Ikuo Matsui

Novel substrate specificity of a membrane-bound β-glycosidase from the hyperthermophilic archaeon Pyrococcus horikoshii

A β‐glycosidase gene homolog of Pyrococcus horikoshii (BGPh) was successfully expressed in Escherichia coli. The enzyme was localized in a membrane fraction and solubilized with 2.5% Triton X‐100 at 85°C for 15 min. The optimum pH was 6.0 and the optimum temperature was over 100°C, respectively. BGPh stability was dependent on the presence of Triton X‐100, the enzymes half‐life at 90°C (pH 6.0) was 15 h. BGPh has a novel substrate specificity with k cat/K m values high enough for hydrolysis of β‐D‐Glcp derivatives with long alkyl chain at the reducing end and low enough for the hydrolysis of β‐linked glucose dimer more hydrophilic than aryl‐ or alkyl‐β‐D‐Glcp.

Crystal structure of a core domain of stomatin from Pyrococcus horikoshii Illustrates a novel trimeric and coiled-coil fold.

Stomatin is a major integral membrane protein of human erythrocytes, the absence of which is associated with a form of hemolytic anemia known as hereditary stomatocytosis. However, the function of stomatin is not fully understood. An open reading frame, PH1511, from the hyperthermophilic archaeon Pyrococcus horikoshii encodes p-stomatin, a prokaryotic stomatin. Here, we report the first crystal structure of a stomatin ortholog, the core domain of the p-stomatin PH1511p (residues 56-234 of PH1511p, designated as PhSto(CD)). PhSto(CD) forms a novel homotrimeric structure. Three alpha/beta domains form a triangle of about 50 A on each side, and three alpha-helical segments of about 60 A in length extend from the apexes of the triangle. The alpha/beta domain of PhSto(CD) is partly similar in structure to the band-7 domain of mouse flotillin-2. While the alpha/beta domain is relatively rigid, the alpha-helical segment shows conformational flexibility, adapting to the neighboring environment. One alpha-helical segment forms an anti-parallel coiled coil with another alpha-helical segment from a symmetry-related molecule. The alpha-helical segment shows a heptad repeat pattern, and mainly hydrophobic residues form a coiled-coil interface. According to chemical cross-linking experiments, PhSto(CD) would be able to assemble into an oligomeric form. The coiled-coil fold observed in the crystal probably contributes to self-association.

Implication for buried polar contacts and ion pairs in hyperthermostable enzymes

Understanding the structural basis of thermostability and thermoactivity, and their interdependence, is central to the successful future exploitation of extremophilic enzymes in biotechnology. However, the structural basis of thermostability is still not fully characterized. Ionizable residues play essential roles in proteins, modulating protein stability, folding and function. The dominant roles of the buried polar contacts and ion pairs have been reviewed by distinguishing between the inside polar contacts and the total intramolecular polar contacts, and by evaluating their contribution as molecular determinants for protein stability using various protein structures from hyperthermophiles, thermophiles and mesophilic organisms. The analysis revealed that the remarkably increased number of internal polar contacts in a monomeric structure probably play a central role in enhancing the melting temperature value up to 120 °C for hyperthermophilic enzymes from the genus Pyrococcus. These results provide a promising contribution for improving the thermostability of enzymes by modulating buried polar contacts and ion pairs.

Novel Bifunctional Hyperthermostable Carboxypeptidase/Aminoacylase from Pyrococcus horikoshii OT3

ABSTRACT Genome sequencing of the thermophilic archaeon Pyrococcus horikoshii OT3 revealed a gene which had high sequence similarity to the gene encoding the carboxypeptidase ofSulfolobus solfataricus and also to that encoding the aminoacylase from Bacillus stearothermophilus. The gene from P. horikoshii comprises an open reading frame of 1,164 bp with an ATG initiation codon and a TGA termination codon, encoding a 43,058-Da protein of 387 amino acid residues. However, some of the proposed active-site residues for carboxypeptidase were not found in this gene. The gene was overexpressed in Escherichia coli with the pET vector system, and the expressed enzyme had high hydrolytic activity for both carboxypeptidase and aminoacylase at high temperatures. The enzyme was stable at 90°C, with the highest activity above 95°C. The enzyme contained one bound zinc ion per one molecule that was essential for the activity. The results of site-directed mutagenesis of Glu367, which corresponds to the essential Glu270 in bovine carboxypeptidase A and the essential Glu in other known carboxypeptidases, revealed that Glu367 was not essential for this enzyme. The results of chemical modification of the SH group and site-directed mutagenesis of Cys102 indicated that Cys102 was located at the active site and was related to the activity. From these findings, it was proven that this enzyme is a hyperthermostable, bifunctional, new zinc-dependent metalloenzyme which is structurally similar to carboxypeptidase but whose hydrolytic mechanism is similar to that of aminoacylase. Some characteristics of this enzyme suggested that carboxypeptidase and aminoacylase might have evolved from a common origin.

An increase in the transglycosylation activity of Saccharomycopsis α-amylase altered by site-directed mutagenesis

The 84th tryptophan residue in Saccharomycopsis alpha-amylase molecule was replaced by a leucine residue and the resulting site-directed mutant, W84L enzyme, showed an increase in transglycosylation activity. At a 40% digestion point of maltoheptaose (G7), for example, maltooligosaccharide products larger than maltodecaose (G10) amounted to approx. 60% of the total product from the mutant enzyme reaction, whereas no such large products were observed in the native enzyme reaction. Analysis of the reaction products from p-nitrophenyl maltooligosaccharides indicated that these large products were formed by addition of the hydrolysis products on the nonreducing end side to the starting intact substrates. These results suggest that the tryptophan residue located at subsite 3 of the enzyme plays an important role not only to hold the substrate, but also to liberate the hydrolysis products from the substrate binding pocket.

A hyperthermostable D-ribose-5-phosphate isomerase from Pyrococcus horikoshii characterization and three-dimensional structure.

A gene homologous to D-ribose-5-phosphate isomerase (EC 5.3.1.6) was found in the genome of Pyrococcus horikoshii. D-ribose-5-phosphate isomerase (PRI) is of particular metabolic importance since it catalyzes the interconversion between the ribose and ribulose forms involved in the pentose phosphate cycle and in the process of photosynthesis. The gene consisting of 687 bp was overexpressed in Escherichia coli, and the resulting enzyme showed activity at high temperatures with an optimum over 90 degrees C. The crystal structures of the enzyme, free and in complex with D-4-phosphoerythronic acid inhibitor, were determined. PRI is a tetramer in the crystal and in solution, and each monomer has a new fold consisting of two alpha/beta domains. The 3D structures and the characterization of different mutants indicate a direct or indirect catalytic role for the residues E107, D85, and K98.

Molecular Structure and Novel DNA Binding Sites Located in Loops of Flap Endonuclease-1 from Pyrococcus horikoshii

The crystal structure of flap endonuclease-1 fromPyrococcus horikoshii (phFEN-1) was determined to a resolution of 3.1 Å. The active cleft of the phFEN-1 molecule is formed with one large loop and four small loops. We examined the function of the conserved residues and positively charged clusters on these loops by kinetic analysis with 45 different mutants. Arg40 and Arg42 on small loop 1, a cluster Lys193–Lys195 on small loop 2, and two sites, Arg94 and Arg118-Lys119, on the large loop were identified as binding sites. Lys87 on the large loop may play significant roles in catalytic reaction. Furthermore, we successfully elucidated the function of the four DNA binding sites that form productive ES complexes specific for each endo- or exo-type hydrolysis, probably by bending the substrates. For the endo-activity, Arg94 and Lys193–Lys195 located at the top and bottom of the molecule were key determinants. For the exo-activity, all four sites were needed, but Arg118-Lys119 was dominant. The major binding sites for both the nick substrate and double-stranded DNA might be the same.

A mutant α-amylase with enhanced activity specific for short substrates

The 210th lysine (K210) at the active site in Saccharomycopsis fibuligera α‐amylase was altered to arginine (R) or asparagine (N) by site‐directed mutagenesis. Replacement of K210 by R strengthened the 7th and weakened the 8th subsite affinities. K210 was found to contribute to both the 8th and the 7th subsites. The catalytic activity of the K210R enzyme for the hydrolysis of maltose (G2) was three‐times higher than that of the native enzyme due to an increase in the affinity of the 7th subsite adjacent to the catalytic site, whereas the activity of the K210N enzyme for G2 was decreased to 1% of that of the native enzyme by a reduction in the 7th subsite affinity.

Multi-functional roles of a histidine residue in human pancreatic α -amylase

Abstract Functional roles of histidine residues at the active site in human pancreatic α -amylase were examined by protein engineering. Three histidine residues at 101, 201, and 299 were converted to asparagine residues, respectively. It was found that His201 played multi-functional roles concerning so many functions; substrate binding, control of optimum pH, change in substrate specificity, activation by chloride ion, and inhibition by a proteinaceous inhibitor.

A Novel Thermostable Membrane Protease Forming an Operon with a Stomatin Homolog from the Hyperthermophilic Archaebacterium Pyrococcus horikoshii

Membrane-bound proteases play several important roles in protein quality control and regulation. In the genome of the hyperthermophilic archaebacterium Pyrococcus horikoshii, the open reading frames PH1510 and PH1511 are homologous to the genes nfed (nodulation formation efficiency D) and stomatin, respectively, and probably form an operon. The nfed proteins are putative membrane proteins, and the N-terminal region shows homology to ClpP-type serine proteases. Stomatin is one of the major integral membrane proteins of human erythrocytes, and its absence is associated with the hemolytic anemia known as hereditary stomatocytosis. Thus, the N-terminal region of PH1510 (1510-N, residues 16–236) was expressed and purified. From activity staining and SDS-PAGE analysis using fluorescein isothiocyanate-casein, 1510-N was identified as a thermostable endo-type protease. From site-directed mutagenesis, the conserved Ser-97 and Lys-138 are involved in proteolysis and, therefore, PH1510 is probably a serine protease with a catalytic Ser-Lys dyad. The sites of cleavage by 1510-N are rich in hydrophobic residues. The site P1 (position –1 relative to the cleavage site) is mainly leucine. P4 and P4′ are mainly hydrophobic residues. Interestingly, the 1510-N protease cleaves the C-terminal hydrophobic region of PH1511. From this result and the probability of an operon, PH1510 probably functions in cooperation with PH1511. It is hypothesized that the cleavage of the stomatin-homolog PH1511 by the PH1510 protease causes an ion channel to open.