Biomedical subjects

Haruo Suzuki

Publications and source records attributed to Haruo Suzuki.

10 recordsLinked to original sources

PubMed2006

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.

Amino Acid Oxidoreductases↗

PubMed2005

A problem in multivariate analysis of codon usage data and a possible solution.

Multivariate analyses are often used to identify major trends of variation in synonymous codon usage among genes. These analyses need to be performed on properly normalized codon usage data to avoid biases masking this synonymous variation, i.e., gene length, amino acid usage, and codon degeneracy; however, previous studies have failed to do so. In this paper, we demonstrate that the use of alternative normalized data (called 'relative adaptiveness' in the literature) can avoid all these biases and furthermore, can identify more trends of variation among genes, including GC-ending codon usage, GT-ending codon usage, and gene expression level.

Amino Acids↗

PubMed2005

Crystal structure of heterotetrameric sarcosine oxidase from Corynebacterium sp. U-96.

Sarcosine oxidase from Corynebacterium sp. U-96 is a heterotetrameric enzyme. Here we report the crystal structures of the enzyme in complex with dimethylglycine and folinic acid. The alpha subunit is composed of two domains, contains NAD(+), and binds folinic acid. The beta subunit contains dimethylglycine, FAD, and FMN, and these flavins are approximately 10A apart. The gamma subunit is in contact with two domains of alpha subunit and has possibly a folate-binding structure. The delta subunit contains a single atom of zinc and has a Cys(3)His zinc finger structure. Based on the structures determined and on the previous works, the structure-function relationship on the heterotetrameric sarcosine oxidase is discussed.

Binding Sites↗

PubMed2005

A comprehensive software suite for the analysis of cDNAs.

We have developed a comprehensive software suite for bioinformatics research of cDNAs; it is aimed at rapid characterization of the features of genes and the proteins they code. Methods implemented include the detection of translation initiation and termination signals, statistical analysis of codon usage, comparative study of amino acid composition, comparative modeling of the structures of product proteins, prediction of alternative splice forms, and metabolic pathway reconstruction.

Alternative Splicing↗

PubMed2005

Site-directed mutagenesis study of the antibody 2D7 which catalyzes a reaction for insertion of Cu2+ into mesoporphyrin.

Monoclonal antibody 2D7 generated against a transition-state analog N-methyl mesoporphyrin catalyzes a reaction for insertion of a cupric ion into mesoporphyrin. To investigate amino acid residues responsible for the catalytic activity, site-directed mutagenesis of the amino acid residues in the third complementarity determining region of the heavy chain (CDRH3) was performed on the antigen-binding fragment (Fab) of the antibody. Recombinant Fab mutants, in which Arg95 is replaced with Ala (R95A), Asp96 with Asn (D96N) and Met97 with Gly (M97G), were examined in terms of the catalytic efficiency of the reaction (k/K(S)) and the dissociation constant for N-methyl mesoporphyrin binding (K(d)) and these values were compared with those of the wild type. The k/K(S) values of the R95A and D96N mutants were 0.96% and 1.0% of that of the wild type, respectively, whereas the M97G mutant had no detectable catalytic activity. The K(d) values of the R95A and D96N mutants were 165 and 69 times that of the wild type, respectively, while that of the M97G mutant was similar to that of the wild type. The relationship between the k/K(S) and 1/K(d) values in the wild type and the R95A and D96N mutants suggests that Arg95 and Asp96 are responsible for stabilizing the transition-state in the catalytic reaction. The results of the M97G mutant allow us to propose that Met97 plays an important role in the catalytic activity probably due to a subtle and specific conformation of the antibody.

Antibodies, Monoclonal↗

PubMed2005

Corynebacterium sp. U-96 contains a cluster of genes of enzymes for the catabolism of sarcosine to pyruvate.

The sarcosine oxidase gene and nearby genes from Corynebacterium sp. U-96 were determined. The genes for serine hydroxymethyltransferase, the beta, delta, alpha, and gamma subunits of sarcosine oxidase, serine dehydratase, and 10-formyltetrahydrofolate hydrolase are arranged in this order. This suggests that the bacteria contain a cluster of genes for the catabolism of sarcosine to pyruvate. The possibility that the gene cluster is a merit for the cellular energy demands of the bacteria is discussed. Functional expression of sarcosine oxidase in Escherichia coli was accomplished, but the beta subunit and the betadelta complex were expressed at a low level as a soluble protein.

Bacterial Proteins↗

PubMed2004

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.

Amino Acid Sequence↗

PubMed2004

The 'weighted sum of relative entropy': a new index for synonymous codon usage bias.

Shannon entropy from information theory has been applied to estimate the degree of deviation from equal usage of synonymous codons; however, previous attempts have failed to take into account all three aspects of amino acid usage, i.e. (i) the number of distinct amino acids, (ii) their relative frequencies, and (iii) their degree of codon degeneracy. A new index taking into account all of these aspects is proposed. The index, designated as the 'weighted sum of relative entropy' (E(w)), is defined as the sum of the relative entropy of each amino acid weighted by its relative frequency in the sequence. In this paper, we demonstrate that E(w) allows us to avoid some amino acid usage biases and can yield results contradictory to those obtained by previous methods.

Algorithms↗

PubMed2004

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.

Amino Acid Oxidoreductases↗

PubMed2002

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.

Bacterial Proteins↗