Hiroki Tsuruta

Identification and cloning of a gene encoding tannase (tannin acylhydrolase) from Lactobacillus plantarum ATCC 14917T

The gene tanLpl, encoding a novel tannase enzyme (TanLpl), has been cloned from Lactobacillus plantarum ATCC 14917(T). This is the first report of a tannase gene cloned from a bacterial source other than from Staphylococcus lugdunensis, which has been reported elsewhere. The open reading frame of tanLpl, spanning 1410 bp, encoded a 469-amino-acid protein that showed 28.8% identity to the tannase of S. lugdunensis with several commonly conserved sequences. These sequences could not be found in putative tannases reported for other bacteria and fungi. TanLpl was expressed in Escherichia coli DH5alpha from a pGEM-T expression system and purified. SDS-PAGE analysis indicated that purified TanLpl was a monomer polypeptide of approximately 50 kDa in size. Subsequent enzymatic characterization revealed that TanLpl was most active in an alkaline pH range at 40 degrees C, which was quite different from that observed for a fungal tannase of Aspergillus oryzae. In addition, the Michaelis-Menten constant of TanLpl was markedly lower than that of A. oryzae tannase. The evidence suggests that TanLpl should be classified into a novel family of tannases.

Functional analysis of the cucumisin propeptide as a potent inhibitor of its mature enzyme

Cucumisin is a subtilisin-like serine protease (subtilase) that is found in the juice of melon fruits (Cucumis melo L.). It is synthesized as a preproprotein consisting of a signal peptide, NH2-terminal propeptide, and 67-kDa protease domain. We investigated the role of this propeptide (88 residues) in the cucumisin precursor. Complementary DNAs encoding the propeptides of cucumisin, two other plant subtilases (Arabidopsis ARA12 and rice RSP1), and bacterial subtilisin E were expressed in Escherichia coli independently of their mature enzymes. The cucumisin propeptide strongly inhibited cucumisin in a competitive manner with a Ki value of 6.2 ± 0.55 nm. Interestingly, cucumisin was also strongly inhibited by ARA12 and RSP1 propeptides but not by the subtilisin E propeptide. In contrast, the propeptides of cucumisin, ARA12, and RSP1 did not inhibit subtilisin. Deletion analysis clearly showed that two hydrophobic regions, Asn32–Met38 and Gly97–Leu103, in the cucumisin propeptide were important for its inhibitory activity. Site-directed mutagenesis also confirmed the role of a Val36-centerd hydrophobic cluster within the Asn32–Met38 region in cucumisin inhibition. Circular dichroism spectroscopy revealed that the cucumisin propeptide had a secondary structure without a cognate protease domain and that the thermal unfolding of the propeptide at 90 °C was only partial and reversible. A tripeptide, Ile35-Val36-Tyr37, in the Asn32–Met38 region was thought to contribute toward the formation of a proper secondary structure necessary for cucumisin inhibition. This is the first report on the function and structural information of the propeptide of a plant serine protease.

Characteristics of Psychrophilic Alkaline Phosphatase

The phosphatase of a psychrophile (Shewanella sp.) was purified by ammonium sulfate fractionation, followed by sequential column chromatographies. The purified enzyme was electrophoretically homogeneous on native- and SDS-PAGE. Its molecular weight was 41,826 by its amino acid composition. The enzyme had its optimal pH for the activity at 9.8, and a broad substrate specificity to dephosphorylate ATP, pyrophosphate, glycerophosphate, and so on. Its activity was increased by metal ions including Mg(2+), Mn(2+), and Co(2+). The maximal activity was observed at 40°C, and the enzyme at 0°C showed 39% of activity at 40°C. The enzyme, however, tended to lose its activity at 20°C and pH 9.8. These results indicated that purified enzyme was an alkaline phosphatase with characteristics; high catalytic efficiency at low temperature and gradual inactivation at an intermediate temperature.

A Major Latex-Like Protein Is a Key Factor in Crop Contamination by Persistent Organic Pollutants

A latex-like protein binds and transports polychlorinated biphenyls into the aerial part of plants. This is the first report, to our knowledge, to reveal important factors by which members of the Cucurbitaceae family, such as cucumber (Cucumis sativus), watermelon (Citrullus lanatus), melon (Cucumis melo), pumpkin (Cucurbita pepo), squash (C. pepo), and zucchini (C. pepo), are selectively polluted with highly toxic hydrophobic contaminants, including organochlorine insecticides and dioxins. Xylem sap of C. pepo ssp. pepo, which is a high accumulator of hydrophobic compounds, solubilized the hydrophobic compound pyrene into the aqueous phase via some protein(s). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of xylem sap of two C. pepo subspecies revealed that the amount of 17-kD proteins in C. pepo ssp. pepo was larger than that in C. pepo ssp. ovifera, a low accumulator, suggesting that these proteins may be related to the translocation of hydrophobic compounds. The protein bands at 17 kD contained major latex-like proteins (MLPs), and the corresponding genes MLP-PG1, MLP-GR1, and MLP-GR3 were cloned from the C. pepo cultivars Patty Green and Gold Rush. Expression of the MLP-GR3 gene in C. pepo cultivars was positively correlated with the band intensity of 17-kD proteins and bioconcentration factors toward dioxins and dioxin-like compounds. Recombinant MLP-GR3 bound polychlorinated biphenyls immobilized on magnetic beads, whereas recombinant MLP-PG1 and MLP-GR1 did not. These results indicate that the high expression of MLP-GR3 in C. pepo ssp. pepo plants and the existence of MLP-GR3 in their xylem sap are related to the efficient translocation of hydrophobic contaminants. These findings should be useful for decreasing the contamination of fruit of the Cucurbitaceae family as well as the phytoremediation of hydrophobic contaminants.

Cloning of Cold-active Alkaline Phosphatase Gene of a Psychrophile, Shewanella sp., and Expression of the Recombinant Enzyme

A psychrophilic alkaline phosphatase (EC 3.1.3.1) from Shewanella sp. is a cold-active enzyme that has high catalytic activity at low temperature [Ishida et al. (1998) Biosci. Biotechnol. Biochem., 62, 2246–2250]. Here, we identified the nucleotide sequence of a gene encoding the enzyme after cloning with the polymerase chain reaction (PCR) and inverted PCR techniques. The deduced amino acid sequence of the enzyme contained conserved amino acids found among mesophilic alkaline phosphatases and showed some structural characteristics including a high content of hydrophobic amino acid residues and the lack of single α-helix compared with the alkaline phosphatase of Escherichia coli, which were possibly efficient for catalytic reaction at low temperatures. The recombinant enzyme expressed in E. coli was purified to homogeneity with the molecular mass of 41 kDa. The recombinant enzyme had a specific activity of 1,500 units/mg and had high catalytic activity at low temperatures.

Crystal Structure of Cold-Active Alkaline Phosphatase from the Psychrophile Shewanella sp.

The crystal structure of a cold-active alkaline phosphatase from a psychrophile, Shewanella sp. (SCAP), was solved at 2.2 Å. A refined model showed a homodimer with six metal-ligand sites. The arrangement of the catalytic residues resembled those of alkaline phosphatases (APs), suggesting that the reaction mechanism of SCAP was fundamentally identical to those of other APs. SCAP had two distinct structural features: (i) a loop with Arg122 that bound to the phosphate moiety of the substrate suffered no constraints from the linkage to other secondary structures, and (ii) Mg3-ligand His109 was considered to undergo repulsive effect with neighboring Trp228. The local flexibility led by these features might be an important factor in the high catalytic efficiency of SCAP at low temperatures.

Polymorphism and distribution of putative cell-surface adhesin-encoding ORFs among human fecal isolates of Bifidobacterium longum subsp. longum

The polymorphism of ORFs encoding putative cell-surface adhesins was investigated in Bifidobacterium longum subsp. longum. Firstly, we performed a PCR assay targeting 15 ORFs encoding putative adhesion proteins, which included 8 ORFs with a sortase targeting LPXTG motif, in 42 strains of different pulsotypes isolated from fecal samples from 12 human individuals. We found a variability in the presence of an ORF, BL0675, which encodes a putative fimbrial subunit protein. We sequenced ORFs corresponding to BL0675 in the 42 strains and adjacent ORFs corresponding to BL0674 and BL0676. The results indicated that ORFs corresponding to BL0675 were highly polymorphic with five variant types (i.e. A-, B-, C-, D-, and E-types). Meanwhile, BL0674 and BL0676, which encode an additional putative fimbrial subunit protein and a fimbrial-associated sortase-like protein, were highly conserved. Subsequent quantitative polymerase chain reaction (qPCR) assays targeting the variant types in 89 human fecal samples revealed that A-type was the most commonly distributed (74.2%), followed by B-type (59.6%), D-type (31.5%), E-type (32.6%) and C-type (5.6% prevalence). Since BL0675 is considered to be a fimbrial protein with glycoprotein-binding ability, the proteins encoded by the five variant types of BL0675 may have specific binding properties to various carbohydrate structures expressed on the human intestinal wall, thereby allowing B. longum to colonize the intestine in a host-specific manner.

The role of group bulkiness in the catalytic activity of psychrophile cold-active protein tyrosine phosphatase

The cold‐active protein tyrosine phosphatase found in psychrophilic Shewanella species exhibits high catalytic efficiency at low temperatures as well as low thermostability, both of which are characteristics shared by many cold‐active enzymes. The structure of cold‐active protein tyrosine phosphatase is notable for the presence of three hydrophobic sites (termed the CA, Zn‐1 and Zn‐2 sites) behind the loop structures comprising the catalytic region. To identify the structural components responsible for specific enzyme characteristics, we determined the structure of wild‐type cold‐active protein tyrosine phosphatase at high resolution (1.1 Å) and measured the catalytic efficiencies of enzymes containing mutations in the three hydrophobic sites. The bulkiness of the amino acid side chains in the core region of the Zn‐1 site strongly affects the thermostability and the catalytic efficiency at low temperatures. The mutant enzyme I115M possessed a higher kcat at low temperatures. Elucidation of the crystal structure of I115M at a resolution of 1.5 Å revealed that the loop structures involved in retaining the nucleophilic group and the acid catalyst are more flexible than in the wild‐type enzyme.

Complexing of Green Tea Catechins with Food Constituents and Degradation of the Complexes by Lactobacillus plantarum

Complexing of green tea catechins with food constituents and their hydrolysis by tannase-producing Lactobacillus plantarum strains, were investigated. Our observations indicated that 1) epigallocatechin gallate (EGCg) and other catechin galloyl esters bound with food ingredients (i.e., proteins) to form a complex that is likely to be unabsorbable through the intestinal wall, whereas most catechins not esterified with gallic acid (GA) remain in free form, not complexing with food ingredients; 2) tannase activity of L. plantarum is strain dependent, possibly grouped into those with high tannase activity hydrolyzing EGCg to epigallocatechin and GA and those with the low activity; and 3) L. plantarum strains with high tannase activity are capable of hydrolyzing not only intact EGCg but also EGCg and other catechin galloyl esters complexed with dietary proteins to free non-galloyl ester catechins and GA. The evidence suggests that L. plantarum with high tannase activity, if it colonizes the human intestine, would release free non-galloyl-ester catechins and GA that are readily absorbed through the human intestinal epithelia from the complexes, thereby ensuring maximum delivery of the bioactive polyphenols of green tea to the host.

Characterisitics and Gene Cloning of Phospholipase D of the Psychrophile, Shewanella sp.

Phospholipase D, with a molecular mass of 64 kDa, was purified from the psychrophile, Shewanella sp. The enzyme showed maximal activity at pH 7.8 and 40 °C in the presence of the Ca2+-ion, and its activity at 10 °C was 6.5% of maximum. The enzyme exhibited high activity to the non-micelle form of phosphatidylcholine in an aqueous solution containing water miscible alcohols such as methanol, ethanol, iso-propanol, and n-propanol. Nucleotide sequencing of the enzyme gene yielded a deduced amino acid sequence, which showed 36.2% identity to that of Streptomyces chromofuscus phopsholipase D alone. The low sequence similarity to other phopsholipase D enzymes suggests that the purified enzyme might be a novel phospholipase D.