scholarly journals Active-site- and substrate-specificity of Thermoanaerobium Tok6-B1 pullulanase

1987 ◽  
Vol 246 (2) ◽  
pp. 537-541 ◽  
Author(s):  
A R Plant ◽  
R M Clemens ◽  
H W Morgan ◽  
R M Daniel
Keyword(s):  
Kinetic Model ◽  
Substrate Specificity ◽  
Chain Length ◽  
Carboxy Group ◽  
Active Site ◽  
Product Formation ◽  
Inactivation Kinetics ◽  
Short Chain Length ◽  
Ph Dependent ◽  
Hydrolysis Of

Thermoanaerobium Tok6-B1 pullulanase (EC 3.2.1.41) was active on alpha 1-6-glucosidic linkages of pullulan, amylopectin and glycogen and the alpha 1-4 linkages of amylose, amylopectin and glycogen but not of pullulan. Hydrolysis of short-chain-length malto-oligosaccharides (seven or fewer glucose residues) yielded maltose as product. Pullulan hydrolysis was pH-dependent and a plot of log(V/Km) versus pH implied a carboxy group with pKa 4.3 at the active site. Modification with 1-(3-dimethylaminopropyl)-3-ethylcarbodi-imide (EDAC) confirmed this view, and analysis of the order of reaction and inactivation kinetics suggested the presence of a single carboxy group at a catalytic centre of the active site. EDAC-mediated inhibition of pullulan alpha 1-6-bond hydrolysis was relieved by amylose or pullulan. Similarly both pullulan and amylose protected the activity directed at alpha 1-4 bonds of amylose from EDAC inhibition. When both amylose and pullulan were simultaneously present, the observed rate of product formation closely fitted a kinetic model in which both substrates were hydrolysed at the same active site.

scholarly journals Altering the Substrate Specificity of Polyhydroxyalkanoate Synthase 1 Derived from Pseudomonas putida GPo1 by Localized Semirandom Mutagenesis

2004 ◽  
Vol 186 (13) ◽  
pp. 4177-4184 ◽  
Author(s):  
Der-Shyan Sheu ◽  
Chia-Yin Lee
Keyword(s):  
Substrate Specificity ◽  
Pseudomonas Putida ◽  
Chain Length ◽  
Ralstonia Eutropha ◽  
Pha Synthase ◽  
Multiple Point ◽  
Evolution System ◽  
Degenerate Primers ◽  
Short Chain Length ◽  
Single Polymer Chain

ABSTRACT The substrate specificity of polyhydroxyalkanoate (PHA) synthase 1 (PhaC1 Pp , class II) from Pseudomonas putida GPo1 (formerly known as Pseudomonas oleovorans GPo1) was successfully altered by localized semirandom mutagenesis. The enzyme evolution system introduces multiple point mutations, designed on the basis of the conserved regions of the PHA synthase family, by using PCR-based gene fragmentation with degenerate primers and a reassembly PCR. According to the opaqueness of the colony, indicating the accumulation of large amounts of PHA granules in the cells, 13 PHA-accumulating candidates were screened from a mutant library, with Pseudomonas putida GPp104 PHA− as the host. The in vivo substrate specificity of five candidates, L1-6, D7-47, PS-A2, PS-C2, and PS-E1, was evaluated by the heterologous expression in Ralstonia eutropha PHB−4 supplemented with octanoate. Notably, the amount of 3-hydroxybutyrate (short-chain-length [SCL] 3-hydroxyalkanoate [3-HA] unit) was drastically increased in recombinants that expressed evolved mutant enzymes L1-6, PS-A2, PS-C2, and PS-E1 (up to 60, 36, 50, and 49 mol%, respectively), relative to the amount in the wild type (12 mol%). Evolved enzyme PS-E1, in which 14 amino acids had been changed and which was heterologously expressed in R. eutropha PHB−4, not only exhibited broad substrate specificity (49 mol% SCL 3-HA and 51 mol% medium-chain-length [MCL] 3-HA) but also conferred the highest PHA production (45% dry weight) among the candidates. The 3-HA and MCL 3-HA units of the PHA produced by R. eutropha PHB−4/pPS-E1 were randomly copolymerized in a single polymer chain, as analytically confirmed by acetone fractionation and the 13C nuclear magnetic resonance spectrum.

Length of the active-site crossover loop defines the substrate specificity of ubiquitin C-terminal hydrolases for ubiquitin chains

2011 ◽  
Vol 441 (1) ◽  
pp. 143-149 ◽  
Author(s):  
Zi-Ren Zhou ◽  
Yu-Hang Zhang ◽  
Shuai Liu ◽  
Ai-Xin Song ◽  
Hong-Yu Hu
Keyword(s):  
Substrate Specificity ◽  
Active Site ◽  
Dependent Manner ◽  
Deubiquitinating Enzymes ◽  
Chain Flexibility ◽  
Isopeptide Bond ◽  
Short Loop ◽  
The One ◽  
Isopeptidase Activity ◽  
Hydrolysis Of

UCHs [Ub (ubiquitin) C-terminal hydrolases] are a family of deubiquitinating enzymes that are often thought to only remove small C-terminal peptide tails from Ub adducts. Among the four UCHs identified to date, neither UCH-L3 nor UCH-L1 can catalyse the hydrolysis of isopeptide Ub chains, but UCH-L5 can when it is present in the PA700 complex of the proteasome. In the present paper, we report that the UCH domain of UCH-L5, different from UCH-L1 and UCH-L3, by itself can process the K48-diUb (Lys48-linked di-ubiquitin) substrate by cleaving the isopeptide bond between two Ub units. The catalytic specificity of the four UCHs is dependent on the length of the active-site crossover loop. The UCH domain with a long crossover loop (usually >14 residues), such as that of UCH-L5 or BAP1 [BRCA1 (breast cancer early-onset 1)-associated protein 1], is able to cleave both small and large Ub derivatives, whereas the one with a short loop can only process small Ub derivatives. We also found that elongation of the crossover loop enables UCH-L1 to have isopeptidase activity for K48-diUb in a length-dependent manner. Thus the loop length of UCHs defines their substrate specificity for diUb chains, suggesting that the chain flexibility of the crossover loop plays an important role in determining its catalytic activity and substrate specificity for cleaving isopeptide Ub chains.

Substrate specificities of poly(hydroxyalkanoate)-degrading bacteria and active site studies on the extracellular poly(3-hydroxyoctanoic acid) depolymerase of Pseudomonas fluorescens GK13

1995 ◽  
Vol 41 (13) ◽  
pp. 170-179 ◽  
Author(s):  
Andreas Schirmer ◽  
Claudia Matz ◽  
Dieter Jendrossek
Keyword(s):  
Pseudomonas Fluorescens ◽  
Chain Length ◽  
Active Site ◽  
Consensus Sequence ◽  
Serine Hydrolases ◽  
Short Chain Length ◽  
Substrate Specificities ◽  
Medium Chain Length ◽  
Degrading Bacteria ◽  
The Family

The isolation of poly(3-hydroxyoctanoic acid)- and poly(6-hydroxyhexanoic acid)-degrading bacteria yielded 28 strains with abilities to degrade various polymers. The most versatile strains hydrolyzed five different polyesters comprising short chain length and medium chain length poly(hydroxyalkanoates). The new isolates together with previously isolated poly(hydroxyalkanoate)-degrading bacteria were classified into 11 groups with respect to their polymer-degrading specificities. All PHA depolymerases studied so far have been characterized by the lipase consensus sequence Gly-X-Ser-X-Gly in their amino acid sequence, which is a known sequence for serine hydrolases. When we replaced the central residue, Ser-172, in the corresponding sequence Gly-Ile-Ser-Ser-Gly of the extracellular poly(3-hydroxyoctanoic acid) depolymerase of Pseudomonas fluorescens GK13, with alanine the enzyme lost its activity completely. This result of the mutational experiment indicates that the poly(3-hydroxyoctanoic acid) depolymerase belongs to the family of serine hydrolases.Key words: poly(hydroxyalkanoates), PHA depolymerases, serine hydrolases, substrate specificity, Pseudomonas fluorescens.

scholarly journals Amino Acid Residues That Contribute to Substrate Specificity of Class A β-Lactamase SME-1

2005 ◽  
Vol 49 (8) ◽  
pp. 3421-3427 ◽  
Author(s):  
Fahd K. Majiduddin ◽  
Timothy Palzkill
Keyword(s):  
Substrate Specificity ◽  
Active Site ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Class A ◽  
Study Support ◽  
Functional Mutants ◽  
Hydrolysis Of ◽  
Traditional Class

ABSTRACT Carbapenem antibiotics are used as antibiotics of last resort because they possess a broad spectrum of antimicrobial activity and are not easily hydrolyzed by β-lactamases. Recently, class A enzymes, such as the SME-1, NMC-A, and IMI-1 β-lactamases, have been identified with the capacity to hydrolyze carbapenem antibiotics. Traditional class A β-lactamases, such as TEM-1 and SHV-1, are unable to hydrolyze carbapenem antibiotics and exhibit some differences in sequence from those that are able to hydrolyze carbapenem antibiotics. The positions that differ may contribute to the unique substrate specificity of the class A carbapenemase SME-1. Codons in the SME-1 gene representing residues 104, 105, 132, 167, 237, and 241 were randomized by site-directed mutagenesis, and functional mutants were selected for the ability to hydrolyze imipenem, ampicillin, or cefotaxime. Although several positions are important for hydrolysis of β-lactam antibiotics, no single position was found to uniquely contribute to carbapenem hydrolysis. The results of this study support a model whereby the carbapenemase activity of SME-1 is due to a highly distributed set of interactions that subtly alter the structure of the active-site pocket.

scholarly journals Processivity, Synergism, and Substrate Specificity of Thermobifida fusca Cel6B

2009 ◽  
Vol 75 (21) ◽  
pp. 6655-6661 ◽  
Author(s):  
Thu V. Vuong ◽  
David B. Wilson
Keyword(s):  
Substrate Specificity ◽  
Microcrystalline Cellulose ◽  
Filter Paper ◽  
Active Site ◽  
Carboxymethyl Cellulose ◽  
Inverse Relationship ◽  
Biomass Conversion ◽  
Thermobifida Fusca ◽  
Effective Strategy ◽  
Hydrolysis Of

ABSTRACT A relationship between processivity and synergism has not been reported for cellulases, although both characteristics are very important for hydrolysis of insoluble substrates. Mutation of two residues located in the active site tunnel of Thermobifida fusca exocellulase Cel6B increased processivity on filter paper. Surprisingly, mixtures of the Cel6B mutant enzymes and T. fusca endocellulase Cel5A did not show increased synergism or processivity, and the mutant enzyme which had the highest processivity gave the poorest synergism. This study suggests that improving exocellulase processivity might be not an effective strategy for producing improved cellulase mixtures for biomass conversion. The inverse relationship between the activities of many of the mutant enzymes with bacterial microcrystalline cellulose and their activities with carboxymethyl cellulose indicated that there are differences in the mechanisms of hydrolysis for these substrates, supporting the possibility of engineering Cel6B to target selected substrates.

scholarly journals Substrate specificity and kinetic properties of α-galactosidases from Vicia faba

1969 ◽  
Vol 115 (1) ◽  
pp. 47-54 ◽  
Author(s):  
P. M. Dey ◽  
J. B. Pridham
Keyword(s):  
Catalytic Activity ◽  
Substrate Specificity ◽  
Kinetic Parameters ◽  
Vicia Faba ◽  
Active Site ◽  
Effect Of Temperature ◽  
Kinetic Properties ◽  
Photo Oxidation ◽  
Hydrolysis Of ◽  
Enzyme I

1. The hydrolysis of a variety of galactosides and other glycosides by α-galactosidases I and II of Vicia faba was studied. 2. The effect of temperature on kinetic parameters was also examined. 3. Both enzymes are inhibited by excess of substrate (p-nitrophenyl α-d-galactoside); with enzyme I this is competitive and is caused by the galactosyl moiety. 4. Enzyme I is inhibited by oligosaccharides possessing terminal non-reducing galactose residues and to a smaller extent by l-arabinose and d-fucose. 5. The effect of pH on Km and Vmax. values suggests that carboxyl and imidazole groups are involved in the catalytic activity of enzyme I. 6. Photo-oxidation experiments with enzyme I also suggest that an imidazole group is present at the active site.

scholarly journals Role of PhaC Type I and Type II Enzymes during PHA Biosynthesis

2018 ◽  
Author(s):  
Valeria Mezzolla ◽  
Oscar Fernando D'Urso ◽  
Palmiro Poltronieri
Keyword(s):  
Chain Length ◽  
Chemical Properties ◽  
Product Formation ◽  
Class I ◽  
Type I ◽  
Class Iii ◽  
Short Chain Length ◽  
Medium Chain ◽  
Medium Chain Length

PHA synthases (PhaC) are grouped into four classes based on the kinetics and mechanisms of reaction. The grouping of PhaC enzymes into four classes is dependent on substrate specificity, according to the preference in forming short chain length (scl) or medium chain length (mcl) polymers: class I, class III, and class IV produce scl-PHAs depending on propionate, butyrate, valerate and hexanoate precursors, while class II phaC synthesize mcl-PHAs based on the alkane (C6 to C14) precursors. PHA synthases of class I, in particular PhaCCs from Chromobacterium USM2 and PhaCCn/RePhaC1 from Cupriavidus necator/R. eutropha, have been analysed and the crystal structures of the C-domains have been determined. PhaCCn/RePhaC1 was also studied by small angle X-ray scattering (SAXS) analysis. Models have been proposed for dimerization, catalysis mechanism, substrate recognition and affinity, product formation and product egress route. The assays based on amino acid substitution by mutagenesis have been useful to validate the hypothesis on the role of amino acids in catalysis and in accommodation of bulky substrates, for the synthesis of PHB co-polymers and medium chain length-PHA polymers with optimized chemical properties.

scholarly journals Control of Substrate Specificity by Active-Site Residues in Nitrobenzene Dioxygenase

2006 ◽  
Vol 72 (3) ◽  
pp. 1817-1824 ◽  
Author(s):  
Kou-San Ju ◽  
Rebecca E. Parales
Keyword(s):  
Hydrogen Bond ◽  
Substrate Specificity ◽  
Active Site ◽  
Product Formation ◽  
Oxidation Products ◽  
Site Directed Mutagenesis ◽  
Initial Reaction ◽  
Active Site Residues ◽  
Aromatic Rings ◽  
Α Subunit

ABSTRACT Nitrobenzene 1,2-dioxygenase from Comamonas sp. strain JS765 catalyzes the initial reaction in nitrobenzene degradation, forming catechol and nitrite. The enzyme also oxidizes the aromatic rings of mono- and dinitrotoluenes at the nitro-substituted carbon, but the basis for this specificity is not understood. In this study, site-directed mutagenesis was used to modify the active site of nitrobenzene dioxygenase, and the contribution of specific residues in controlling substrate specificity and enzyme performance was evaluated. The activities of six mutant enzymes indicated that the residues at positions 258, 293, and 350 in the α subunit are important for determining regiospecificity with nitroarene substrates and enantiospecificity with naphthalene. The results provide an explanation for the characteristic specificity with nitroarene substrates. Based on the structure of nitrobenzene dioxygenase, substitution of valine for the asparagine at position 258 should eliminate a hydrogen bond between the substrate nitro group and the amino group of asparagine. Up to 99% of the mononitrotoluene oxidation products formed by the N258V mutant were nitrobenzyl alcohols rather than catechols, supporting the importance of this hydrogen bond in positioning substrates in the active site for ring oxidation. Similar results were obtained with an I350F mutant, where the formation of the hydrogen bond appeared to be prevented by steric interference. The specificity of enzymes with substitutions at position 293 varied depending on the residue present. Compared to the wild type, the F293Q mutant was 2.5 times faster at oxidizing 2,6-dinitrotoluene while retaining a similar Km for the substrate based on product formation rates and whole-cell kinetics.

An active-site carboxyl group in liquefying α-amylase: specific chemical modification

1984 ◽  
Vol 4 (7) ◽  
pp. 613-619 ◽  
Author(s):  
Sunil Kochhar ◽  
R. D. Dua
Keyword(s):  
Chemical Modification ◽  
Active Site ◽  
Amylase Activity ◽  
Enzymic Activity ◽  
Complete Loss ◽  
Inactivation Kinetics ◽  
Carboxyl Group ◽  
Specific Chemical ◽  
Specific Chemical Modification ◽  
Ph Dependent

Bacillus amyloliquefaciens α-amylase activity is pH-dependent and the plot log (Vmax/Km) versus pH implicated a carboxyl group of aspartic acid/glutamic acid at the active site. Chemical modification of α-amylase with EDC confirmed this view. Further, analysis of inactivation kinetics showed that modification of a single carboxyl group led to complete loss of the enzymic activity.

scholarly journals Hydrolysis of rat chylomicron acylglycerols: a kinetic model

1981 ◽  
Vol 22 (3) ◽  
pp. 506-513 ◽  
Author(s):  
D M Foster ◽  
M Berman
Keyword(s):  
Kinetic Model ◽  
Hydrolysis Of
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