Degradation and Adsorption Characteristics of PHB Depolymerase As Revealed by Kinetics of Mutant Enzymes with Amino Acid Substitution in Substrate-Binding Domain

2010 ◽  
Vol 11 (1) ◽  
pp. 113-119 ◽  
Author(s):  
Tomohiro Hiraishi ◽  
Naoya Komiya ◽  
Nobuhiko Matsumoto ◽  
Hideki Abe ◽  
Masahiro Fujita ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Substitution ◽  
Substrate Binding ◽  
Binding Domain ◽  
Phb Depolymerase ◽  
Adsorption Characteristics ◽  
Kinetics Of ◽  
Substrate Binding Domain

scholarly journals Effects of Mutations in the Substrate-Binding Domain of Poly[(R)-3-Hydroxybutyrate] (PHB) Depolymerase from Ralstonia pickettii T1 on PHB Degradation

2006 ◽  
Vol 72 (11) ◽  
pp. 7331-7338 ◽  
Author(s):  
Tomohiro Hiraishi ◽  
Yoko Hirahara ◽  
Yoshiharu Doi ◽  
Mizuo Maeda ◽  
Seiichi Taguchi
Keyword(s):  
High Frequency ◽  
High Throughput Screening ◽  
Substrate Binding ◽  
Random Mutagenesis ◽  
Hydrolytic Activity ◽  
Binding Domain ◽  
Hydroxyl Groups ◽  
Phb Depolymerase ◽  
Ralstonia Pickettii ◽  
Substrate Binding Domain

ABSTRACT Poly[(R)-3-hydroxybutyrate] (PHB) depolymerase from Ralstonia pickettii T1 (PhaZRpiT1) adsorbs to denatured PHB (dPHB) via its substrate-binding domain (SBD) to enhance dPHB degradation. To evaluate the amino acid residues participating in dPHB adsorption, PhaZRpiT1 was subjected to a high-throughput screening system consisting of PCR-mediated random mutagenesis targeted to the SBD gene and a plate assay to estimate the effects of mutations in the SBD on dPHB degradation by PhaZRpiT1. Genetic analysis of the isolated mutants with lowered activity showed that Ser, Tyr, Val, Ala, and Leu residues in the SBD were replaced by other residues at high frequency. Some of the mutant enzymes, which contained the residues replaced at high frequency, were applied to assays of dPHB degradation and adsorption, revealing that those residues are essential for full activity of both dPHB degradation and adsorption. These results suggested that PhaZRpiT1 adsorbs on the surface of dPHB not only via hydrogen bonds between hydroxyl groups of Ser in the enzyme and carbonyl groups in the PHB polymer but also via hydrophobic interaction between hydrophobic residues in the enzyme and methyl groups in the PHB polymer. The L441H enzyme, which displayed lower dPHB degradation and adsorption abilities, was purified and applied to a dPHB degradation assay to compare it with the wild-type enzyme. The kinetic analysis of the dPHB degradation suggested that lowering the affinity of the SBD towards dPHB causes a decrease in the dPHB degradation rate without the loss of its hydrolytic activity for the polymer chain.

scholarly journals Sequence Analysis of Amplified DNA Fragments Containing the Region Encoding the Putative Lipase Substrate-Binding Domain and Genotyping of Aeromonas hydrophila

2004 ◽  
Vol 70 (1) ◽  
pp. 145-151 ◽  
Author(s):  
Noboru Watanabe ◽  
Koji Morita ◽  
Tomoko Furukawa ◽  
Taki Manzoku ◽  
Eiko Endo ◽  
...  
Keyword(s):  
Amino Acid ◽  
Nucleotide Sequence ◽  
Amino Acid Sequence ◽  
Aeromonas Hydrophila ◽  
Substrate Binding ◽  
Binding Domain ◽  
Dna Fragments ◽  
Environmental Isolates ◽  
Sequence Patterns ◽  
Substrate Binding Domain

ABSTRACT DNA fragments were amplified by PCR from all tested strains of Aeromonas hydrophila, A. caviae, and A. sobria with primers designed based on sequence alignment of all lipase, phospholipase C, and phospholipase A1 genes and the cytotonic enterotoxin gene, all of which have been reported to have the consensus region of the putative lipase substrate-binding domain. All strains showed lipase activity, and all amplified DNA fragments contained a nucleotide sequence corresponding to the substrate-binding domain. Thirty-five distinct nucleotide sequence patterns and 15 distinct deduced amino acid sequence patterns were found in the amplified DNA fragments from 59 A. hydrophila strains. The deduced amino acid sequences of the amplified DNA fragments from A. caviae and A. sobria strains had distinctive amino acids, suggesting a species-specific sequence in each organism. Furthermore, the amino acid sequence patterns appear to differ between clinical and environmental isolates among A. hydrophila strains. Some strains whose nucleotide sequences were identical to one another in the amplified region showed an identical DNA fingerprinting pattern by repetitive extragenic palindromic sequence-PCR genotyping. These results suggest that A. hydrophila, and also A. caviae and A. sobria strains, have a gene encoding a protein with lipase activity. Homologs of the gene appear to be widely distributed in Aeromonas strains, probably associating with the evolutionary genetic difference between clinical and environmental isolates of A. hydrophila. Additionally, the distinctive nucleotide sequences of the genes could be attributed to the genotype of each strain, suggesting that their analysis may be helpful in elucidating the genetic heterogeneity of Aeromonas.

scholarly journals Interdomain communication in the molecular chaperone DnaK

2003 ◽  
Vol 369 (3) ◽  
pp. 627-634 ◽  
Author(s):  
Wanjiang HAN ◽  
Philipp CHRISTEN
Keyword(s):  
Substrate Binding ◽  
Peptide Binding ◽  
Binding Domain ◽  
Intrinsic Fluorescence ◽  
Tryptophan Residue ◽  
Atpase Domain ◽  
Chaperone Dnak ◽  
The One ◽  
Kinetics Of ◽  
Substrate Binding Domain

DnaK, a heat-shock protein 70 (Hsp70) homologue in Escherichia coli, possesses a single tryptophan residue in its ATPase domain. Changes in the intrinsic fluorescence of DnaK offer a simple means not only to follow the binding of ATP and of ADP plus the co-chaperone GrpE to the ATPase domain, but also to investigate the kinetics of peptide binding to the substrate-binding domain of ATP·DnaK and GrpE-liganded ADP·DnaK. Addition of ATP or of ADP plus GrpE to nucleotide-free DnaK resulted in a similar decrease in intrinsic fluorescence, indicating similar open conformations of the ATPase domain under these two conditions. Binding of peptide increased the intrinsic fluorescence of both ATP·DnaK and ADP·DnaK·GrpE and rendered their spectra similar to the spectrum of ADP·DnaK with closed conformation of the ATPase domain. These results, together with the differential kinetics of peptide binding to ADP·DnaK on the one hand, and to ATP·DnaK or ADP·DnaK·GrpE on the other, suggest that ligands for either domain, i.e. ATP or ADP plus GrpE for the ATPase domain and peptides for the substrate-binding domain, shift the conformational equilibrium of both domains of DnaK towards the open and closed forms, respectively, in a concerted and parallel manner.

scholarly journals Entropic Inhibition: How the Activity of a AAA+ Machine Is Modulated by Its Substrate-Binding Domain

2021 ◽  
Author(s):  
Marija Iljina ◽  
Hisham Mazal ◽  
Pierre Goloubinoff ◽  
Inbal Riven ◽  
Gilad Haran
Keyword(s):  
Substrate Binding ◽  
Binding Domain ◽  
Substrate Binding Domain

scholarly journals Flexible Linkers Leash the Substrate Binding Domain of SspB to a Peptide Module that Stabilizes Delivery Complexes with the AAA+ ClpXP Protease

2003 ◽  
Vol 12 (2) ◽  
pp. 355-363 ◽  
Author(s):  
David A Wah ◽  
Igor Levchenko ◽  
Gabrielle E Rieckhof ◽  
Daniel N Bolon ◽  
Tania A Baker ◽  
...  
Keyword(s):  
Substrate Binding ◽  
Binding Domain ◽  
Substrate Binding Domain

scholarly journals An atomistic view of Hsp70 allosteric crosstalk: from the nucleotide to the substrate binding domain and back

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Federica Chiappori ◽  
Ivan Merelli ◽  
Luciano Milanesi ◽  
Giorgio Colombo ◽  
Giulia Morra
Keyword(s):  
Substrate Binding ◽  
Binding Domain ◽  
Substrate Binding Domain

Rotary and Unidirectional Metal Shadowing of VAT: Localization of the Substrate-Binding Domain

2000 ◽  
Vol 132 (2) ◽  
pp. 162-168 ◽  
Author(s):  
Beate Rockel ◽  
Reinhard Guckenberger ◽  
Heinz Gross ◽  
Peter Tittmann ◽  
Wolfgang Baumeister
Keyword(s):  
Substrate Binding ◽  
Binding Domain ◽  
Substrate Binding Domain

Studies on the effect of the J-domain on the substrate binding domain (SBD) of Hsp70 using a chimeric human J-SBD polypeptide

2019 ◽  
Vol 124 ◽  
pp. 111-120 ◽  
Author(s):  
Ana O. Tiroli-Cepeda ◽  
Thiago V. Seraphim ◽  
Glaucia M.S. Pinheiro ◽  
Denio E.P. Souto ◽  
Lauro T. Kubota ◽  
...  
Keyword(s):  
Substrate Binding ◽  
Binding Domain ◽  
J Domain ◽  
Substrate Binding Domain
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