Cloning and Characterization of the Polyhydroxybutyrate Depolymerase Gene of Pseudomonas stutzeri and Analysis of the Function of Substrate-Binding Domains

ABSTRACT The extracellular polyhydroxybutyrate (PHB) depolymerase gene (phaZPst ) of Pseudomonas stutzeriwas cloned and sequenced. phaZPst was composed of 1,728 bp encoding a protein of 576 amino acids. Analyses of the N-terminal amino acid sequence and the matrix-assisted laser desorption/ionization–time-of-flight (MALDI-TOF) mass spectrum of the purified enzyme showed that the mature enzyme consisted of 538 amino acids with a deduced molecular mass of 57,506 Da. Analysis of the deduced amino acid sequence of the protein revealed a domain structure containing a catalytic domain, putative linker region, and two putative substrate-binding domains (SBDI and SBDII). The putative linker region was similar to the repeating units of the cadherin-like domain of chitinase A from Vibrio harveyi and chitinase B fromClostridium paraputrificum. The binding characteristics of SBDs to poly([R]-3-hydroxybutyrate) [P(3HB)] and chitin granules were characterized by using fusion proteins of SBDs with glutathione S -transferase (GST). These GST fusion proteins with SBDII and SBDI showed binding activity toward P(3HB) granules but did not bind on chitin granules. It has been suggested that the SBDs of the depolymerase interact specifically with the surface of P(3HB). In addition, a kinetic analysis for the enzymatic hydrolysis of 3-hydroxybutyrate oligomers of various sizes has suggested that the catalytic domain of the enzyme recognizes at least two monomeric units as substrates.

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Characterization of the extracellular poly(3-hydroxybutyrate) depolymerase ofComamonassp. and of its structural gene

Canadian Journal of Microbiology ◽

10.1139/m95-183 ◽

1995 ◽

Vol 41 (13) ◽

pp. 160-169 ◽

Cited By ~ 52

Author(s):

Dieter Jendrossek ◽

Martina Backhaus ◽

Meike Andermann

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Amino Acid Sequence ◽

Binding Site ◽

Catalytic Domain ◽

Substrate Binding ◽

Catalytic Triad ◽

Reading Frame ◽

Phb Depolymerase ◽

Substrate Binding Site

The poly(3-hydroxybutyrate) (PHB) depolymerase structural gene of Comamonas sp. (phaZCsp) was cloned in Escherichia coli and identified by halo formation on PHB-containing solid medium. The nucleotide sequence of a 1719 base pair MboI fragment was determined and contained one large open reading frame (ORF1, 1542 base pairs). This open reading frame encoded the precursor of the PHB depolymerase (514 amino acids; Mr, 53 095), and the deduced amino acid sequence was in agreement with the N-terminal amino acid sequence of the purified PHB depolymerase from amino acid 26 onwards. Analysis of the deduced amino acid sequence revealed a domain structure of the protein: a signal peptide that was 25 amino acids long was followed by a catalytic domain of about 300 amino acids, a fibronectin type III (Fn3) modul sequence, and a putative PHB-specific substrate-binding site. By comparison of the primary structure with that of other polyhydroxyalkanoate (PHA) depolymerases, the catalytic domain apparently contained a catalytic triad of serine, histidine, and aspartate. In addition, a conserved region resembling the oxyanion hole of lipases was present. The catalytic domain was linked to a C-terminal putative substrate-binding site by a sequence about 90 amino acids long resembling the Fn3 modul of fibronectin and other eukaryotic extracellular matrix proteins. A threonine-rich region, which was found in four of five PHA depolymerases of Pseudomonas lemoignei, was not present in the Comamonas sp. depolymerase. The similarities with and differences from other PHA depolymerases are discussed.Key words: biodegradable polymer, poly(3-hydroxybutyrate) depolymerase, serine hydrolase, catalytic triad, Comamonas sp., fibronectin type III modul, substrate-binding site.

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Analysis of Htra Gene from Zebrafish (Danio Rerio)

Indonesian Journal of Biotechnology ◽

10.22146/ijbiotech.7554 ◽

2015 ◽

Vol 10 (2) ◽

Author(s):

M. Murwantoko ◽

Chio Oka ◽

Masashi Kawaichi

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Amino Acid Sequence ◽

Danio Rerio ◽

Serine Proteases ◽

Catalytic Domain ◽

Fruit Fly ◽

Wide Range ◽

Igf Binding ◽

Bacterial Homolog

HtrA which is characterized by the combination of a trypsin-like catalytic domain with at least one C-terminalPDZ domain is a highly conserved family of serine proteases found in a wide range of organisms. However theidentified HtrA family numbers varies among spesies, for example the number of mammalian, Eschericia coli,fruit fly-HtrA family are 4, 3 and 1 gene respectively. One gene is predicted exist in zebrafish. Since no completeinformation available on zebrafish HtrA, in this paper zebrafish HtrA (zHtrA) gene was analyzed. The zHtrA isbelonged to HtrA1 member and predicted encodes 478 amino acids with a signal peptide, a IGF binding domain,a Kazal-type inhibitor domain in the up stream of HtrA-bacterial homolog. At the amino acid sequence the zHtrA1showed the 69%, 69%, 68%, 54% and 54% with the rat HtrA1, mouse HtrA1, human HtrA1, human HtrA3 andmouse HtrA4 respectively. The zHtrA1 is firstly expressed at 60 hpf and mainly in the vertebral rudiments in thetail region.

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Substrate-binding domains of glycanases from Streptomyces lividans: characterization of a new family of xylan-binding domains

Biochemical Journal ◽

10.1042/bj3300041 ◽

1998 ◽

Vol 330 (1) ◽

pp. 41-45 ◽

Cited By ~ 36

Author(s):

Claude DUPONT ◽

Martin ROBERGE ◽

François SHARECK ◽

Rolf MOROSOLI ◽

Dieter KLUEPFEL

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Substrate Binding ◽

Streptomyces Lividans ◽

Cellulose Binding Domain ◽

Binding Domains ◽

New Family ◽

Cellulose Binding ◽

Sequence Similarities

The substrate-binding domains of six glycanases from Streptomyces lividans were investigated to determine their specificity towards cellulose and xylan. Based upon amino acid sequence similarities, four of the six domains could be assigned to existing cellulose-binding domain families. However, the binding domains of xylanase A and arabinofuranosidase B could not be classified in any of the known families and should therefore be classified as members of a new family. Evidence is also presented that this new family is one of true xylan-binding domains.

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Differential scanning fluorimetric analysis of the amino-acid binding to taste receptor using a model receptor protein, the ligand-binding domain of fish T1r2a/T1r3

10.1101/670828 ◽

2019 ◽

Author(s):

Takashi Yoshida ◽

Norihisa Yasui ◽

Yuko Kusakabe ◽

Chiaki Ito ◽

Miki Akamatsu ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Ligand Binding ◽

Taste Receptor ◽

Thermal Stabilization ◽

Receptor Protein ◽

Binding Characteristics ◽

Melting Temperatures ◽

Binding Domains ◽

Amino Acid Binding

AbstractTaste receptor type 1 (T1r) is responsible for the perception of essential nutrients, such as sugars and amino acids, and evoking sweet and umami (savory) taste sensations. T1r receptors recognize many of the taste substances at their extracellular ligand-binding domains (LBDs). In order to detect a wide array of taste substances in the environment, T1r receptors often possess broad ligand specificities. However, the entire ranges of chemical spaces and their binding characteristics to any T1rLBDs have not been extensively analyzed. In this study, we exploited the differential scanning fluorimetry (DSF) to medaka T1r2a/T1r3LBD, a current sole T1rLBD heterodimer amenable for recombinant preparation, and analyzed their thermal stabilization by adding various amino acids. The assay showed that the agonist amino acids induced thermal stabilization and shifted the melting temperatures (Tm) of the protein. An agreement between the DSF results and the previous biophysical assay was observed, suggesting that DSF can detect ligand binding at the orthosteric-binding site in T1r2a/T1r3LBD. The assay further demonstrated that most of the tested L-amino acids, but no D-amino acid, induced Tm shifts of T1r2a/T1r3LBD, indicating the broad L-amino acid specificities of the proteins probably with several different manners of recognition. The Tm shifts by each amino acid also showed a fair correlation with the responses exhibited by the full-length receptor, verifying the broad amino-acid binding profiles at the orthosteric site in LBD observed by DSF.

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Structural heterogeneity of Pseudomonas aeruginosa bacterioferritin

Biochemical Journal ◽

10.1042/bj3040493 ◽

1994 ◽

Vol 304 (2) ◽

pp. 493-497 ◽

Cited By ~ 30

Author(s):

G R Moore ◽

F H Kadir ◽

F K al-Massad ◽

N E Le Brun ◽

A J Thomson ◽

...

Keyword(s):

Amino Acids ◽

Pseudomonas Aeruginosa ◽

Amino Acid ◽

Amino Acid Sequence ◽

Structural Heterogeneity ◽

Amino Acid Sequences ◽

Binding Study ◽

Alpha Subunit ◽

Beta Subunits ◽

Binding Characteristics

The subunit composition, amino acid sequence and haem-binding characteristics of bacterioferritin (BFR) from Pseudomonas aeruginosa have been studied. Unlike other BFRs, P. aeruginosa BFR was found to contain two subunit types, designated alpha and beta, which differed considerably in their amino acid sequences. The N-terminal 69 and 55 amino acids of the alpha and beta subunits respectively were determined. The alpha subunit differed most from other BFRs. The two subunits were present in variable proportions in different preparations. The maximum stoichiometry of haem binding was found to be sample-dependent and to be different from the previously reported one per subunit [Kadir and Moore (1990) FEBS Lett. 271, 141-143]. This previous haem-binding study was shown to have been carried out with damaged protein, which contained both normal alpha and beta subunits and shorter versions of these that appeared to have been produced by cleavage of the normal subunits. The possibility that aging processes degrade ferritins and affect their haem-binding characteristics is discussed.

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New α-l-arabinofuranosidase produced by Streptomyces lividans: cloning and DNA sequence of the abfB gene and characterization of the enzyme

Biochemical Journal ◽

10.1042/bj3220845 ◽

1997 ◽

Vol 322 (3) ◽

pp. 845-852 ◽

Cited By ~ 37

Author(s):

Patrick VINCENT ◽

François SHARECK ◽

Claude DUPONT ◽

Rolf MOROSOLI ◽

Dieter KLUEPFEL

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Dna Sequence ◽

Catalytic Domain ◽

Expression System ◽

Prolonged Incubation ◽

Linker Region ◽

Xylanolytic Activity ◽

Homologous Expression

A fully secreted α-l-arabinofuranosidase was cloned from the homologous expression system of Streptomyces lividans.The gene, located upstream adjacent to the previously described xylanase A gene, was sequenced. It is divergently transcribed from the xlnAgene and the two genes are separated by an intercistronic region of 391 nt which contains a palindromic AT-rich sequence. The deduced amino acid sequence of the protein shows that the enzyme contains a distinct catalytic domain which is linked to a specific xylan-binding domain by a linker region. The purified enzyme has a specific arabinofuranose-debranching activity on xylan from Gramineae,acts synergistically with the S. lividansxylanases and binds specifically to xylan. From small arabinoxylo-oligosides, it liberates arabinose and, after prolonged incubation, the purified enzyme exhibits some xylanolytic activity as well.

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Gene Cloning and Characterization of the Very Large NAD-Dependent l-Glutamate Dehydrogenase from the Psychrophile Janthinobacterium lividum, Isolated from Cold Soil

Journal of Bacteriology ◽

10.1128/jb.00496-07 ◽

2007 ◽

Vol 189 (15) ◽

pp. 5626-5633 ◽

Cited By ~ 15

Author(s):

Ryushi Kawakami ◽

Haruhiko Sakuraba ◽

Toshihisa Ohshima

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Amino Acid Sequence ◽

Gene Cloning ◽

Glutamate Dehydrogenase ◽

Catalytic Domain ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Streptomyces Clavuligerus ◽

Janthinobacterium Lividum

ABSTRACT NAD-dependent l-glutamate dehydrogenase (NAD-GDH) activity was detected in cell extract from the psychrophile Janthinobacterium lividum UTB1302, which was isolated from cold soil and purified to homogeneity. The native enzyme (1,065 kDa, determined by gel filtration) is a homohexamer composed of 170-kDa subunits (determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Consistent with these findings, gene cloning and sequencing enabled deduction of the amino acid sequence of the subunit, which proved to be comprised of 1,575 amino acids with a combined molecular mass of 169,360 Da. The enzyme from this psychrophile thus appears to belong to the GDH family characterized by very large subunits, like those expressed by Streptomyces clavuligerus and Pseudomonas aeruginosa (about 180 kDa). The entire amino acid sequence of the J. lividum enzyme showed about 40% identity with the sequences from S. clavuligerus and P. aeruginosa enzymes, but the central domains showed higher homology (about 65%). Within the central domain, the residues related to substrate and NAD binding were highly conserved, suggesting that this is the enzyme's catalytic domain. In the presence of NAD, but not in the presence of NADP, this GDH exclusively catalyzed the oxidative deamination of l-glutamate. The stereospecificity of the hydride transfer to NAD was pro-S, which is the same as that of the other known GDHs. Surprisingly, NAD-GDH activity was markedly enhanced by the addition of various amino acids, such as l-aspartate (1,735%) and l-arginine (936%), which strongly suggests that the N- and/or C-terminal domains play regulatory roles and are involved in the activation of the enzyme by these amino acids.

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Identification and Characterization of a Novel Class of Extracellular Poly(3-Hydroxybutyrate) Depolymerase from Bacillus sp. Strain NRRL B-14911

Applied and Environmental Microbiology ◽

10.1128/aem.06069-11 ◽

2011 ◽

Vol 77 (22) ◽

pp. 7924-7932 ◽

Cited By ~ 10

Author(s):

Wan-Ting Ma ◽

Ju-Hui Lin ◽

Hui-Ju Chen ◽

Syuan-Yi Chen ◽

Gwo-Chyuan Shaw

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Sequence Similarity ◽

Amino Acid Sequence Similarity ◽

Content Type ◽

Linker Region ◽

Phb Depolymerase ◽

Binding Domains ◽

Significant Amino Acid Sequence ◽

Significant Amino Acid

ABSTRACTThe catalytic, linker, and denatured poly(3-hydroxybutyrate) (dPHB)-binding domains of bacterial extracellular PHB depolymerases (PhaZs) are classified into several different types. We now report a novel class of extracellular PHB depolymerase fromBacillussp. strain NRRL B-14911. Its catalytic domain belongs to type 1, whereas its putative linker region neither possesses the sequence features of the three known types of linker domains nor exhibits significant amino acid sequence similarity to them. Instead, this putative linker region can be divided into two distinct linker domains of novel types: LD1 and LD2. LD1 shows significant amino acid sequence similarity to certain regions of a large group of PHB depolymerase-unrelated proteins. LD2 and its homologs are present in a small group of PhaZs. The remaining C-terminal portion of this PhaZ can be further divided into two distinct domains: SBD1 and SBD2. Each domain showed strong binding to dPHB, and there is no significant sequence similarity between them. Each domain neither possesses the sequence features of the two known types of dPHB-binding domains nor shows significant amino acid sequence similarity to them. These unique features indicate the presence of two novel and distinct types of dPHB-binding domains. Homologs of these novel domains also are present in the extracellular PhaZ ofBacillus megateriumand the putative extracellular PhaZs ofBacillus pseudofirmusandBacillussp. strain SG-1. TheBacillussp. NRRL B-14911 PhaZ appears to be a representative of a novel class of extracellular PHB depolymerases.

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Complete Covalent Structure of Human Platelet Factor 4

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657071 ◽

1979 ◽

Vol 42 (05) ◽

pp. 1652-1660 ◽

Cited By ~ 4

Author(s):

Francis J Morgan ◽

Geoffrey S Begg ◽

Colin N Chesterman

Keyword(s):

Amino Acids ◽

Molecular Weight ◽

Amino Acid ◽

Amino Acid Sequence ◽

Human Platelet ◽

Platelet Factor 4 ◽

Platelet Factor ◽

Disulphide Bonds ◽

Covalent Structure

SummaryThe amino acid sequence of the subunit of human platelet factor 4 has been determined. Human platelet factor 4 consists of identical subunits containing 70 amino acids, each with a molecular weight of 7,756. The molecule contains no methionine, phenylalanine or tryptophan. The proposed amino acid sequence of PF4 is: Glu-Ala-Glu-Glu-Asp-Gly-Asp-Leu-Gln-Cys-Leu-Cys-Val-Lys-Thr-Thr-Ser- Gln-Val-Arg-Pro-Arg-His-Ile-Thr-Ser-Leu-Glu-Val-Ile-Lys-Ala-Gly-Pro-His-Cys-Pro-Thr-Ala-Gin- Leu-Ile-Ala-Thr-Leu-Lys-Asn-Gly-Arg-Lys-Ile-Cys-Leu-Asp-Leu-Gln-Ala-Pro-Leu-Tyr-Lys-Lys- Ile-Ile-Lys-Lys-Leu-Leu-Glu-Ser. From consideration of the homology with p-thromboglobulin, disulphide bonds between residues 10 and 36 and between residues 12 and 52 can be inferred.

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Chitosanase from Streptomyces sp. strain N174: a comparative review of its structure and function

Biochemistry and Cell Biology ◽

10.1139/o97-079 ◽

1997 ◽

Vol 75 (6) ◽

pp. 687-696 ◽

Cited By ~ 20

Author(s):

Tamo Fukamizo ◽

Ryszard Brzezinski

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Substrate Binding ◽

Structure And Function ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Amino Acid Residues ◽

Streptomyces Sp ◽

Binding Cleft ◽

And Function

Novel information on the structure and function of chitosanase, which hydrolyzes the beta -1,4-glycosidic linkage of chitosan, has accumulated in recent years. The cloning of the chitosanase gene from Streptomyces sp. strain N174 and the establishment of an efficient expression system using Streptomyces lividans TK24 have contributed to these advances. Amino acid sequence comparisons of the chitosanases that have been sequenced to date revealed a significant homology in the N-terminal module. From energy minimization based on the X-ray crystal structure of Streptomyces sp. strain N174 chitosanase, the substrate binding cleft of this enzyme was estimated to be composed of six monosaccharide binding subsites. The hydrolytic reaction takes place at the center of the binding cleft with an inverting mechanism. Site-directed mutagenesis of the carboxylic amino acid residues that are conserved revealed that Glu-22 and Asp-40 are the catalytic residues. The tryptophan residues in the chitosanase do not participate directly in the substrate binding but stabilize the protein structure by interacting with hydrophobic and carboxylic side chains of the other amino acid residues. Structural and functional similarities were found between chitosanase, barley chitinase, bacteriophage T4 lysozyme, and goose egg white lysozyme, even though these proteins share no sequence similarities. This information can be helpful for the design of new chitinolytic enzymes that can be applied to carbohydrate engineering, biological control of phytopathogens, and other fields including chitinous polysaccharide degradation. Key words: chitosanase, amino acid sequence, overexpression system, reaction mechanism, site-directed mutagenesis.

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