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Aspergillus oryzae RIB40 formate oxidase has Arg87 and Arg554 near the formyl group and O(4) atom of 8-formyl-flavin adenine dinucleotide (FAD), respectively, with Asp396 neighbouring Arg554. Herein, we probed the roles of these three residues in modification of FAD to 8-formyl-FAD. Replacement of Arg87 or Arg554 with Lys or Ala decreased and abolished the modification, respectively. Replacement of Asp396 with Ala or Asn lowered the modification rate. The observation of unusual effects of maintaining pH 7.0 on the modification in R87K, R554K and D396 variants indicates initial and subsequent processes with different pH dependencies. Comparison of the initial process at pH 4.5 and 7.0 suggests that the microenvironment around Arg87 and the protonation state of Asp396 affect the initial process in the native enzyme. Comparison of the crystal structures of native and R554 variants showed that the replacements had minimal effect on catalytic site structure. The positively charged Arg87 might contribute to the formation of an anionic quinone-methide tautomer intermediate, while the positively charged Arg554, in collaboration with the negatively charged Asp396, might stabilize this intermediate and form a hydrogen bonding network with the N(5)/O(4) region, thereby facilitating efficient FAD modification. [ABSTRACT FROM AUTHOR]

Alanine racemase of Bacillus stearothermophilus has been proposed to catalyze alanine racemization by means of two catalytic bases: lysine 39 (K39) abstracting specifically the α-hydrogen of D-alanine and tyrosine 265 (Y265) playing the corresponding role for the antipode L-alanine. The role of K39 as indicated has already been verified Watanabe, A., Kurokawa, Y., Yoshimura, T., Kurihara, T., Soda, K., and Esaki, N. (1999) J. Biol. Chenu 274, 4189–4194. We here present evidence for the functioning of Y265 as the base catalyst specific to L-alanine. The Y265↑Ala mutant enzyme (Y265A), like Y265S and Y265F, was a poor catalyst for alanine racemization. However, Y265A and Y265S catalyzed trans-amination with D-alanine much more rapidly than the wild-type enzyme, and the bound coenzyme, pyridoxal 5'-phosphate (PLP), was converted to pyridoxamine 5' -phosphate (PMP). The rate of transamination catalyzed by Y265F was about 9% of that by the wild-type enzyme. However, Y265A, Y265S, and Y265F were similar in that L-alanine was inert as a substrate in transamination. The apo-form of the wild-type enzyme catalyzes the abstraction of tritium non-specifically from both (4'S)- and (4'R)-[4'-3H]PMP in the presence of pyruvate. In contrast, apo-Y265A abstracts tritium virtually from only the R-isomer. This indicates that the side-chain of Y265 abstracts the α-hydrogen of L-alanine and transfers it supra-facially to the pro-S position at C-4' of PMP. Y265 is the counterpart residue to K39 that transfers the α-hydrogen of D-alanine to the pro-R position of PMP. [ABSTRACT FROM AUTHOR]

The crystal and molecular structure of Lipase II from Rhizopus niveus was analyzed using X-ray single crystal diffraction data at a resolution of 2.2 Å. The structure was refined than R-factor of 0.19 for all available data. This lipase was purified and crystallized as Lipase I, which contains two polypeptide chains combined through non-covalent interaction. However, during crystal growth, Lipase I was converted to Lipase II, which consists of a single polypeptide chain of 269 amino acid residues, by limited proteolysis. The structure of Lipase II shows a typical α/β hydrolase fold containing the so-called nucleophilic elbow. The catalytic center of this enzyme is analogous to those of other neutral lipases and serine proteases. This catalytic center is sheltered by an α-helix lid, which appears in neutral lipases, opening the active site at the oil-water interface. [ABSTRACT FROM AUTHOR]

N-Acyl-D-glucosamine 2-epimerase from porcine kidney, which was cloned and expressed in Escherichia coli, was crystallized by the vapor-diffusion method, using polyethylene glycol and ammonium acetate as precipitants. The crystals were resistant to X-ray radiation damage and diffracted to more than 2.0 Å resolution. The diffraction pattern indicated that the crystals belong to the orthorhombic system, space group P21,21 21 with unit-cell dimensions of a=78.1, b=97.2, and c=100.7 Å. It is supposed that the asymmetric unit consists of two N-acyl-D-glucosamine 2-epimerase molecules. Collection of data on the native crystals indicated that they are suitable for X-ray structural analysis. [ABSTRACT FROM AUTHOR]

Aspergillus oryzae RIB40 formate oxidase has Arg87 and Arg554 near the formyl group and O(4) atom of 8-formyl-flavin adenine dinucleotide (FAD), respectively, with Asp396 neighbouring Arg554. Herein, we probed the roles of these three residues in modification of FAD to 8-formyl-FAD. Replacement of Arg87 or Arg554 with Lys or Ala decreased and abolished the modification, respectively. Replacement of Asp396 with Ala or Asn lowered the modification rate. The observation of unusual effects of maintaining pH 7.0 on the modification in R87K, R554K and D396 variants indicates initial and subsequent processes with different pH dependencies. Comparison of the initial process at pH 4.5 and 7.0 suggests that the microenvironment around Arg87 and the protonation state of Asp396 affect the initial process in the native enzyme. Comparison of the crystal structures of native and R554 variants showed that the replacements had minimal effect on catalytic site structure. The positively charged Arg87 might contribute to the formation of an anionic quinone-methide tautomer intermediate, while the positively charged Arg554, in collaboration with the negatively charged Asp396, might stabilize this intermediate and form a hydrogen bonding network with the N(5)/O(4) region, thereby facilitating efficient FAD modification., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)