UV-vis spectra, the changes were compensated by the substrate or product. The Ser ligand is important for the structure around the catalytic cavity, but is not essential for catalysis. The betaY72F mutant exhibited no activity. The structure of the betaY72F mutant was highly conserved but was found to be the inactivated state, with alphaCys114-SO(H) oxidized to Cys-SO(2) (-), suggesting that betaTyr72 affected the electronic state of the Fe center. The catalytic mechanism is discussed on the basis of the results obtained.

Structural basis for catalytic activation of thiocyanate hydrolase involving metal-ligated cysteine modification.

Thiocyanate hydrolase (SCNase) is a member of a family of nitrile hydratase proteins, each of which contains a unique noncorrin cobalt center with two post-translationally modified cysteine ligands, cysteine-sulfenic acid or -sulfenate (Cys-SO(H)), and cysteine-sulfininate (Cys-SO(2)(-)), respectively. We have found that a partially matured recombinant SCNase was activated during storage. The crystal structures of SCNase before and after storage demonstrated that Cys-SO(2)(-) modification of gammaCys131 proceeded to completion prior to storage, while Cys-SO(H) modification of gammaCys133 occurred during storage. SCNase activity was suppressed when gammaCys133 was further oxidized to Cys-SO(2)(-). The correlation between the catalytic activity and the extent of the gammaCys133 modification indicates that the cysteine sulfenic acid modification of gammaCys133 is of primary importance in determining the activity of SCNase.

Structure of aspartate racemase complexed with a dual substrate analogue, citric acid, and implications for the reaction mechanism.

Pyrococcus horikoshii OT3 aspartate racemase (PhAspR) catalyzes the interconversion between L- and D-aspartate. The X-ray structure of PhAspR revealed a pseudo mirror-symmetric distribution of the residues around its active site, which is very reasonable for its chiral substrates, L-aspartate and D-aspartate. In this study, we have determined the crystal structure of an inactive mutant PhAspR complexed with a citric acid (Cit) at a resolution of 2.0 A. Cit contains the substrate analogue moieties of both L- and D-aspartate and exhibits a low competitive inhibition activity against PhAspR. In the structure, Cit binds to the catalytic site of PhAspR, which induced the conformational change to close the active site. The distance between the thiolates was estimated to be 7.4 A, representing a catalytic state and the substrate binding modes of PhAspR. Two conserved basic residues, Arg48 and Lys164, seem to be indispensable for PhAspR activity. Arg48 is thought to be responsible for recognizing carboxyl groups of the substrates L-/D-aspartates and stabilizing a reaction intermediate, and Lys164 is responsible for stabilizing a closed state structure. In this structure, the L-aspartate moiety of Cit is likely to take the substrate position of the PhAspR-substrate complex, which is very similar to that of Glutamate racemase. There is also another possibility that the two substrate analogue moieties of the bound Cit reflect the binding modes of both L- and D-aspartates. Based on the PhAspR-Cit complex structure, the reaction mechanism of aspartate racemase was elucidated.

Structure of thiocyanate hydrolase: a new nitrile hydratase family protein with a novel five-coordinate cobalt(III) center.

Thiocyanate hydrolase (SCNase) of Thiobacillus thioparus THI115 is a cobalt(III)-containing enzyme catalyzing the degradation of thiocyanate to carbonyl sulfide and ammonia. We determined the crystal structures of the apo- and native SCNases at a resolution of 2.0 A. SCNases in both forms had a conserved hetero-dodecameric structure, (alphabetagamma)(4). Four alphabetagamma hetero-trimers were structurally equivalent. One alphabetagamma hetero-trimer was composed of the core domain and the betaN domain, which was located at the center of the molecule and linked the hetero-trimers with novel quaternary interfaces. In both the apo- and native SCNases, the core domain was structurally conserved between those of iron and cobalt-types of nitrile hydratase (NHase). Native SCNase possessed the post-translationally modified cysteine ligands, gammaCys131-SO(2)H and gammaCys133-SOH like NHases. However, the low-spin cobalt(III) was found to be in the distorted square-pyramidal geometry, which had not been reported before in any protein. The size as well as the electrostatic properties of the substrate-binding pocket was totally different from NHases with respect to the charge distribution and the substrate accessibility, which rationally explains the differences in the substrate preference between SCNase and NHase.

Successful PEGylation of hollow encapsulin nanoparticles from Rhodococcus erythropolis N771 without affecting their disassembly and reassembly properties.

We developed a hollow PEGylated encapsulin nanoparticle from Rhodococcus erythropolis N771. The hollow engineered encapsulin nanoparticles with His-Tag and Lys residues on the surface were constructed by means of genetic recombination. The Lys residues on the particle surface were successfully PEGylated with a PEG derivative, methoxy-PEG-SCM. Consequently, we demonstrated that the hollow PEGylated engineered encapsulin nanoparticle could successfully disassemble or reassemble even after PEGylation in the presence or absence of a protein denaturing agent. The nanoparticle obtained in the present study has the potential to incorporate hydrophilic compounds in the internal cavity of the particle by reversibly controllable disassembly and reassembly. The hollow PEGylated encapsulin nanoparticle can be used as a drug carrier for the delivery of hydrophilic biopolymers in future medical applications.

HSP60 possesses a GTPase activity and mediates protein folding with HSP10.

The mammalian molecular chaperone, HSP60, plays an essential role in protein homeostasis through mediating protein folding and assembly. The structure and ATP-dependent function of HSP60 has been well established in recent studies. After ATP, GTP is the major cellular nucleotide. In this paper, we have investigated the role of GTP in the activity of HSP60. It was found that HSP60 has different properties with respect to allostery, complex formation and protein folding activity depending on the nucleoside triphosphate present. The presence of GTP slightly affected the ATPase activity of HSP60 during protein folding. These results provide clues as to the functional mechanism of the HSP60-HSP10 complex.

Functional expression of thiocyanate hydrolase is promoted by its activator protein, P15K.

Thiocyanate hydrolase (SCNase) is a cobalt-containing enzyme with a post-translationally modified cysteine ligand, gammaCys131-SO(2)H. When the SCNase alpha, beta and gamma subunits were expressed in Escherichia coli, the subunits assembled to form a hetero-dodecamer, (alphabetagamma)(4), like native SCNase but exhibited no catalytic activity. Metal analysis indicated that SCNase was expressed as an apo-form irrespective of the presence of cobalt in the medium. On the contrary, SCNase co-expressed with P15K, encoded just downstream of SCNase genes, in cobalt-enriched medium under the optimized condition (SCNase((+P15K))) possessed 0.86 Co atom/alphabetagamma trimer and exhibited 78% of the activity of native SCNase. SCNase((+P15K)) showed a UV-Vis absorption peak characteristic of the SCNase cobalt center. About 70% of SCNase((+P15K)) had the gammaCys131-SO(2)H modification. These results indicate that SCNase((+P15K)) is the active holo-SCNase. P15K is likely to promote the functional expression of SCNase probably by assisting the incorporation of cobalt ion.

Catalytic Mechanism of Nitrile Hydratase Subsequent to Cyclic Intermediate Formation: A QM/MM Study.

The catalytic mechanism of an Fe-containing nitrile hydratase (NHase) subsequent to the formation of a cyclic intermediate was investigated using a hybrid quantum mechanics/molecular mechanics (QM/MM) method. We identified the following mechanism: (i) proton transfer from ßTyr72 to the substrate via αSer113, and cleavage of the S-O bond of αCys114-SO(-) and formation of a disulfide bond between αCys109 and αCys114; (ii) direct attack of a water molecule on the sulfur atom of αCys114, which resulted in the generation of both an imidic acid and a renewed sulfenic cysteine; and (iii) isomerization of the imidic acid to the amide. In addition, to clarify the role of ßArg56K, which is one of the essential amino residues in the enzyme, we analyzed a ßR56K mutant in which ßArg56 was replaced by Lys. The results suggest that ßArg56 is necessary for the formation of disulfide intermediate by stabilizing the cleavage of the S-O bond via a hydrogen bond with the oxygen atom of αCys114-SO(-).