scholarly journals Key Residues for Controlling Enantioselectivity of Halohydrin Dehalogenase from Arthrobacter sp. Strain AD2, Revealed by Structure-Guided Directed Evolution

2012 ◽  
Vol 78 (8) ◽  
pp. 2631-2637 ◽  
Author(s):  
Lixia Tang ◽  
Xuechen Zhu ◽  
Huayu Zheng ◽  
Rongxiang Jiang ◽  
Maja Majerić Elenkov
Keyword(s):  
Active Site ◽  
Specific Activity ◽  
Saturation Mutagenesis ◽  
Active Site Residues ◽  
Agrobacterium Radiobacter ◽  
Content Type ◽  
Halohydrin Dehalogenase ◽  
Aromatic Substrates ◽  
Key Residues ◽  
Selection Of

ABSTRACTHalohydrin dehalogenase fromAgrobacterium radiobacterAD1 (HheC) is a valuable tool in the preparation ofRenantiomers of epoxides and β-substituted alcohols. In contrast, the halohydrin dehalogenase fromArthrobactersp. AD2 (HheA) shows a lowSenantioselectivity toward most aromatic substrates. Here, three amino acids (V136, L141, and N178) located in the two neighboring active-site loops of HheA were proposed to be the key residues for controlling enantioselectivity. They were subjected to saturation mutagenesis aimed at evolving anS-selective enzyme. This led to the selection of two outstanding mutants (the V136Y/L141G and N178A mutants). The double mutant displayed an inverted enantioselectivity (fromSenantioselectivity [ES] = 1.7 toRenantioselectivity [ER] = 13) toward 2-chloro-1-phenylethanol without compromising enzyme activity. Strikingly, the N178A mutant showed a large enantioselectivity improvement (ES> 200) and a 5- to 6-fold-enhanced specific activity toward (S)-2-chloro-1-phenylethanol. Further analysis revealed that those mutations produced some interference for the binding of nonfavored enantiomers which could account for the observed enantioselectivities. Our work demonstrated that those three active-site residues are indeed crucial in modulating the enantioselectivity of HheA and that a semirational design strategy has great potential for rapid creation of novel industrial biocatalysts.

scholarly journals Exploring the Enantioselective Mechanism of Halohydrin Dehalogenase from Agrobacterium radiobacter AD1 by Iterative Saturation Mutagenesis

2015 ◽  
Vol 81 (8) ◽  
pp. 2919-2926 ◽  
Author(s):  
Chao Guo ◽  
Yanpu Chen ◽  
Yu Zheng ◽  
Wei Zhang ◽  
Yunwen Tao ◽  
...  
Keyword(s):  
Catalytic Efficiency ◽  
Significant Loss ◽  
Saturation Mutagenesis ◽  
Active Site Residues ◽  
Agrobacterium Radiobacter ◽  
Content Type ◽  
Halohydrin Dehalogenase ◽  
Aromatic Substrates ◽  
Chiral Epoxides ◽  
Iterative Saturation Mutagenesis

ABSTRACTHalohydrin dehalogenase fromAgrobacterium radiobacterAD1 (HheC) shows great potential in producing valuable chiral epoxides and β-substituted alcohols. The wild-type (WT) enzyme displays a highR-enantiopreference toward most aromatic substrates, whereas noS-selective HheC has been reported to date. To obtain more enantioselective enzymes, seven noncatalytic active-site residues were subjected to iterative saturation mutagenesis (ISM). After two rounds of screening aspects of both activity and enantioselectivity (E), three outstanding mutants (Thr134Val/Leu142Met, Leu142Phe/Asn176His, and Pro84Val/Phe86Pro/Thr134Ala/Asn176Ala mutants) with divergent enantioselectivity were obtained. The two double mutants displayed approximately 2-fold improvement inR-enantioselectivity toward 2-chloro-1-phenylethanol (2-CPE) without a significant loss of enzyme activity compared with the WT enzyme. Strikingly, the Pro84Val/Phe86Pro/Thr134Ala/Asn176Ala mutant showed an inverted enantioselectivity (from anERof 65 [WT] to anESof 101) and approximately 100-fold-enhanced catalytic efficiency toward (S)-2-CPE. Molecular dynamic simulation and docking analysis revealed that the phenyl side chain of (S)-2-CPE bound at a different location than that of itsR-counterpart; those mutations generated extra connections for the binding of the favored enantiomer, while the eliminated connections reduced binding of the nonfavored enantiomer, all of which could contribute to the observed inverted enantiopreference.

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.

scholarly journals Regioisomerization of Antimalarial Drug WR99210 Explains the Inactivity of a Commercial Stock

2020 ◽  
Vol 65 (1) ◽  
pp. e01385-20
Author(s):  
T. Parks Remcho ◽  
Sravanthi D. Guggilapu ◽  
Phillip Cruz ◽  
Glenn A. Nardone ◽  
Gavin Heffernan ◽  
...  
Keyword(s):  
Active Site ◽  
Antimalarial Drug ◽  
High Performance ◽  
Drug Candidate ◽  
Resonance Spectroscopy ◽  
Content Type ◽  
Computational Docking ◽  
Layer Chromatography ◽  
Commercial Stock ◽  
Selection Of

ABSTRACTWR99210, a former antimalarial drug candidate now widely used for the selection of Plasmodium transfectants, selectively targets the parasite’s dihydrofolate reductase thymidine synthase bifunctional enzyme (DHFR-TS) but not human DHFR, which is not fused with TS. Accordingly, WR99210 and plasmids expressing the human dhfr gene have become valued tools for the genetic modification of parasites in the laboratory. Concerns over the ineffectiveness of WR99210 from some sources encouraged us to investigate the biological and chemical differences of supplies from two different companies (compounds 1 and 2). Compound 1 proved effective at low nanomolar concentrations against Plasmodium falciparum parasites, whereas compound 2 was ineffective, even at micromolar concentrations. Intact and fragmented mass spectra indicated identical molecular formulae of the unprotonated (free base) structures of compounds 1 and 2; however, the compounds displayed differences by thin-layer chromatography, reverse-phase high-performance liquid chromatography, and UV-visible spectroscopy, indicating important isomeric differences. Structural evaluations by 1H, 13C, and 15N nuclear magnetic resonance spectroscopy confirmed compound 1 as WR99210 and compound 2 as a dihydrotriazine regioisomer. Induced fit computational docking models showed that compound 1 binds tightly and specifically in the P. falciparum DHFR active site, whereas compound 2 fits poorly to the active site in loose and varied orientations. Stocks and concentrates of WR99210 should be monitored for the presence of regioisomer 2, particularly when they are not supplied as the hydrochloride salt or are exposed to basic conditions that may promote rearrangement. Absorption spectroscopy can serve for assays of the unrearranged and rearranged triazines.

scholarly journals Protein Engineering of Toluene-o-Xylene Monooxygenase from Pseudomonas stutzeri OX1 for Synthesizing 4-Methylresorcinol, Methylhydroquinone, and Pyrogallol

2004 ◽  
Vol 70 (6) ◽  
pp. 3253-3262 ◽  
Author(s):  
G�n�l Vardar ◽  
Thomas K. Wood
Keyword(s):  
Active Site ◽  
High Performance ◽  
Agar Plate ◽  
Pseudomonas Stutzeri ◽  
Saturation Mutagenesis ◽  
Dna Shuffling ◽  
Natural Substrate ◽  
Wild Type ◽  
Active Site Residues ◽  
Derivatives Of

ABSTRACT Toluene-o-xylene monooxygenase (ToMO) from Pseudomonas stutzeri OX1 oxidizes toluene to 3- and 4-methylcatechol and oxidizes benzene to form phenol; in this study ToMO was found to also form catechol and 1,2,3-trihydroxybenzene (1,2,3-THB) from phenol. To synthesize novel dihydroxy and trihydroxy derivatives of benzene and toluene, DNA shuffling of the alpha-hydroxylase fragment of ToMO (TouA) and saturation mutagenesis of the TouA active site residues I100, Q141, T201, and F205 were used to generate random mutants. The mutants were initially identified by screening with a rapid agar plate assay and then were examined further by high-performance liquid chromatography and gas chromatography. Several regiospecific mutants with high rates of activity were identified; for example, Escherichia coli TG1/pBS(Kan)ToMO expressing the F205G TouA saturation mutagenesis variant formed 4-methylresorcinol (0.78 nmol/min/mg of protein), 3-methylcatechol (0.25 nmol/min/mg of protein), and methylhydroquinone (0.088 nmol/min/mg of protein) from o-cresol, whereas wild-type ToMO formed only 3-methylcatechol (1.1 nmol/min/mg of protein). From o-cresol, the I100Q saturation mutagenesis mutant and the M180T/E284G DNA shuffling mutant formed methylhydroquinone (0.50 and 0.19 nmol/min/mg of protein, respectively) and 3-methylcatechol (0.49 and 1.5 nmol/min/mg of protein, respectively). The F205G mutant formed catechol (0.52 nmol/min/mg of protein), resorcinol (0.090 nmol/min/mg of protein), and hydroquinone (0.070 nmol/min/mg of protein) from phenol, whereas wild-type ToMO formed only catechol (1.5 nmol/min/mg of protein). Both the I100Q mutant and the M180T/E284G mutant formed hydroquinone (1.2 and 0.040 nmol/min/mg of protein, respectively) and catechol (0.28 and 2.0 nmol/min/mg of protein, respectively) from phenol. Dihydroxybenzenes were further oxidized to trihydroxybenzenes with different regiospecificities; for example, the I100Q mutant formed 1,2,4-THB from catechol, whereas wild-type ToMO formed 1,2,3-THB (pyrogallol). Regiospecific oxidation of the natural substrate toluene was also checked; for example, the I100Q mutant formed 22% o-cresol, 44% m-cresol, and 34% p-cresol, whereas wild-type ToMO formed 32% o-cresol, 21% m-cresol, and 47% p-cresol.

Active-site mutations impairing the catalytic function of the catalytic subunit of human protein phosphatase 2A permit baculovirus-mediated overexpression in insect cells

2001 ◽  
Vol 357 (1) ◽  
pp. 225-232 ◽  
Author(s):  
Timothy MYLES ◽  
Karsten SCHMIDT ◽  
David R. H. EVANS ◽  
Peter CRON ◽  
Brian A. HEMMINGS
Keyword(s):  
Active Site ◽  
Protein Phosphatase ◽  
Insect Cells ◽  
Specific Activity ◽  
Catalytic Subunit ◽  
Regulatory Control ◽  
Wild Type ◽  
Active Site Residues ◽  
Catalytic Function

Members of the phosphoprotein phosphatase (PPP) family of protein serine/threonine phosphatases, including protein phosphatase (PP)1, PP2A and PP2B, share invariant active-site residues that are critical for catalytic function [Zhuo, Clemens, Stone and Dixon (1994) J. Biol. Chem. 269, 26234–26238]. Mutation of the active-site residues Asp88 or His118 within the human PP2A catalytic subunit (PP2Ac)α impaired catalytic activity in vitro; the D88N and H118N substitutions caused a 9- and 23-fold reduction in specific activity respectively, when compared with wild-type recombinant PP2Ac, indicating an important role for these residues in catalysis. Consistent with this, the D88N and H118N substituted forms failed to provide PP2A function in vivo, because, unlike wild-type human PP2Acα, neither substituted for the endogenous PP2Ac enzyme of budding yeast. Relative to wild-type PP2Ac, the active-site mutants were dramatically overexpressed in High Five® insect cells using the baculovirus system. Milligram quantities of PP2Ac were purified from 1×109 High Five cells and the kinetic constants for dephosphorylation of the peptide RRA(pT)VA (single-letter amino-acid notation) by PP2Ac (Km = 337.5μM; kcat = 170s−1) and D88N (Km = 58.4μM; kcat = 2s−1) were determined. The results show that the substitution impairs catalysis severely without a significant effect on substrate binding, consistent with the PPP catalytic mechanism. Combination of the baculovirus and yeast systems provides a strategy whereby the structure–function of PP2Ac may be fully explored, a goal which has previously proven difficult, owing to the stringent auto-regulatory control of PP2Ac protein levels in vivo.

scholarly journals Structural snapshots of OxyR reveal the peroxidatic mechanism of H2O2 sensing

2018 ◽  
Vol 115 (50) ◽  
pp. E11623-E11632 ◽  
Author(s):  
Brandán Pedre ◽  
David Young ◽  
Daniel Charlier ◽  
Álvaro Mourenza ◽  
Leonardo Astolfi Rosado ◽  
...  
Keyword(s):  
Active Site ◽  
Promoter Region ◽  
Structural Transition ◽  
Quaternary Structure ◽  
Antioxidant Response ◽  
Active Site Residues ◽  
Disulfide Formation ◽  
Dimerization Interface ◽  
H2o2 Sensing ◽  
Key Residues

Hydrogen peroxide (H2O2) is a strong oxidant capable of oxidizing cysteinyl thiolates, yet only a few cysteine-containing proteins have exceptional reactivity toward H2O2. One such example is the prokaryotic transcription factor OxyR, which controls the antioxidant response in bacteria, and which specifically and rapidly reduces H2O2. In this study, we present crystallographic evidence for the H2O2-sensing mechanism and H2O2-dependent structural transition of Corynebacterium glutamicum OxyR by capturing the reduced and H2O2-bound structures of a serine mutant of the peroxidatic cysteine, and the full-length crystal structure of disulfide-bonded oxidized OxyR. In the H2O2-bound structure, we pinpoint the key residues for the peroxidatic reduction of H2O2, and relate this to mutational assays showing that the conserved active-site residues T107 and R278 are critical for effective H2O2 reduction. Furthermore, we propose an allosteric mode of structural change, whereby a localized conformational change arising from H2O2-induced intramolecular disulfide formation drives a structural shift at the dimerization interface of OxyR, leading to overall changes in quaternary structure and an altered DNA-binding topology and affinity at the catalase promoter region. This study provides molecular insights into the overall OxyR transcription mechanism regulated by H2O2.

Site Saturation Mutagenesis of Active Site Residues of β-Lactamase

Proteins ◽  
1987 ◽  
pp. 521-528 ◽  
Author(s):  
Steve C. Schultz ◽  
Steven S. Carroll ◽  
John H. Richards
Keyword(s):  
Active Site ◽  
Saturation Mutagenesis ◽  
Active Site Residues ◽  
Site Saturation

2-Tier Bacterial and In Vitro Selection of Active and Methotrexate-Resistant Variants of Human Dihydrofolate Reductase

2008 ◽  
Vol 13 (6) ◽  
pp. 504-514 ◽  
Author(s):  
Elena Fossati ◽  
Jordan P. Volpato ◽  
Lucie Poulin ◽  
Vanessa Guerrero ◽  
David-Antoine Dugas ◽  
...  
Keyword(s):  
Dihydrofolate Reductase ◽  
Active Site ◽  
Drug Target ◽  
In Vitro Selection ◽  
Fold Increase ◽  
Active Site Residues ◽  
20 Nm ◽  
Human Dihydrofolate Reductase ◽  
Selection Of

We report a rapid and reliable 2-tier selection and screen for detection of activity as well as drug-resistance in mutated variants of a clinically-relevant drug-target enzyme. Human dihydrofolate reductase point-mutant libraries were subjected to a 1st-tier bacterial complementation assay, such that bacterial propagation served as an indicator of enzyme activity. Alternatively, when selection was performed in the presence of the inhibitor methotrexate (MTX), propagation indicated MTX resistance. The selected variants were then subjected to a 2nd-tier in vitro screen in 96-well plate format using crude bacterial lysate. Conditions were defined to establish a threshold for activity or for MTX resistance. The 2nd-tier assay allowed rapid detection of the best variants among the leads and provided reliable estimates of relative reactivity, ( kcat) and IC50MTX. Screening saturation libraries of active-site positions 7, 15, 24, 70, and 115 revealed a variety of novel mutations compatible with reactivity as well as 2 novel MTX-resistant variants: V115A and V115C. Both variants displayed KiMTX = 20 nM, a 600-fold increase relative to the wild-type. We also present preliminary results from screening against further antifolates following simple modifications of the protocol. The flexibility and robustness of this method will provide new insights into interactions between ligands and active-site residues of this clinically relevant human enzyme. ( Journal of Biomolecular Screening 2008:504-514)

scholarly journals Protein Engineering of Epoxide Hydrolase from Agrobacterium radiobacter AD1 for Enhanced Activity and Enantioselective Production of (R)-1-Phenylethane-1,2-Diol

2005 ◽  
Vol 71 (7) ◽  
pp. 3995-4003 ◽  
Author(s):  
Lingyun Rui ◽  
Li Cao ◽  
Wilfred Chen ◽  
Kenneth F. Reardon ◽  
Thomas K. Wood
Keyword(s):  
Active Site ◽  
Epoxide Hydrolase ◽  
Styrene Oxide ◽  
Saturation Mutagenesis ◽  
Dna Shuffling ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Agrobacterium Radiobacter ◽  
Enantiomeric Ratio ◽  
Enhanced Activity

ABSTRACT DNA shuffling and saturation mutagenesis of positions F108, L190, I219, D235, and C248 were used to generate variants of the epoxide hydrolase of Agrobacterium radiobacter AD1 (EchA) with enhanced enantioselectivity and activity for styrene oxide and enhanced activity for 1,2-epoxyhexane and epoxypropane. EchA variant I219F has more than fivefold-enhanced enantioselectivity toward racemic styrene oxide, with the enantiomeric ratio value (E value) for the production of (R)-1-phenylethane-1,2-diol increased from 17 for the wild-type enzyme to 91, as well as twofold-improved activity for the production of (R)-1-phenylethane-1,2-diol (1.96 ± 0.09 versus 1.04 ± 0.07 μmol/min/mg for wild-type EchA). Computer modeling indicated that this mutation significantly alters (R)-styrene oxide binding in the active site. Another three variants from EchA active-site engineering, F108L/C248I, I219L/C248I, and F108L/I219L/C248I, also exhibited improved enantioselectivity toward racemic styrene oxide in favor of production of the corresponding diol in the (R) configuration (twofold enhancement in their E values). Variant F108L/I219L/C248I also demonstrated 10-fold- and 2-fold-increased activity on 5 mM epoxypropane (24 ± 2 versus 2.4 ± 0.3 μmol/min/mg for the wild-type enzyme) and 5 mM 1,2-epoxyhexane (5.2 ± 0.5 versus 2.6 ± 0.0 μmol/min/mg for the wild-type enzyme). Both variants L190F (isolated from a DNA shuffling library) and L190Y (created from subsequent saturation mutagenesis) showed significantly enhanced activity for racemic styrene oxide hydrolysis, with 4.8-fold (8.6 ± 0.3 versus 1.8 ± 0.2 μmol/min/mg for the wild-type enzyme) and 2.7-fold (4.8 ± 0.8 versus 1.8 ± 0.2 μmol/min/mg for the wild-type enzyme) improvements, respectively. L190Y also hydrolyzed 1,2-epoxyhexane 2.5 times faster than the wild-type enzyme.

scholarly journals Active-Site Protonation States in an Acyl-Enzyme Intermediate of a Class A β-Lactamase with a Monobactam Substrate

2016 ◽  
Vol 61 (1) ◽  
Author(s):  
Venu Gopal Vandavasi ◽  
Patricia S. Langan ◽  
Kevin L. Weiss ◽  
Jerry M. Parks ◽  
Jonathan B. Cooper ◽  
...  
Keyword(s):  
Active Site ◽  
Catalytic Mechanism ◽  
Active Site Residues ◽  
Hydrogen Atoms ◽  
Content Type ◽  
X Ray Crystallography ◽  
General Base ◽  
Class A ◽  
Extended Spectrum ◽  
Enzyme Intermediate

ABSTRACT The monobactam antibiotic aztreonam is used to treat cystic fibrosis patients with chronic pulmonary infections colonized by Pseudomonas aeruginosa strains expressing CTX-M extended-spectrum β-lactamases. The protonation states of active-site residues that are responsible for hydrolysis have been determined previously for the apo form of a CTX-M β-lactamase but not for a monobactam acyl-enzyme intermediate. Here we used neutron and high-resolution X-ray crystallography to probe the mechanism by which CTX-M extended-spectrum β-lactamases hydrolyze monobactam antibiotics. In these first reported structures of a class A β-lactamase in an acyl-enzyme complex with aztreonam, we directly observed most of the hydrogen atoms (as deuterium) within the active site. Although Lys 234 is fully protonated in the acyl intermediate, we found that Lys 73 is neutral. These findings are consistent with Lys 73 being able to serve as a general base during the acylation part of the catalytic mechanism, as previously proposed.

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