Shin-ichi Sakasegawa

Bilirubin Oxidase Activity of Bacillus subtilis CotA

ABSTRACT The spore coat protein CotA from Bacillus subtilis was previously identified as a laccase. We have now found that CotA also shows strong bilirubin oxidase activity and markedly higher affinity for bilirubin than conventional bilirubin oxidase. This is the first characterization of bilirubin oxidase activity in a bacterial protein.

Molecular cloning and expression of uricase gene from Arthrobacter globiformis in Escherichia coli and characterization of the gene product.

Arthrobacter globiformis FERM BP-360 produces uricase (urate oxidase; EC 1.7.3.3) intracellularly. A genomic library of the bacterium, prepared in the plasmid vector pUC118, was screened with probes based on the amino acid sequence of the purified uricase. We found that a chimeric plasmid in the library, designated pUOD1, carries a 2.0-kb DNA insert from the Arthrobacter DNA that hybridizes with the probe. The DNA insert contains an ORF consisting of 302 amino acids with a calculated molecular mass of 33,858. The protein translated from the ORF displays the highest identity (67%) to uricase from a bacterium, Cellulomonas flavigena. X-ray fluorescence analysis showed that the Arthrobacter uricase contains copper ion. However, we found that the catalytic activity of uricase is inhibited by the excessive addition of copper ion. Although the production of A. globiformis uricase is induced by the addition of uric acid to the culture medium, Escherichia coli harboring pUOD1 produced 20-fold higher uricase than the original Arthrobacter strain, even without an inducer.

A novel nucleoside kinase from Burkholderia thailandensis: a member of the phosphofructokinase B-type family of enzymes.

The genome of the mesophilic Gram‐negative bacterium Burkholderia thailandensis contains an open reading frame (i.e. the Bth_I1158 gene) that has been annotated as a putative ribokinase and PFK‐B family member. Notably, although the deduced amino acid sequence of the gene showed only 29% similarity to the recently identified nucleoside kinase from hyperthermophilic archaea Methanocaldococcus jannaschii, 15 of 17 residues reportedly involved in the catalytic activity of M. jannaschii nucleoside kinase were conserved. The gene was cloned and functionally overexpressed in Rhodococcus erythropolis, and the purified enzyme was characterized biochemically. The substrate specificity of the enzyme was unusually broad for a bacterial PFK‐B protein, and the specificity extended not only to purine and purine‐analog nucleosides but also to uridine. Inosine was the most effective phosphoryl acceptor, with the highest kcat/Km value (80 s−1·mm−1) being achieved when ATP served as the phosphoryl donor. By contrast, this enzyme exhibited no activity toward ribose, indicating that the recombinant enzyme was a nucleoside kinase rather than a ribokinase. To our knowledge, this is the first detailed analysis of a bacterial nucleoside kinase in the PFK‐B family.

Colorimetric inorganic pyrophosphate assay using a double cycling enzymatic method

Pyruvate phosphate dikinase (PPDK, EC 2.7.9.1) from the hyperthermophile Thermotoga maritima was biochemically characterized with the aim of establishing a colorimetric assay for inorganic pyrophosphate (PPi). When heterologously expressed in Escherichia coli, T. maritima PPDK (TmPPDK) was far more stable any other PPDK reported so far: it retained >90% of its activity after incubation for 1 h at 80°C, and >80% of its activity after incubation for 20 min at pHs ranging from 6.5 to 10.5 (50°C). In contrast to PPDKs from protozoa and plants, this TmPPDK showed very long-term stability at low temperature: full activity was retained even after storage for at least 2 years at 4°C. TmPPDK was successfully applied to a novel colorimetric PPi assay, which employed (i) a PPi cycling reaction using TmPPDK and nicotinamide mononucleotide adenylyltransferase (EC 2.7.7.1) from Saccharomyces cerevisiae and (ii) a NAD cycling reaction to accumulate reduced nitroblue tetrazolium (diformazan). This enabled detection of 0.2 μM PPi, making this method applicable for preliminary measurement of PPi levels in PCR products in an automatic clinical analyzer.

Structures of Burkholderia thailandensis nucleoside kinase: implications for the catalytic mechanism and nucleoside selectivity

The nucleoside kinase (NK) from the mesophilic Gram-negative bacterium Burkholderia thailandensis (BthNK) is a member of the phosphofructokinase B (Pfk-B) family and catalyzes the Mg(2+)- and ATP-dependent phosphorylation of a broad range of nucleosides such as inosine (INO), adenosine (ADO) and mizoribine (MZR). BthNK is currently used in clinical practice to measure serum MZR levels. Here, crystal structures of BthNK in a ligand-free form and in complexes with INO, INO-ADP, MZR-ADP and AMP-Mg(2+)-AMP are described. The typical homodimeric architecture of Pfk-B enzymes was detected in three distinct conformational states: an asymmetric dimer with one subunit in an open conformation and the other in a closed conformation (the ligand-free form), a closed conformation (the binary complex with INO) and a fully closed conformation (the other ternary and quaternary complexes). The previously unreported fully closed structures suggest the possibility that Mg(2+) might directly interact with the β- and γ-phosphates of ATP to maintain neutralization of the negative charge throughout the reaction. The nucleoside-complex structures also showed that the base moiety of the bound nucleoside is partly exposed to the solvent, thereby enabling the recognition of a wide range of nucleoside bases. Gly170 is responsible for the solvent accessibility of the base moiety and is assumed to be a key residue for the broad nucleoside recognition of BthNK. Remarkably, the G170Q mutation increases the specificity of BthNK for ADO. These findings provide insight into the conformational dynamics, catalytic mechanism and nucleoside selectivity of BthNK and related enzymes.

Stabilization of Flavobacterium meningosepticum glycerol kinase by introduction of a hydrogen bond

The thermostability of Flavobacterium meningosepticum glycerol kinase was increased by the change from Ser329 to Asp [Protein Eng., 14, 663-667 (2001)]. Based on a three-dimensional structure model of the mutant, we have postulated that a new charged-neutral hydrogen bond was formed between Asp329 and Ser414, and the formation of the hydrogen bond contributed to the stabilization of the tertiary structure and increased thermostability of the mutant enzyme. If the postulation is the case, FGK thermostabilization would be possible similarly by the single amino acid substitution from Ser414 to another amino acid which could form the hydrogen bond with Ser329. We did a single amino acid substitution of the wild-type enzyme from Ser414 to Asn. As we expected, S414N showed comparable thermostability to that of S329D. On the other hand, a difference in kinetic properties for ATP between S414N and S329D was observed.

Enzymatic assay method for measuring mizoribine levels in serum.

A sensitive and specific method for assaying serum mizoribine levels that can be applied to general automatic clinical analyzers was developed. Regression analysis of the enzymatic assay (y) vs. the HPLC method (x) produced the following relation: y=0.964x+0.090 (n=262, Sy, x=6.37 ng/mL).

Development of an enzyme cycling method by a purine nucleoside phosphorylase for assaying inorganic phosphate

We have developed a novel enzymatic cycling method that uses purine nucleoside phosphorylase (PNP) (EC 2.4.2.1) from Bacillus sp. to measure inorganic phosphate. The method utilizes the reversibility of the PNP reaction, in which the forward and reverse reactions are catalyzed in the presence of an excess amount of inosine and guanine, respectively, a principle similar to that previously demonstrated with creatine kinase (CK). Real-time detection was accomplished by coupling the reaction with commercially available xanthine dehydrogenase (EC 1.17.1.4) in the presence of NAD+. The efficiency of the cycling reaction per unit of enzyme (U ml−1) was remarkably higher than the CK.

Sphingomyelinase C from Streptomyces sp. A9107: Unusual primary structure for bacterial sphingomyelinase C

A sphingomyelinase C (SMase) was identified in the culture supernatant of Streptomyces sp. A9107 (S-SMase). Although S-SMase seems to be a typical bacterial SMase, the primary structure of S-SMase was unusual for known bacterial SMase. The gene was functionally overexpressed in the culture medium of recombinant Rhodococcus erythropolis.