Shotaro Yamaguchi

X-ray analysis of bilirubin oxidase from Myrothecium verrucaria at 2.3 Å resolution using a twinned crystal

Bilirubin oxidase (BOD), a multicopper oxidase found in Myrothecium verrucaria, catalyzes the oxidation of bilirubin to biliverdin. Oxygen is the electron acceptor and is reduced to water. BOD is used for diagnostic analysis of bilirubin in serum and has attracted considerable attention as an enzymatic catalyst for the cathode of biofuel cells that work under neutral conditions. Here, the crystal structure of BOD is reported for the first time. Blue bipyramid-shaped crystals of BOD obtained in 2-methyl-2,4-pentanediol (MPD) and ammonium sulfate solution were merohedrally twinned in space group P6(3). Structure determination was achieved by the single anomalous diffraction (SAD) method using the anomalous diffraction of Cu atoms and synchrotron radiation and twin refinement was performed in the resolution range 33-2.3 A. The overall organization of BOD is almost the same as that of other multicopper oxidases: the protein is folded into three domains and a total of four copper-binding sites are found in domains 1 and 3. Although the four copper-binding sites were almost identical to those of other multicopper oxidases, the hydrophilic Asn residue (at the same position as a hydrophobic residue such as Leu in other multicopper oxidases) very close to the type I copper might contribute to the characteristically high redox potential of BOD.

Crystal structures of protein-glutaminase and its pro forms converted into enzyme-substrate complex

Background: Protein glutaminase (PG) catalyzes deamination of Gln residues in proteins. Results: The structures of mature and pro forms and a pro form mutant reveal that the side chain of Gln-47 of mutant A47Q mimics the protein substrate of PG. Conclusion: Gln-47 of A47Q forms an S-acyl covalent intermediate with the catalytic Cys. Significance: PG shares a common catalytic mechanism with transglutaminase and cysteine protease. Protein glutaminase, which converts a protein glutamine residue to a glutamate residue, is expected to be useful as a new food-processing enzyme. The crystal structures of the mature and pro forms of the enzyme were refined at 1.15 and 1.73 Å resolution, respectively. The overall structure of the mature enzyme has a weak homology to the core domain of human transglutaminase-2. The catalytic triad (Cys-His-Asp) common to transglutaminases and cysteine proteases is located in the bottom of the active site pocket. The structure of the recombinant pro form shows that a short loop between S2 and S3 in the proregion covers and interacts with the active site of the mature region, mimicking the protein substrate of the enzyme. Ala-47 is located just above the pocket of the active site. Two mutant structures (A47Q-1 and A47Q-2) refined at 1.5 Å resolution were found to correspond to the enzyme-substrate complex and an S-acyl intermediate. Based on these structures, the catalytic mechanism of protein glutaminase is proposed.

Crystal structure of enzyme–substrate complex of protein-glutaminase obtained by the mutant of pro-enzyme

Protein-glutaminase, which converts glutamine residues in proteins or peptides to glutamic acid residues, is expected to see wide use as a new food processing enzyme. Deamidation of proteins can improve their solubility, emulsifying activity, forming activity, and other functional properties by increasing the number of negative charges. We have reported the crystal structures of a mature protein-glutaminase with 185 amino acid residues refined at 1.15 Å resolution and a recombinant pro-enzyme with 299 amino acid residues refined at 1.5 Å resolution [1]. The enzyme has a catalytic triad, Cys-His-Asp conserved in transglutaminases and cysteine proteases. We found that a short loop around Ala 47 in the pro-region covers and interacts with the active site. In order to elucidate the catalytic mechanism of this enzyme, crystal structures of A47Q mutant were deternined in a various conditions. We concluded that the side chain of Gln 47 forms a covalent bond with catalytic Cys156/S in the absence of ammonium ion but it forms a non-covalent ES complex in the presence of ammonium ion. We also found an oxidized Cys156 forming a covalent bond with Arg 159 in the presence of pottasium ion, which indicate the inactivation of this enzyme by oxidation. The structure of A47Q mutant provides insights into the catalytic mechanism of the enzyme which forms a covalent S-acyl intermediate before release of ammonia.

Structure and Function Analysis of Geobacillus Starch Antistaling Enzyme

Glycosyltransferase from Geobacillus sp. (SAS) is expected to see wide use as a starch antistaling enzyme in food including bread and rice products. The enzyme is thought to transfer maltotriose (G3) unit into non-reducing ends of sarch with unknown likage except for usual alpha-1,4 linkage. SAS was crystallized by sitting drop vapar diffusion method in 14~28% PEG4000 (w/v), 10mM CaCl2, 0.1M NaAC at pH 4.6 and 20 ̊C for 1 month. The obtained crystals belong to a space group of P6522 with cell dimensions of a = b = 112 and c = 320 Å. The crystals were soaked in various oligomaltosaccharides (G1, G2, G3, G4, G5 and G6) for 15 min before flash cooling. The diffraction data of each complex were collected at beam-lines of BL26B1, BL38B1 and BL44XU in SPring-8. The crystal data were collected with 97-99 % completeness and Rmerge of 0.07-0.09 up to 1.6-2.3 Å resolution. The structures were determined by molecular replacement with cyclodextrin glucanotransferase (CGTase, PDB 1CYG) as a search model and were refined with PHENIX. The refined models of SAS/sugars contain one molecule of SAS comprising 733 amino acid residues, 5-8 calcium ions, 543-1141 water molecules and several sugars with R = 0.15-0.19 and Rfree = 0.16-0.23 for the data up to 1.6-2.3 Å resolution. SAS has almost the same overall structure with the CGTase except for several loops in the catalytic domain A. They share a similar active site except for subsite +3 where the non-reducing ends of the oligosaccharides bind. G1 bound to subsite +3, indicating +3 site has the highest affinity to G1. Only G3 was found to bind at subsites +3 ~ +1 when G3, G5 and G6 were soaked, whereas G4 bound at subsites +3 ~ -1 when G4 was soaked. From the clear density map of the bound G4, the bound glucose residue at subsute -1 is found to have alpha-1,6 linkage, indicating the product of this transglucosidase.