Engineering of ωTransaminase for Effective Production of Chiral Amines

2020 ◽  
Vol 17 (6) ◽  
pp. 2827-2832
Author(s):  
Mitra Mirzaei ◽  
Per Berglund
Keyword(s):  
Active Site ◽  
Rational Design ◽  
Enzyme Stability ◽  
Building Blocks ◽  
Site Directed Mutagenesis ◽  
Saturation Mutagenesis ◽  
Rational Approach ◽  
Chiral Amines ◽  
Enzyme Specificity ◽  
Screening Assay

ωTransaminases are pyridoxal-5-phosphat (PLP) dependent enzymes having the ability to catalyze the transference of an amino group to a keto compound. These enzymes are used for production of chiral amines which are important building blocks in pharmaceutical industry. There is often a need to improve enzyme properties such as enzyme stability, enzyme specificity and to decrease substrate-product inhibition. Here, protein engineering was applied to improve the enzyme activity of the enzyme from Chromobacterium violaceum Rational-design and site-directed mutagenesis were applied on position of (W60) in the active site of the enzyme. Different mutated enzyme variants such as W60H, W60F and W60Y were made. Also, the enantiopreference of the wild type enzyme was reversed to produce (R)-chiral amines. For this aim, a screening assay was followed by semi-rational approach and saturation mutagenesis in the active site of the enzyme. Creating the mutated enzyme libraries resulted to obtaining two enzyme variants. Their properties were low enantiopreference towards formations of (R)-enantiopreference and low specific constant ratio between fast and slow enantiomers (Evalue around one).

scholarly journals Dissecting the evolvability landscape of the CalB active site toward aromatic substrates

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Yossef López de los Santos ◽  
Ying Lian Chew-Fajardo ◽  
Guillaume Brault ◽  
Nicolas Doucet
Keyword(s):  
Active Site ◽  
Rational Design ◽  
Interaction Network ◽  
Saturation Mutagenesis ◽  
Rational Approach ◽  
Library Size ◽  
Aromatic Substrates ◽  
Enzyme Substrate ◽  
Residue Interaction ◽  
Enzyme Recognition

Abstract A key event in the directed evolution of enzymes is the systematic use of mutagenesis and selection, a process that can give rise to mutant libraries containing millions of protein variants. To this day, the functional analysis and identification of active variants among such high numbers of mutational possibilities is not a trivial task. Here, we describe a combinatorial semi-rational approach to partly overcome this challenge and help design smaller and smarter mutant libraries. By adapting a liquid medium transesterification assay in organic solvent conditions with a combination of virtual docking, iterative saturation mutagenesis, and residue interaction network (RIN) analysis, we engineered lipase B from P. antarctica (CalB) to improve enzyme recognition and activity against the bulky aromatic substrates and flavoring agents methyl cinnamate and methyl salicylate. Substrate-imprinted docking was used to target active-site positions involved in enzyme-substrate and enzyme-product complexes, in addition to identifying ‘hot spots’ most likely to yield active variants. This iterative semi-rational design strategy allowed selection of CalB variants exhibiting increased activity in just two rounds of site-saturation mutagenesis. Beneficial replacements were observed by screening only 0.308% of the theoretical library size, illustrating how semi-rational approaches with targeted diversity can quickly facilitate the discovery of improved activity variants relevant to a number of biotechnological applications.

How carbonyl reductases control stereoselectivity: Approaching the goal of rational design

2010 ◽  
Vol 82 (1) ◽  
pp. 117-128 ◽  
Author(s):  
Dunming Zhu ◽  
Ling Hua
Keyword(s):  
Rational Design ◽  
Reaction Medium ◽  
Site Directed Mutagenesis ◽  
Saturation Mutagenesis ◽  
Rational Approach ◽  
Reaction Conditions ◽  
In Silico Docking ◽  
Enzyme Substrate ◽  
Hydrophobic Binding ◽  
Binding Pockets

Although "Prelog’s rule" and "two hydrophobic binding pockets" model have been used to predict and explain the stereoselectivity of enzymatic ketone reduction, the molecular basis of stereorecognition by carbonyl reductases has not been well understood. The stereoselectivity is not only determined by the structures of enzymes and substrates, but also affected by the reaction conditions such as temperature and reaction medium. Structural analysis coupled with site-directed mutagenesis of stereocomplementary carbonyl reductases readily reveals the key elements of controlling stereoselectivity in these enzymes. In our studies, enzyme-substrate docking and molecular modeling have been engaged to understand the enantioselectivity diversity of the carbonyl reductase from Sporobolomyces salmonicolor (SSCR), and to guide site-saturation mutagenesis for altering the enantioselectivity of this enzyme. These studies provide valuable information for our understanding of how the residues involved in substrate binding affect the orientation of bound substrate, and thus control the reaction stereoselectivity. The in silico docking-guided semi-rational approach should be a useful methodology for discovery of new carbonyl reductases.

scholarly journals Main Structural Targets for Engineering Lipase Substrate Specificity

Catalysts ◽  
2020 ◽  
Vol 10 (7) ◽  
pp. 747
Author(s):  
Samah Hashim Albayati ◽  
Malihe Masomian ◽  
Siti Nor Hasmah Ishak ◽  
Mohd Shukuri bin Mohamad Ali ◽  
Adam Leow Thean ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Rational Design ◽  
Molecular Mechanisms ◽  
Experimental Studies ◽  
Random Mutagenesis ◽  
Binding Pocket ◽  
Amino Acid Sequences ◽  
Site Directed Mutagenesis ◽  
Saturation Mutagenesis ◽  
Process Conditions

Microbial lipases represent one of the most important groups of biotechnological biocatalysts. However, the high-level production of lipases requires an understanding of the molecular mechanisms of gene expression, folding, and secretion processes. Stable, selective, and productive lipase is essential for modern chemical industries, as most lipases cannot work in different process conditions. However, the screening and isolation of a new lipase with desired and specific properties would be time consuming, and costly, so researchers typically modify an available lipase with a certain potential for minimizing cost. Improving enzyme properties is associated with altering the enzymatic structure by changing one or several amino acids in the protein sequence. This review detailed the main sources, classification, structural properties, and mutagenic approaches, such as rational design (site direct mutagenesis, iterative saturation mutagenesis) and direct evolution (error prone PCR, DNA shuffling), for achieving modification goals. Here, both techniques were reviewed, with different results for lipase engineering, with a particular focus on improving or changing lipase specificity. Changing the amino acid sequences of the binding pocket or lid region of the lipase led to remarkable enzyme substrate specificity and enantioselectivity improvement. Site-directed mutagenesis is one of the appropriate methods to alter the enzyme sequence, as compared to random mutagenesis, such as error-prone PCR. This contribution has summarized and evaluated several experimental studies on modifying the substrate specificity of lipases.

Crystallographic analysis of 1,2,3-trichloropropane biodegradation by the haloalkane dehalogenase DhaA31

2014 ◽  
Vol 70 (2) ◽  
pp. 209-217 ◽  
Author(s):  
Maryna Lahoda ◽  
Jeroen R. Mesters ◽  
Alena Stsiapanava ◽  
Radka Chaloupkova ◽  
Michal Kuty ◽  
...  
Keyword(s):  
Active Site ◽  
Rational Design ◽  
Chloride Ions ◽  
Saturation Mutagenesis ◽  
Hydrolytic Cleavage ◽  
Wild Type ◽  
Haloalkane Dehalogenase ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Resolution Structure

Haloalkane dehalogenases catalyze the hydrolytic cleavage of carbon–halogen bonds, which is a key step in the aerobic mineralization of many environmental pollutants. One important pollutant is the toxic and anthropogenic compound 1,2,3-trichloropropane (TCP). Rational design was combined with saturation mutagenesis to obtain the haloalkane dehalogenase variant DhaA31, which displays an increased catalytic activity towards TCP. Here, the 1.31 Å resolution crystal structure of substrate-free DhaA31, the 1.26 Å resolution structure of DhaA31 in complex with TCP and the 1.95 Å resolution structure of wild-type DhaA are reported. Crystals of the enzyme–substrate complex were successfully obtained by adding volatile TCP to the reservoir after crystallization at pH 6.5 and room temperature. Comparison of the substrate-free structure with that of the DhaA31 enzyme–substrate complex reveals that the nucleophilic Asp106 changes its conformation from an inactive to an active state during the catalytic cycle. The positions of three chloride ions found inside the active site of the enzyme indicate a possible pathway for halide release from the active site through the main tunnel. Comparison of the DhaA31 variant with wild-type DhaA revealed that the introduced substitutions reduce the volume and the solvent-accessibility of the active-site pocket.

scholarly journals Role of the conserved amino acids of the ‘SDN’ loop (Ser130, Asp131 and Asn132) in a class A β-lactamase studied by site-directed mutagenesis

1990 ◽  
Vol 271 (2) ◽  
pp. 399-406 ◽  
Author(s):  
F Jacob ◽  
B Joris ◽  
S Lepage ◽  
J Dusart ◽  
J M Frère
Keyword(s):  
Active Site ◽  
Enzyme Stability ◽  
Enzymic Activity ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Mutant Proteins ◽  
Bond Forming ◽  
Class A ◽  
Active Site Cavity

Ser130, Asp131 and Asn132 (‘SDN’) are highly conserved residues in class A beta-lactamases forming one wall of the active-site cavity. All three residues of the SDN loop in Streptomyces albus G beta-lactamase were modified by site-directed mutagenesis. The mutant proteins were expressed in Streptomyces lividans, purified from culture supernatants and their kinetic parameters were determined for several substrates. Ser130 was substituted by Asn, Ala and Gly. The first modification yielded an almost totally inactive protein, whereas the smaller-side-chain mutants (A and G) retained some activity, but were less stable than the wild-type enzyme. Ser130 might thus be involved in maintaining the structure of the active-site cavity. Mutations of Asp131 into Glu and Gly proved to be highly detrimental to enzyme stability, reflecting significant structural perturbations. Mutation of Asn132 into Ala resulted in a dramatically decreased enzymic activity (more than 100-fold) especially toward cephalosporin substrates, kcat. being the most affected parameter, which would indicate a role of Asn132 in transition-state stabilization rather than in ground-state binding. Comparison of the N132A and the previously described N132S mutant enzymes underline the importance of an H-bond-forming residue at position 132 for the catalytic process.

Reengineering of subtilisin Carlsberg for oxidative resistance

2013 ◽  
Vol 394 (1) ◽  
pp. 79-87 ◽  
Author(s):  
Ljubica Vojcic ◽  
Dragana Despotovic ◽  
Karl-Heinz Maurer ◽  
Martin Zacharias ◽  
Marco Bocola ◽  
...  
Keyword(s):  
Active Site ◽  
Enzyme Stability ◽  
Saturation Mutagenesis ◽  
Wild Type ◽  
Subtilisin Carlsberg ◽  
Oxidative Resistance ◽  
Production Of Hydrogen ◽  
Chemical Oxidants ◽  
Current Article

Abstract Mild bleaching conditions by in situ production of hydrogen peroxide or peroxycarboxylic acid is attractive for pulp, textile, and cosmetics industries. The enzymatic generation of chemical oxidants is often limited by enzyme stability. The subtilisin Carlsberg variant T58A/L216W/M221 is a promiscuous protease that was improved in producing peroxycarboxylic acids. In the current article, we identified two amino acid positions (Trp216 and Met221) that are important for the oxidative resistance of subtilisin Carlsberg T58A/L216W/M221. Site-saturation mutagenesis at positions Trp216 and Met221, which are located close to the active site, resulted in variants M4 (T58/W216M/M221) and M6 (T58A/W216L/M221C). Variants M4 (T58/W216M/M221) and M6 (T58A/W216L/M221C) have a 2.6-fold (M4) and 1.5-fold (M6) increased oxidative resistance and 1.4-fold increased kcat values for peroxycarboxylic acid formation, compared with wild-type subtilisin Carlsberg.

scholarly journals Hydroxylation of Steroids by a Microbial Substrate-Promiscuous P450 Cytochrome (CYP105D7): Key Arginine Residues for Rational Design

2019 ◽  
Vol 85 (23) ◽  
Author(s):  
Bingbing Ma ◽  
Qianwen Wang ◽  
Haruo Ikeda ◽  
Chunfang Zhang ◽  
Lian-Hua Xu
Keyword(s):  
Active Site ◽  
Rational Design ◽  
Molecular Mechanisms ◽  
Fold Increase ◽  
Site Directed Mutagenesis ◽  
Cytochrome P450 Monooxygenases ◽  
Content Type ◽  
Conversion Rates ◽  
Arginine Residues

ABSTRACT Our previous study showed that CYP105D7, a substrate-promiscuous P450, catalyzes the hydroxylation of 1-deoxypentalenic acid, diclofenac, naringenin, and compactin. In this study, 14 steroid compounds were screened using recombinant Escherichia coli cells harboring genes encoding CYP105D7 and redox partners (Pdx/Pdr, RhFRED, and FdxH/FprD), and the screening identified steroid A-ring 2β- and D-ring 16β-hydroxylation activity. Wild-type CYP105D7 was able to catalyze the hydroxylation of five steroids (testosterone, progesterone, 4-androstene-3,17-dione, adrenosterone, and cortisone) with low (<10%) conversion rates. Structure-guided site-directed mutagenesis of arginine residues around the substrate entrance and active site showed that the R70A and R190A single mutants and an R70A/R190A double mutant exhibited greatly enhanced conversion rates for steroid hydroxylation. For the conversion of testosterone in particular, the R70A/R190A mutant's kcat/Km values increased 1.35-fold and the in vivo conversion rates increased significantly by almost 9-fold with high regio- and stereoselectivity. Molecular docking analysis revealed that when Arg70 and Arg190 were replaced with alanine, the volume of the substrate access and binding pocket increased 1.08-fold, which might facilitate improvement of the hydroxylation efficiency of steroids. IMPORTANCE Cytochrome P450 monooxygenases (P450s) are able to introduce oxygen atoms into nonreactive hydrocarbon compounds under mild conditions, thereby offering significant advantages compared to chemical catalysts. Promiscuous P450s with broad substrate specificity and reaction diversity have significant potential for applications in various fields, including synthetic biology. The study of the function, molecular mechanisms, and rational engineering of substrate-promiscuous P450s from microbial sources is important to fulfill this potential. Here, we present a microbial substrate-promiscuous P450, CYP105D7, which can catalyze hydroxylation of steroids. The loss of the bulky side chains of Arg70 and Arg190 in the active site and substrate entrance resulted in an up to 9-fold increase in the substrate conversion rate. These findings will support future rational and semirational engineering of P450s for applications as biocatalysts.

Unprecedented Role of The N73-F124 Pair in The Staphylococcus equorum MnSOD Activity

2020 ◽  
Vol 16 ◽  
Author(s):  
Debbie Soefie Retnoningrum ◽  
Hiromi Yoshida ◽  
Muthia Dzaky Razani ◽  
Vincencius Felix Meidianto ◽  
Andrian Hartanto ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Active Site ◽  
Manganese Superoxide Dismutase ◽  
Enzyme Stability ◽  
The Other ◽  
Site Directed Mutagenesis ◽  
X Ray Crystallography ◽  
Staphylococcus Equorum ◽  
The Stability

Background:: Bacterial manganese superoxide dismutase (MnSOD) occurs as a dimer, which is responsible for its activity and stability. Thereby, increasing the dimeric strength would increase the enzyme stability while maintaining its activity. Objective:: An N73F substitution was introduced to strengthen interactions between the monomers at the dimer interface. This substitution would introduce a π-stacking interaction between F73 of one monomer to F124 from the other monomer. Method:: Site-directed mutagenesis was carried out to substitute N73 with phenylalanine. The activity of the mutant was qualitative- and quantitatively checked while the stability was evaluated with a fluorescence-based thermal-shift assay. Finally, the structure of the mutant was elucidated by means of X-ray crystallography. Results:: The N73F mutant activity was only ~40% of the wildtype. The N73F mutant showed one TM at 60+1oC while the wildtype has two (at 52-55oC and 63-67oC). The crystal structure of the mutant showed the interactions between F73 from one monomer to F124 from the other monomer. The N73F structure presents an enigma because of no change in the enzyme structure including the active site. Furthermore, N73 and F124 position and interaction are conserved in human MnSOD but with a different location in the amino acid sequence. N73 has a role in the enzyme activity that is likely related to its interaction with F124, which resides in the active site region but has not been considered to participate in the reaction. Conclusion:: The N73F substitution has revealed the unprecedented role of the N73-F124 pair in the enzyme activity.

scholarly journals A single point mutation converts a glutaryl-7-aminocephalosporanic acid acylase into an N-acyl-homoserine lactone acylase

2021 ◽  
Author(s):  
Shereen A. Murugayah ◽  
Gary B. Evans ◽  
Joel D. A. Tyndall ◽  
Monica L. Gerth
Keyword(s):  
Single Point ◽  
Quorum Quenching ◽  
Homoserine Lactone ◽  
Site Directed Mutagenesis ◽  
Saturation Mutagenesis ◽  
Single Amino Acid ◽  
Single Point Mutation ◽  
Acyl Homoserine Lactone ◽  
Signalling Molecules ◽  
Single Amino Acid Residue

Abstract Objective To change the specificity of a glutaryl-7-aminocephalosporanic acid acylase (GCA) towards N-acyl homoserine lactones (AHLs; quorum sensing signalling molecules) by site-directed mutagenesis. Results Seven residues were identified by analysis of existing crystal structures as potential determinants of substrate specificity. Site-saturation mutagenesis libraries were created for each of the seven selected positions. High-throughput activity screening of each library identified two variants—Arg255Ala, Arg255Gly—with new activities towards N-acyl homoserine lactone substrates. Structural modelling of the Arg255Gly mutation suggests that the smaller side-chain of glycine (as compared to arginine in the wild-type enzyme) avoids a key clash with the acyl group of the N-acyl homoserine lactone substrate. Conclusions Mutation of a single amino acid residue successfully converted a GCA (with no detectable activity against AHLs) into an AHL acylase. This approach may be useful for further engineering of ‘quorum quenching’ enzymes.

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