Role of proline residues in conferring thermostability on aqualysin I.

Abstract:

:To understand the molecular basis of the thermostability of a thermophilic serine protease aqualysin I from Thermus aquaticus YT-1, we introduced mutations at Pro5, Pro7, Pro240 and Pro268, which are located on the surface loops of aqualysin I, by changing these amino acid residues into those found at the corresponding locations in VPR, a psychrophilic serine protease from Vibrio sp. PA-44. All mutants were expressed stably and exhibited essentially the same specific activity as wild-type aqualysin I at 40 degrees C. The P240N mutant protein had similar thermostability to wild-type aqualysin I, but P5N and P268T showed lower thermostability, with a half-life at 90 degrees C of 15 and 30 min, respectively, as compared to 45 min for the wild-type enzyme. The thermostability of P7I was decreased even more markedly, and the mutant protein was rapidly inactivated at 80 degrees C and even at 70 degrees C, with half-lives of 10 and 60 min, respectively. Differential scanning calorimetry analysis showed that the transition temperatures of wild-type enzyme, P5N, P7I, P240N and P268T were 93.99 degrees C, 83.45 degrees C, 75.66 degrees C, 91.78 degrees C and 86.49 degrees C, respectively. These results underscore the importance of the proline residues in the N- and C-terminal regions of aqualysin I in maintaining the integrity of the overall protein structure at elevated temperatures.

journal_name

J Biochem

journal_title

Journal of biochemistry

authors

Sakaguchi M,Matsuzaki M,Niimiya K,Seino J,Sugahara Y,Kawakita M

doi

10.1093/jb/mvm025

subject

Has Abstract

pub_date

2007-02-01 00:00:00

pages

213-20

issue

2

eissn

0021-924X

issn

1756-2651

pii

mvm025

journal_volume

141

pub_type

杂志文章
  • Cloning and expression of aminopeptidase P gene from Escherichia coli HB101 and characterization of expressed enzyme.

    abstract::The aminopeptidase P gene in Escherichia coli HB101 was cloned into the plasmid pBR322. Introduction of the hybrid plasmid, pAPP01, into the E. coli DH1 resulted in an 8-fold increase of aminopeptidase P activity as compared with that of the host. The enzyme was purified by series of chromatographies on DEAE-Sephadex,...

    journal_title:Journal of biochemistry

    pub_type: 杂志文章

    doi:10.1093/oxfordjournals.jbchem.a122430

    authors: Yoshimoto T,Murayama N,Honda T,Tone H,Tsuru D

    更新日期:1988-07-01 00:00:00

  • Serine racemase is involved in d-aspartate biosynthesis.

    abstract::d-Aspartate is found in the nervous and reproductive system and participates in various physiological roles. While several lines of evidence suggest that this amino acid has an endogenous origin, the enzyme responsible for mammalian d-Asp biosynthesis has not yet been identified. We show that mammalian serine racemase...

    journal_title:Journal of biochemistry

    pub_type: 杂志文章

    doi:10.1093/jb/mvw043

    authors: Ito T,Hayashida M,Kobayashi S,Muto N,Hayashi A,Yoshimura T,Mori H

    更新日期:2016-12-01 00:00:00

  • Characterization of novel acetyltransferases found in budding and fission yeasts that detoxify a proline analogue, azetidine-2-carboxylic acid.

    abstract::We recently found that budding yeast Saccharomyces cerevisiae sigma1278b, but not genome project strain S288C, has a gene conferring resistance to L-azetidine-2-carboxylic acid (AZC), a toxic four-membered ring analogue of L-proline. Also, the gene, designated as MPR1, encodes a novel acetyltransferase that detoxifies...

    journal_title:Journal of biochemistry

    pub_type: 杂志文章

    doi:10.1093/jb/mvg003

    authors: Nomura M,Nakamori S,Takagi H

    更新日期:2003-01-01 00:00:00

  • Differential changes in relative levels of arachidonic acid in major phospholipids from rat tissues during the progression of diabetes.

    abstract::Changes in the fatty acid composition of choline glycerophospholipid (CGP) and ethanolamine glycerophospholipid (EGP) in the liver, heart, kidney, testis, spleen, and brain of rats were examined at various stages of streptozotocin-induced diabetes. Diabetes of prolonged duration (8 weeks) caused profound alterations i...

    journal_title:Journal of biochemistry

    pub_type: 杂志文章

    doi:10.1093/oxfordjournals.jbchem.a124351

    authors: Hu Q,Ishii E,Nakagawa Y

    更新日期:1994-03-01 00:00:00

  • Crystal structure of serine dehydrogenase from Escherichia coli: important role of the C-terminal region for closed-complex formation.

    abstract::Serine dehydrogenase from Escherichia coli is a homotetrameric enzyme belonging to the short-chain dehydrogenase/reductase (SDR) family. This enzyme catalyses the NADP(+)-dependent oxidation of serine to 2-aminomalonate semialdehyde. The enzyme shows a stereospecificity for β-(3S)-hydroxy acid as a substrate; however,...

    journal_title:Journal of biochemistry

    pub_type: 杂志文章

    doi:10.1093/jb/mvr024

    authors: Yamazawa R,Nakajima Y,Mushiake K,Yoshimoto T,Ito K

    更新日期:2011-06-01 00:00:00

  • Regulation of TGF-β family signalling by ubiquitination and deubiquitination.

    abstract::Members of the transforming growth factor-β (TGF-β) family, including TGF-βs, activin and bone morphogenetic proteins (BMPs), are multifunctional proteins that regulate a wide variety of cellular responses, such as proliferation, differentiation, migration and apoptosis. TGF-β family signalling is mainly mediated by m...

    journal_title:Journal of biochemistry

    pub_type: 杂志文章,评审

    doi:10.1093/jb/mvt097

    authors: Imamura T,Oshima Y,Hikita A

    更新日期:2013-12-01 00:00:00

  • Intermolecular cross-linking of a novel rice Kinesin k16 motor domain with a photoreactive ATP derivative.

    abstract::A fluorescent photoreactive ATP derivative, 2'(3')-O-(4-benzoylbenzoyl)-1,N(6)-etheno-ATP (Bz(2)-epsilonATP), was synthesized and reacted with the rice kinesin K16 motor domain (K16MD). In the presence of ADP or ATP, UV irradiation of the K16MD solution containing Bz(2)-epsilonATP resulted in a new 100 kDa band, which...

    journal_title:Journal of biochemistry

    pub_type: 杂志文章

    doi:10.1093/jb/mvj098

    authors: Umeki N,Mitsui T,Koike Y,Maruta S

    更新日期:2006-05-01 00:00:00

  • The effects of mutations at position 253 on the thermostability of the Bacillus subtilis 3-isopropylmalate dehydrogenase subunit interface.

    abstract::3-Isopropylmalate dehydrogenase (IPMDH) is a dimeric enzyme with a strongly hydrophobic core that is composed of residues from four alpha-helices. We replaced Glu253, which is found in the hydrophobic core and is part of the subunit interface of the Bacillus subtilis (Bs) IPMDH, with several other amino acids to probe...

    journal_title:Journal of biochemistry

    pub_type: 杂志文章

    doi:10.1093/jb/mvm082

    authors: Ohkuri T,Yamagishi A

    更新日期:2007-06-01 00:00:00

  • Myoclonus, motor deficits, alterations in emotional responses and monoamine metabolism in epsilon-sarcoglycan deficient mice.

    abstract::Mutations of epsilon-sarcoglycan gene (SGCE) have been implicated in myoclonus-dystonia (M-D), a movement disorder. To determine the pathophysiology of M-D, we produced Sgce knockout mice and found that the knockout mice exhibited myoclonus, motor impairments, hyperactivity, anxiety, depression, significantly higher l...

    journal_title:Journal of biochemistry

    pub_type: 杂志文章

    doi:10.1093/jb/mvj138

    authors: Yokoi F,Dang MT,Li J,Li Y

    更新日期:2006-07-01 00:00:00

  • Apoptosis of CTLL-2 cells induced by an immunosuppressant, ISP-I, is caspase-3-like protease-independent.

    abstract::In our previous study, the sphingosine-like immunosuppressant ISP-1 was shown to induce apoptosis in the mouse cytotoxic T cell line CTLL-2. In this study, we characterized the ISP-1-induced apoptotic pathway. Although caspase-3-like protease activity increases concomitantly with ISP-1-induced apoptosis in CTLL-2 cell...

    journal_title:Journal of biochemistry

    pub_type: 杂志文章

    doi:10.1093/oxfordjournals.jbchem.a002886

    authors: Yamaji T,Nakamura S,Takematsu H,Kawasaki T,Kozutsumi Y

    更新日期:2001-04-01 00:00:00

  • Perturbation of phospholipid metabolism by erucic acid in male Sprague-Dawley rat heart.

    abstract::Erucic acid was incorporated into cardiac phosphatidylserine and hepatic and cardiac sphingomyelin of male Sprague-Dawley rat. The fatty acid compositions of mitochondrial and microsomal phospholipids were similar in both liver and heart. The effect of a low fat diet and a diet containing erucic acid on the fatty acid...

    journal_title:Journal of biochemistry

    pub_type: 杂志文章

    doi:10.1093/oxfordjournals.jbchem.a133921

    authors: Ishinaga M,Sato J,Kitagawa Y,Sugimoto E,Kito M

    更新日期:1982-07-01 00:00:00

  • LncRNA MEG3 inhibits the progression of prostate cancer by facilitating H3K27 trimethylation of EN2 through binding to EZH2.

    abstract::This study aims to study the effects of intra-nuclear lncRNA MEG3 on the progression of prostate cancer and the underlying mechanisms. Expressions of relative molecules were detected by Quantitative real time PCR (qRT-PCR) and western blot. Chromatin immunoprecipitation and RNA immunoprecipitation (RIP) assays were us...

    journal_title:Journal of biochemistry

    pub_type: 杂志文章

    doi:10.1093/jb/mvz097

    authors: Zhou Y,Yang H,Xia W,Cui L,Xu R,Lu H,Xue D,Tian Z,Ding T,Cao Y,Shi Q,He X

    更新日期:2020-03-01 00:00:00

  • Purification and properties of growth inhibitor from normal rabbit serum.

    abstract::It was previously found that rabbit serum contains a growth-inhibitory substance for a tumorigenic rat liver cell line RSV-BRL. In the present study, the growth inhibitor was purified from normal rabbit serum to show a homogeneous protein band with a molecular weight (Mr) of 56 k on SDS-polyacrylamide gel electrophore...

    journal_title:Journal of biochemistry

    pub_type: 杂志文章

    doi:10.1093/oxfordjournals.jbchem.a123597

    authors: Kimura T,Miyazaki K,Mashima K,Yamashita J,Horio T,Kakuno T

    更新日期:1991-09-01 00:00:00

  • Nucleotide sequence analysis of human beta-globin gene by the quantification method: mutations in 3'-splice junction sequence and beta-thalassemia.

    abstract::The nucleotide sequence at the intron-exon junction in the human beta-globin gene was analyzed by the quantification method (categorical discriminant analysis) proposed previously. Using the sample score of a 16-nucleotide sequence at a 3'-splice junction, we studied to what extent such a sequence contains the 3'-spli...

    journal_title:Journal of biochemistry

    pub_type: 杂志文章

    doi:

    authors: Iida Y

    更新日期:1990-12-01 00:00:00

  • 5-Aminoimidazole-4-carboxyamide-1-β-D-ribofranoside stimulates the rat enhancer of split- and hairy-related protein-2 gene via atypical protein kinase C lambda.

    abstract::The 5'-AMP-activated protein kinase (AMPK) functions as a cellular energy sensor. 5-Aminoimidazole-4-carboxyamide-1-β-D-ribofranoside (AICAR) is a chemical activator of AMPK. In the liver, AICAR suppresses expression of thephosphoenolpyruvate carboxykinase(PEPCK) gene. The rat enhancer of split- and hairy-related prot...

    journal_title:Journal of biochemistry

    pub_type: 杂志文章

    doi:10.1093/jb/mvv116

    authors: Komatsu Y,Yanagisawa Y,Moriizumi M,Tsuchiya Y,Yokouchi H,Otsuka H,Aoyagi M,Tsukada A,Kanai Y,Haneishi A,Takagi K,Asano K,Ono M,Tanaka T,Tomita K,Yamada K

    更新日期:2016-04-01 00:00:00

  • Characterization of a human placental fructose-6-phosphate, 2-kinase/fructose-2,6-bisphosphatase.

    abstract::A full-length cDNA, which encodes a human placental fructose-6-phosphate,2-kinase/ fructose-2,6-bisphosphatase, was constructed and expressed in Escherichia coli. The expressed protein, purified to homogeneity, showed a molecular weight of 58,000 by gel electrophoresis under denaturing conditions, compared to the dedu...

    journal_title:Journal of biochemistry

    pub_type: 杂志文章

    doi:10.1093/oxfordjournals.jbchem.a021719

    authors: Sakakibara R,Kato M,Okamura N,Nakagawa T,Komada Y,Tominaga N,Shimojo M,Fukasawa M

    更新日期:1997-07-01 00:00:00

  • Biochemical identification of the neutral endopeptidase family member responsible for the catabolism of amyloid beta peptide in the brain.

    abstract::Amyloid beta peptide (Abeta) is a physiological peptide that is constantly catabolized in the brain. We previously demonstrated that an endopeptidase sensitive to phosphoramidon and thiorphan conducts the initial rate-limiting proteolysis of Abeta in vivo, but the exact molecular identity of the peptidase(s) has remai...

    journal_title:Journal of biochemistry

    pub_type: 杂志文章

    doi:10.1093/oxfordjournals.jbchem.a022839

    authors: Takaki Y,Iwata N,Tsubuki S,Taniguchi S,Toyoshima S,Lu B,Gerard NP,Gerard C,Lee HJ,Shirotani K,Saido TC

    更新日期:2000-12-01 00:00:00

  • Structure and function of 5S ribosomal ribonucleic acid from Torulopsis utilis. III. Detection of single-stranded regions by digestion with nuclease S1.

    abstract::Identification of single-stranded regions in Torulopsis utilis 5S RNA was attempted by the use of Nuclease S1, a single-strand specific endonuclease. When T. utilis 5S RNA was subjected to prolonged incubation with Nuclease S1, about 50% of the substrate 5S RNA remained as large oligonucleotide "cores." Such Nuclease...

    journal_title:Journal of biochemistry

    pub_type: 杂志文章

    doi:10.1093/oxfordjournals.jbchem.a131566

    authors: Nishikawa K,Takemura S

    更新日期:1977-04-01 00:00:00

  • Purification and properties of epithelial growth inhibitor (EGI) from human platelets: its separation from type beta transforming growth factor (TGF-beta).

    abstract::We previously reported that sera from various kinds of animals contain a protein(s) capable of inhibiting the growth of the non-malignant epithelial cell line derived from Buffalo rat liver (BRL). In the present study, a similar epithelial cell-specific growth inhibitor (EGI) was purified to homogeneity from an acid-e...

    journal_title:Journal of biochemistry

    pub_type: 杂志文章

    doi:10.1093/oxfordjournals.jbchem.a121761

    authors: Huang W,Kimura T,Mashima K,Miyazaki K,Masaki H,Yamashita J,Horio T

    更新日期:1986-09-01 00:00:00

  • Alpha-2-macroglobulin-like protease inhibitor from the egg white of cuban crocodile (Crocodylus rhombifer).

    abstract::A high molecular weight protease inhibitor was purified from the egg white of Cuban crocodile (Crocodylus rhombifer). It inhibited the casein hydrolyzing activity of trypsin, subtilisin and papain. Its native molecular weight was 730,000 and it consisted of four subunits of equal molecular weight, each pair of which w...

    journal_title:Journal of biochemistry

    pub_type: 杂志文章

    doi:10.1093/oxfordjournals.jbchem.a134145

    authors: Ikai A,Kitamoto T,Nishigai M

    更新日期:1983-01-01 00:00:00

  • Thermodynamics of association of egg yolk riboflavin binding protein with 8-substituted riboflavins. Comparison with the egg white protein.

    abstract::The association constants of hen egg yolk riboflavin binding protein with 8-substituted riboflavins were measured using a fluorometric titration method in the range of 10-40 degrees C, and thermodynamic parameters were calculated using ordinary methods. The flavins used were riboflavins whose 8-methyl group was substi...

    journal_title:Journal of biochemistry

    pub_type: 杂志文章

    doi:10.1093/oxfordjournals.jbchem.a133823

    authors: Matsui K,Sugimoto K,Kasai S

    更新日期:1982-04-01 00:00:00

  • Identification of mK1, a true tissue (glandular) kallikrein of mouse submandibular gland: tissue distribution and a comparison of kinin-releasing activity with other submandibular kallikreins.

    abstract::The protein structure, kinin-releasing activity, and tissue distribution of four major proteinases of mouse submandibular gland (mK22, mK9, proteinase F, proteinase P) were studied. When compared with the deduced amino acid sequence of each member of the tissue (glandular) kallikrein gene family, the amino acid sequen...

    journal_title:Journal of biochemistry

    pub_type: 杂志文章

    doi:10.1093/oxfordjournals.jbchem.a124288

    authors: Hosoi K,Tsunasawa S,Kurihara K,Aoyama H,Ueha T,Murai T,Sakiyama F

    更新日期:1994-01-01 00:00:00

  • Affinity chromatography of peptidylarginine deiminase from rabbit skeletal muscle on a column of soybean trypsin inhibitor (Kunitz)-Sepharose.

    abstract::We designed a simple procedure for the purification of peptidylarginine deiminase, which catalyzes the deimination of arginyl residues in protein, from rabbit skeletal muscle using substrate affinity chromatography. Of the immobilized substrate ligands tested, i.e. protamine and soybean trypsin inhibitor (Kunitz) (STI...

    journal_title:Journal of biochemistry

    pub_type: 杂志文章

    doi:10.1093/oxfordjournals.jbchem.a135611

    authors: Takahara H,Okamoto H,Sugawara K

    更新日期:1986-05-01 00:00:00

  • Expression of syndecan-1 and -3 during embryogenesis of the central nervous system in relation to binding with midkine.

    abstract::Midkine (MK) is a 13-kDa heparin-binding growth/differentiation factor, and its interaction with heparan sulfate proteoglycan is important in promotion of neurite outgrowth. This study was performed to reveal the species of syndecans that interact with MK during embryogenesis of the central nervous system in the rat. ...

    journal_title:Journal of biochemistry

    pub_type: 杂志文章

    doi:

    authors: Nakanishi T,Kadomatsu K,Okamoto T,Ichihara-Tanaka K,Kojima T,Saito H,Tomoda Y,Muramatsu T

    更新日期:1997-02-01 00:00:00

  • DCCD-sensitive, Na+-dependent H+-influx process coupled to membrane potential formation in membrane vesicles of Halobacterium halobium.

    abstract::The effects of N,N'-dicyclohexylcarbodiimide (DCCD) on light-induced H+-transport and transmembrane electric potential (delta phi) formation were studied in the membrane vesicles of Halobacterium halobium R1M1. In accordance with our previous finding of the existence of two DCCD-binding components in vesicle membrane ...

    journal_title:Journal of biochemistry

    pub_type: 杂志文章

    doi:10.1093/oxfordjournals.jbchem.a135369

    authors: Murakami N,Konishi T

    更新日期:1985-10-01 00:00:00

  • A 26K fragment of troponin T from rabbit skeletal muscle.

    abstract::A 26K fragment of troponin T, which was produced by endogenous proteases in rabbit skeletal muscle, was isolated by SE-Sephadex column chromatography. This fragment sensitized both superprecipitation and ATPase of actomyosin to calcium ions, to the same extent as troponin T. There was no difference in affinity for tro...

    journal_title:Journal of biochemistry

    pub_type: 杂志文章

    doi:10.1093/oxfordjournals.jbchem.a134740

    authors: Ohtsuki I,Shiraishi F,Suenaga N,Miyata T,Tanokura M

    更新日期:1984-05-01 00:00:00

  • Detection and characterization of p-coumaric acid hydroxylase in mung bean, Vigna mungo, seedlings.

    abstract::A new p-coumaric acid (4-hydroxycinnamic acid) hydroxylase was detected in mung bean seedlings treated with tentoxin, a fungal toxin, in which polyphenol oxidase that hydroxylates a wide variety of monophenols in vitro was completely eliminated. The enzyme required molecular oxygen and showed a pH optimum of 5.0. The ...

    journal_title:Journal of biochemistry

    pub_type: 杂志文章

    doi:10.1093/oxfordjournals.jbchem.a122651

    authors: Kojima M,Takeuchi W

    更新日期:1989-02-01 00:00:00

  • An intermediate of ubiquinone biosynthesis exists in the microsomal fraction of HepG2 cells.

    abstract::We analyzed lipids extracted from human hepatoma HepG2 cells using a high performance liquid chromatograph equipped with a reversed phase column and found a compound with a mass spectrum showing certain diagnostic ion fragments of 1-methoxy-5-polyprenyl-phenol, a known intermediate of ubiquinone biosynthesis. Universa...

    journal_title:Journal of biochemistry

    pub_type: 杂志文章

    doi:10.1093/oxfordjournals.jbchem.a123308

    authors: Kang DC,Takeshige K,Minakami S

    更新日期:1990-12-01 00:00:00

  • Hepato-renal transport of phenolsulfonphthalein in analbuminemic rats: albumin is essential for hepatic compensatory elimination of a nephrophilic ligand in animals with renal dysfunction.

    abstract::To investigate a possible function of plasma albumin in partitioning organic anions into bile and urine, phenolsulfonphthalein (PSP) was administered intravenously and its in vivo fate was studied in normal and analbuminemic mutant rats (NAR). No significant change in the rate of PSP disappearance was observed in bila...

    journal_title:Journal of biochemistry

    pub_type: 杂志文章

    doi:10.1093/oxfordjournals.jbchem.a121693

    authors: Inoue M,Koyama H,Nagase S,Morino Y

    更新日期:1986-07-01 00:00:00

  • Characterization of porcine adrenocortical lysosomes.

    abstract::Porcine adrenocortical lysosomes were characterized by differential centrifugation, acid hydrolase contents, latency of cathepsin D, release of bound acid hydrolases in soluble form, and isopycnic density gradient centrifugation. Cathepsins D and B, beta-N-acetylglucosaminidase, beta-galactosidase and arylsulphatase w...

    journal_title:Journal of biochemistry

    pub_type: 杂志文章

    doi:10.1093/oxfordjournals.jbchem.a133333

    authors: Taguchi S,Watabe S,Kouyama H,Tatsunami S,Yago N

    更新日期:1981-05-01 00:00:00