Enhancement in catalytic activity of Aspergillus niger XynB by selective site-directed mutagenesis of active site amino acids

2017 ◽  
Vol 102 (1) ◽  
pp. 249-260 ◽  
Author(s):  
Xiuyun Wu ◽  
Zhennan Tian ◽  
Xukai Jiang ◽  
Qun Zhang ◽  
Lushan Wang
Keyword(s):  
Amino Acids ◽  
Catalytic Activity ◽  
Aspergillus Niger ◽  
Active Site ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis

scholarly journals The mechanism of action of dd-peptidases: the role of Threonine-299 and -301 in the Streptomyces R61 dd-peptidase

1994 ◽  
Vol 301 (2) ◽  
pp. 477-483 ◽  
Author(s):  
J M Wilkin ◽  
A Dubus ◽  
B Joris ◽  
J M Frère
Keyword(s):  
Amino Acids ◽  
Active Site ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Peptide Substrate ◽  
Binding Properties ◽  
Beta Lactamases ◽  
Penicillin Binding Proteins ◽  
Active Site Cavity

The side chains of residues Thr299 and Thr301 in the Streptomyces R61 DD-peptidase have been modified by site-directed mutagenesis. These amino acids are part of a beta-strand which forms a wall of the active-site cavity. Thr299 corresponds to the second residue of the Lys-Thr(Ser)-Gly triad, highly conserved in active-site beta-lactamases and penicillin-binding proteins (PBPs). Modification of Thr301 resulted only in minor alterations of the catalytic and penicillin-binding properties of the enzyme. No selective decrease of the rate of acylation was observed for any particular class of compounds. By contrast, the loss of the hydroxy group of the residue in position 299 yielded a seriously impaired enzyme. The rates of inactivation by penicillins were decreased 30-50-fold, whereas the reactions with cephalosporins were even more affected. The efficiency of hydrolysis against the peptide substrate was also seriously decreased. More surprisingly, the mutant was completely unable to catalyse transpeptidation reactions. The conservation of an hydroxylated residue in this position in PBPs is thus easily explained by these results.

Site-Directed Mutagenesis of Firefly Luciferase Active Site Amino Acids:  A Proposed Model for Bioluminescence Color†

Biochemistry ◽  
1999 ◽  
Vol 38 (40) ◽  
pp. 13223-13230 ◽  
Author(s):  
Bruce R. Branchini ◽  
Rachelle A. Magyar ◽  
Martha H. Murtiashaw ◽  
Shannon M. Anderson ◽  
Lisa C. Helgerson ◽  
...  
Keyword(s):  
Amino Acids ◽  
Active Site ◽  
Firefly Luciferase ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Proposed Model

Roles of Amino Acids in theEscherichia coliOctaprenyl Diphosphate Synthase Active Site Probed by Structure-Guided Site-Directed Mutagenesis

Biochemistry ◽  
2012 ◽  
Vol 51 (16) ◽  
pp. 3412-3419 ◽  
Author(s):  
Keng-Ming Chang ◽  
Shih-Hsun Chen ◽  
Chih-Jung Kuo ◽  
Chi-Kang Chang ◽  
Rey-Ting Guo ◽  
...  
Keyword(s):  
Amino Acids ◽  
Active Site ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis

scholarly journals Simultaneous Enhancement of Thermostability and Catalytic Activity of a Metagenome-Derived β-Glucosidase Using Directed Evolution for the Biosynthesis of Butyl Glucoside

2019 ◽  
Vol 20 (24) ◽  
pp. 6224 ◽  
Author(s):  
Bangqiao Yin ◽  
Qinyan Hui ◽  
Muhammad Kashif ◽  
Ran Yu ◽  
Si Chen ◽  
...  
Keyword(s):  
Amino Acids ◽  
Catalytic Activity ◽  
Directed Evolution ◽  
Catalytic Efficiency ◽  
Raw Material ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Substitutions ◽  
The Novel ◽  
Wild Type

Butyl glucoside synthesis using bioenzymatic methods at high temperatures has gained increasing interest. Protein engineering using directed evolution of a metagenome-derived β-glucosidase of Bgl1D was performed to identify enzymes with improved activity and thermostability. An interesting mutant Bgl1D187 protein containing five amino acid substitutions (S28T, Y37H, D44E, R91G, and L115N), showed catalytic efficiency (kcat/Km of 561.72 mM−1 s−1) toward ρ-nitrophenyl-β-d-glucopyranoside (ρNPG) that increased by 23-fold, half-life of inactivation by 10-fold, and further retained transglycosidation activity at 50 °C as compared with the wild-type Bgl1D protein. Site-directed mutagenesis also revealed that Asp44 residue was essential to β-glucosidase activity of Bgl1D. This study improved our understanding of the key amino acids of the novel β-glucosidases and presented a raw material with enhanced catalytic activity and thermostability for the synthesis of butyl glucosides.

scholarly journals Targeting active site residues and structural anchoring positions in terpene synthases

2021 ◽  
Vol 17 ◽  
pp. 2441-2449
Author(s):  
Anwei Hou ◽  
Jeroen S Dickschat
Keyword(s):  
Catalytic Activity ◽  
Active Site ◽  
Catalytic Efficiency ◽  
Site Directed Mutagenesis ◽  
Enzyme Function ◽  
Directed Mutagenesis ◽  
Active Site Residues ◽  
Terpene Synthases ◽  
Diterpene Synthase ◽  
Streptomyces Mobaraensis

The sesterterpene synthase SmTS1 from Streptomyces mobaraensis contains several unusual residues in positions that are otherwise highly conserved. Site-directed mutagenesis experiments for these residues are reported that showed different effects, resulting in some cases in an improved catalytic activity, but in other cases in a loss of enzyme function. For other enzyme variants a functional switch was observed, turning SmTS1 from a sesterterpene into a diterpene synthase. This article gives rational explanations for these findings that may generally allow for protein engineering of other terpene synthases to improve their catalytic efficiency or to change their functions.

Site-Directed Mutagenesis of Amino Acids Associated with the Active Site of Rubisco from Anacystis Nidulans

1990 ◽  
pp. 2257-2260
Author(s):  
M. A. Parry ◽  
C. A. Kettleborough ◽  
N. Halford ◽  
A. L. Phillips ◽  
R. Branden ◽  
...  
Keyword(s):  
Amino Acids ◽  
Active Site ◽  
Anacystis Nidulans ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis

scholarly journals Asymmetry in catalysis: ‘unidirectional’ amino acid racemases

2021 ◽  
Vol 43 (1) ◽  
pp. 28-34
Author(s):  
Stephen L. Bearne
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Active Site ◽  
Site Directed Mutagenesis ◽  
Hydroxyl Group ◽  
Directed Mutagenesis ◽  
Biotechnological Applications ◽  
Enzyme Substrate ◽  
Brønsted Base ◽  
Catalytic Dyad

d-Amino acids play widespread structural, functional and regulatory roles in organisms. These d-amino acids often arise through the stereoinversion of the more plentiful l-amino acids catalysed by amino acid racemases and epimerases. Such enzymes are of interest since many are recognized targets for the development of drugs or may be employed industrially in biotransformation reactions. Despite their enzyme–substrate complexes being diastereomers, some racemases and epimerases exhibit a kinetic pseudo-symmetry, binding their enantiomeric or epimeric substrate pairs with roughly equal affinities and catalyzing their stereoinversion with similar turnover numbers. In other cases, this kinetic pseudo-symmetry is absent or may be ‘broken’ by substitution of a catalytic Cys by Ser at the active site of cofactor-independent racemases and epimerases, or by altering the Brønsted base of the catalytic dyad that facilitates deprotonation of the Cys residue. Moreover, a natural Thr-containing l-Asp/Glu racemase was discovered that catalyses ‘unidirectional’ substrate turnover, unlike the typical bidirectional racemases and epimerases. These observations suggest that bidirectional Cys–Cys racemases may be re-engineered into ‘unidirectional’ racemases through substitution of the thiol by a hydroxyl group. Catalysis by such ‘unidirectional’ racemase precursors could then be optimized further by site-directed mutagenesis and directed evolution to furnish useful enzymes for biotechnological applications.

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