Purification, characterization, and molecular cloning of tyrosinase from Pholiota nameko.

Tyrosinase (monophenol, 3,4-dihydroxy L-phenylalanine (L-DOPA):oxygen oxidoreductase, EC 1.14.18.1) was isolated from fruit bodies of Pholiota nameko and purified to homogeneity. The purified enzyme was a monomer with a molecular weight of 42,000 and contained 1.9 copper atoms per molecule. The N-terminal of the purified enzyme could not be detected by Edman degradation, probably due to blocking, while the C-terminal sequence of the enzyme was determined to be -Ala-Ser-Val-Phe-OH. The amino acid sequence deduced by cDNA cloning was made up of 625 amino acid residues and contained two putative copper-binding sites highly conserved in tyrosinases from various organisms. The C-terminal sequence of the purified enzyme did not correspond to that of the deduced sequence, but agreed with Ala384-Ser385-Val386-Phe387 in sequence. When the encoded protein was truncated at Phe387, the molecular weight of the residual protein was calculated to be approximately 42,000. These results suggest that P. nameko tyrosinase is expressed as a proenzyme followed by specific cleavage to produce a mature enzyme.

Kinetic isotope effect of the L-phenylalanine oxidase from Pseudomonas sp. P-501.

Pseudomonas L-phenylalanine oxidase (deaminating and decarboxylating) mainly catalyzes oxygenation when L-phenylalanine is used as the substrate, but oxidation when L-methionine is used as the substrate. Using [C(alpha)-H]-DL-methionine and [C(alpha)-D]-DL-methionine as substrate, the reductive half reaction of FAD cofactor of enzyme has been studied by stopped-flow spectrophotometry. The rate of reduction of FAD cofactor has a kinetic isotope effect (KIE) of 5.4 and 4.1 in the absence and presence of 30% glycerol, respectively. The KIE is independent of temperature, but the rates of the reductive half reaction are dependent on temperature, indicating that thermally induced motion at the active site drives the H-transfer reaction by H-tunneling.

Low pKa lysine residues at the active site of sarcosine oxidase from Corynebacterium sp. U-96.

Sarcosine oxidase from Corynebacterium sp. U-96 is inactivated by iodoacetamide with the modification of two specific residues. Comparing the amino acid sequence and mass spectra of the peptide fragments containing the modified residues with those from the native enzyme, the modified residues were identified to be lysine. The pKa of these residues were estimated to be 8.5 and 6.7 from the pH dependence of inactivation in the presence and absence of the competitive inhibitor, acetate. These estimated pKa values are much lower than that of the epsilon-amino group of lysine residue. There may be unique microenvironments around these residues that activate their -amino groups to be susceptible to iodoacetamide. A possible role of the lysine residue with pKa 6.7 is discussed.

Sequencing and expression of the L-phenylalanine oxidase gene from Pseudomonas sp. P-501. Proteolytic activation of the proenzyme.

The nucleotide sequence encoding L-phenylalanine oxidase (deaminating and decarboxylating) (PAO) from Pseudomonas sp. P-501 was determined. The open reading frame is arranged in the order of prosequence, alpha subunit, dipeptide and beta subunit from the 5'- to 3'-end. Expression of the gene in Escherichia coli showed that without the prosequence, PAO is produced in small quantity as a soluble form with no visible absorption, but with the prosequence (proPAO), PAO is highly expressed and yellow. The purified proPAO contained one mol of FAD per mol of proPAO polypeptide, but had no catalytic activity. Treatment of proPAO with a mixture of Pronase and trypsin converted the noncatalytic proPAO to the catalytic form, and the Pronase-trypsin-treated proPAO showed kinetic and spectral properties comparable to the native enzyme. These results suggest that in Pseudomonas, PAO is expressed as a proenzyme that is processed by proteolysis to the active form.

Cofactors in sarcosine oxidase from Corynebacterium sp. U-96.

Sarcosine oxidase from Corynebacterium sp. U-96 is a heterotetrameric enzyme that was reported to contain 1 mol of covalently bound FAD and 1 mol of non-covalently-bound FAD. This work describes the result of reinvestigation of the cofactors in this enzyme. The enzyme was found to contain 1 mol of non-covalently-bound NAD+, 1 mol of non-covalently-bound FAD, and 1 mol of covalent FMN. The covalent FMN was identified by the mass and amino acid sequence analyses of the flavin peptide.