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

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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Interaction of the neutral amino acid transporter ASCT2 with basic amino acids

Biochemical Journal ◽

10.1042/bcj20190859 ◽

2020 ◽

Vol 477 (8) ◽

pp. 1443-1457

Author(s):

Elias Ndaru ◽

Rachel-Ann A. Garibsingh ◽

Laura Zielewicz ◽

Avner Schlessinger ◽

Christof Grewer

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Binding Site ◽

Substrate Binding ◽

Ph Dependence ◽

Side Chains ◽

Substrate Binding Site ◽

Basic Amino Acids ◽

Voltage Dependent ◽

Diaminopropionic Acid

Glutamine transport across cell membranes is performed by a variety of transporters, including the alanine serine cysteine transporter 2 (ASCT2). The substrate-binding site of ASCT2 was proposed to be specific for small amino acids with neutral side chains, excluding basic substrates such as lysine. A series of competitive inhibitors of ASCT2 with low µM affinity were developed previously, on the basis of the 2,4-diaminobutyric acid (DAB) scaffold with a potential positive charge in the side chain. Therefore, we tested whether basic amino acids with side chains shorter than lysine can interact with the ASCT2 binding site. Molecular docking of L-1,3-diaminopropionic acid (L-DAP) and L-DAB suggested that these compounds bind to ASCT2. Consistent with this prediction, L-DAP and L-DAB, but not ornithine, lysine or D-DAP, elicited currents when applied to ASCT2-expressing cells. The currents were carried by anions and showed the hallmark properties of ASCT2 currents induced by transported substrates. The L-DAP response could be eliminated by a competitive ASCT2 inhibitor, suggesting that binding occurs at the substrate binding site. The KM for L-DAP was weakly voltage dependent. Furthermore, the pH dependence of the L-DAP response showed that the compound can bind in several protonation states. Together, these results suggest that the ASCT2 binding site is able to recognize L-amino acids with short, basic side chains, such as the L-DAP derivative β-N-methylamino-l-Alanine (BMAA), a well-studied neurotoxin. Our results expand the substrate specificity of ASCT2 to include amino acid substrates with positively charged side chains.

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

Applied and Environmental Microbiology ◽

10.1128/aem.65.1.189-197.1999 ◽

1999 ◽

Vol 65 (1) ◽

pp. 189-197 ◽

Cited By ~ 46

Author(s):

Takeshi Ohura ◽

Ken-Ichi Kasuya ◽

Yoshiharu Doi

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Amino Acid Sequence ◽

Fusion Proteins ◽

Catalytic Domain ◽

Substrate Binding ◽

Pseudomonas Stutzeri ◽

Linker Region ◽

Binding Characteristics ◽

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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Homology of Escherichia coli B Glutathione Synthetase with Dihydrofolate Reductase in Amino Acid Sequence and Substrate Binding Site

The Journal of Biochemistry ◽

10.1093/oxfordjournals.jbchem.a121893 ◽

1987 ◽

Vol 101 (1) ◽

pp. 207-215 ◽

Cited By ~ 15

Author(s):

Hiroaki KATO ◽

Mikiko CHIHARA ◽

Takaaki NLSHIOKA ◽

Kousaku MURATA ◽

Akira KIMURA ◽

...

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Amino Acid Sequence ◽

Binding Site ◽

Dihydrofolate Reductase ◽

Substrate Binding ◽

Glutathione Synthetase ◽

Substrate Binding Site ◽

Escherichia Coli B

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Identification of essential lysyl and cysteinyl residues, and the amino acid sequence at the substrate-binding site of retinal oxidase

Biochimica et Biophysica Acta (BBA) - General Subjects ◽

10.1016/0304-4165(94)00170-3 ◽

1995 ◽

Vol 1243 (3) ◽

pp. 431-436 ◽

Cited By ~ 2

Author(s):

Dong-Yang Huang ◽

Yoshiyuki Ichikaw

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Binding Site ◽

Substrate Binding ◽

Substrate Binding Site

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In-silico Investigation of the Interaction between Beta-class Glutathione S-Transferase and Five Antibiotics, namely; Ampicillin, Tetracycline, Chloramphenicol, Ciprofloxacin and Cephalexin

Journal of Applied Sciences and Environmental Management ◽

10.4314/jasem.v25i4.2 ◽

2021 ◽

Vol 25 (4) ◽

pp. 497-502

Author(s):

D. Shehu ◽

S Danlami ◽

M. Ya’u ◽

A. Babandi ◽

H.M. Yakasai ◽

...

Keyword(s):

Amino Acids ◽

Antibiotic Resistance ◽

Molecular Docking ◽

Binding Site ◽

Substrate Binding ◽

Docking Study ◽

Glutathione S Transferase ◽

Molecular Docking Study ◽

Substrate Binding Site ◽

Glutathione S Transferases

Glutathione s-transferases(GSTs) are enzymes involved in the conjugation and deactivation of various xenobiotics including drugs. Thisin-silico study was undertaken in order to investigate the interaction between beta-class glutathione s-transferase and five selected antibiotics, namely; ampicillin, tetracycline, chloramphenicol, ciprofloxacin and cephalexin using molecular docking study. RaptorX server was used to predict the amino acids involved at the binding sitewhile molecular docking study was employed in order to investigate the binding interactions.RaptorX predicted several amino acids which were different from the ones observed in molecular docking because of the variability in the substrate binding site of GSTs however, all the amino acids predicted by RaptorX were also found to be involved in the GSH binding.Lys107, Phe109, Ser110, Leu113, Trp114, His115 and Arg123, Leu168 were the amino acids involved in the binding of various antibiotics to the substrate binding site of the protein while Ala9, Cys10, Leu32, Tyr51, Val52, Pro53, Glu65 and Ala66were involved in the binding of the co-substrate GSH to the binding site of the protein. The results indicated that all the antibiotics showed a good binding affinity with the beta class GST and are therefore capable of deactivating the drugs. With these, finding a beta class GST inhibitors alongside antibiotics during a treatment of diseases will be of beneficial in the current fight against antibiotic resistance.

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Engineering the substrate binding site of the hyperthermostable archaeal endo-β-1,4-galactanase from Ignisphaera aggregans

Biotechnology for Biofuels ◽

10.1186/s13068-021-02025-6 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Sebastian J. Muderspach ◽

Folmer Fredslund ◽

Verena Volf ◽

Jens-Christian Navarro Poulsen ◽

Thomas H. Blicher ◽

...

Keyword(s):

Binding Site ◽

Plant Cell Wall ◽

Biological Diversity ◽

Catalytic Domain ◽

Substrate Binding ◽

Plant Defence ◽

Structural Diversity ◽

Industrial Applications ◽

Glycoside Hydrolases ◽

Substrate Binding Site

Abstract Background Endo-β-1,4-galactanases are glycoside hydrolases (GH) from the GH53 family belonging to the largest clan of GHs, clan GH-A. GHs are ubiquitous and involved in a myriad of biological functions as well as being widely used industrially. Endo-β-1,4-galactanases, in particular hydrolyse galactan and arabinogalactan in pectin, a major component of the primary plant cell wall, with important functions in plant defence and application in the food and other industries. Here, we explore the family’s biological diversity by characterizing the first archaeal and hyperthermophilic GH53 galactanase, and utilize it as a scaffold for engineering enzymes with different product lengths. Results A galactanase gene was identified in the genome of the anaerobic hyperthermophilic archaeon Ignisphaera aggregans, and the isolated catalytic domain expressed and characterized (IaGal). IaGal presents the typical (βα)8 barrel structure of clan GH-A enzymes, with catalytic carboxylates at the end of the 4th and 7th barrel strands. Its activity optimum of at least 95 °C and melting point over 100 °C indicate extreme thermostability, a very advantageous property for industrial applications. If enzyme depletion is reduced, so is the need for re-addition, and thus costs. The main stabilizing features of IaGal compared to other structurally characterized members are π–π and cation–π interactions. The length of the substrate binding site—and thus produced oligosaccharide products—is intermediate compared to previously characterized galactanases. Variants inspired by the structural diversity in the GH53 family were rationally designed to shorten or extend the substrate binding groove, in order to modulate product length. Subsite-deleted variants produced shorter products than IaGal, as do the fungal galactanases inspiring the design. IaGal variants engineered with a longer binding site produced a less expected degradation pattern, though still different from that of wild-type IaGal. All variants remained extremely stable. Conclusions We have characterized in detail the most thermophilic endo-β-1,4-galactanase known to date and successfully engineered it to modify the degradation profile, while maintaining much of its desirable thermostability. This is an important achievement as oligosaccharide products length is an important property for industrial and natural GHs alike.

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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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Effects of Substrate-Binding Site Residues on the Biochemical Properties of a Tau Class Glutathione S-Transferase from Oryza sativa

Genes ◽

10.3390/genes11010025 ◽

2019 ◽

Vol 11 (1) ◽

pp. 25 ◽

Cited By ~ 2

Author(s):

Xue Yang ◽

Jinchi Wei ◽

Zhihai Wu ◽

Jie Gao

Keyword(s):

Amino Acids ◽

Enzyme Activity ◽

Abiotic Stress ◽

Binding Site ◽

Stress Responses ◽

Stress Resistance ◽

Substrate Binding ◽

Biochemical Properties ◽

Biotic And Abiotic Stress ◽

Substrate Binding Site

Glutathione S-transferases (GSTs)—an especially plant-specific tau class of GSTs—are key enzymes involved in biotic and abiotic stress responses. To improve the stress resistance of crops via the genetic modification of GSTs, we predicted the amino acids present in the GSH binding site (G-site) and hydrophobic substrate-binding site (H-site) of OsGSTU17, a tau class GST in rice. We then examined the enzyme activity, substrate specificity, enzyme kinetics and thermodynamic stability of the mutant enzymes. Our results showed that the hydrogen bonds between Lys42, Val56, Glu68, and Ser69 of the G-site and glutathione were essential for enzyme activity and thermal stability. The hydrophobic side chains of amino acids of the H-site contributed to enzyme activity toward 4-nitrobenzyl chloride but had an inhibitory effect on enzyme activity toward 1-chloro-2,4-dinitrobenzene and cumene hydroperoxide. Different amino acids of the H-site had different effects on enzyme activity toward a different substrate, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole. Moreover, Leu112 and Phe162 were found to inhibit the catalytic efficiency of OsGSTU17 to 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, while Pro16, Leu112, and Trp165 contributed to structural stability. The results of this research enhance the understanding of the relationship between the structure and function of tau class GSTs to improve the abiotic stress resistance of crops.

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