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 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 Reductive half-reaction of the H172Q mutant of trimethylamine dehydrogenase: evidence against a carbanion mechanism and assignment of kinetically influential ionizations in the enzyme–substrate complex

1999 ◽  
Vol 341 (2) ◽  
pp. 307-314 ◽  
Author(s):  
Jaswir BASRAN ◽  
Michael J. SUTCLIFFE ◽  
Russ HILLE ◽  
Nigel S. SCRUTTON
Keyword(s):  
Active Site ◽  
Bond Cleavage ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Trimethylamine Dehydrogenase ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Ph Range ◽  
Rate Limiting ◽  
Macroscopic Pka

The reactions of wild-type trimethylamine dehydrogenase (TMADH) and of a His-172 Gln (H172Q) mutant were studied by rapid-mixing stopped-flow spectroscopy over the pH range 6.0-10.5, to address the potential role of His-172 in abstracting a proton from the substrate in a ‘carbanion’ mechanism for C-H bond cleavage. The pH-dependence of the limiting rate for flavin reduction (klim) was studied as a function of pH for the wild-type enzyme with perdeuterated trimethylamine as substrate. The use of perdeuterated trimethylamine facilitated the unequivocal identification of two kinetically influential ionizations in the enzyme-substrate complex, with macroscopic pKa values of 6.5±0.2 and 8.4±0.1. A plot of klim/Kd revealed a bell-shaped curve and two kinetically influential ionizations with macroscopic pKa values of 9.4±0.1 and 10.5±0.1. Mutagenesis of His-172, a potential active-site base and a component of a novel Tyr-His-Asp triad in the active site of TMADH, revealed that the pKa of 8.4±0.1 for the wild-type enzyme-substrate complex represents ionization of the imidazolium side-chain of His-172. H172Q TMADH retains catalytic competence throughout the pH range investigated. At pH 10.5, and in contrast with the wild-type enzyme, flavin reduction in H172Q TMADH is biphasic. The fast phase is dependent on the trimethylamine concentration and exhibits a kinetic isotope effect of about 3; C-H bond cleavage is thus partially rate-limiting. In contrast, the slow phase does not show hyperbolic dependence on substrate concentration, and the observed rate shows no dependence on isotope, revealing that C-H bond cleavage is not rate-limiting. The analysis of H172Q TMADH, together with data recently acquired for the Y169F mutant of TMADH, reveals that C-H bond breakage is not initiated via abstraction of a proton from the substrate by an active-site base. The transfer of reducing equivalents to flavin via a carbanion mechanism is therefore unlikely.

Effect of Modifiers on the Hydrolysis of Basic and Neutral Peptides by Carboxypeptidase B

1975 ◽  
Vol 53 (7) ◽  
pp. 747-757 ◽  
Author(s):  
Graham J. Moore ◽  
N. Leo Benoiton
Keyword(s):  
Active Site ◽  
Active Sites ◽  
Kinetic Behavior ◽  
Carboxypeptidase A ◽  
Phenyllactic Acid ◽  
Carboxypeptidase B ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Basic Peptides ◽  
Hydrolysis Of

The initial rates of hydrolysis of Bz-Gly-Lys and Bz-Gly-Phe by carboxypeptidase B (CPB) are increased in the presence of the modifiers β-phenylpropionic acid, cyclohexanol, Bz-Gly, and Bz-Gly-Gly. The hydrolysis of the tripeptide Bz-Gly-Gly-Phe is also activated by Bz-Gly and Bz-Gly-Gly, but none of these modifiers activate the hydrolysis of Bz-Gly-Gly-Lys, Z-Leu-Ala-Phe, or Bz-Gly-phenyllactic acid by CPB. All modifiers except cyclohexanol display inhibitory modes of binding when present in high concentration.Examination of Lineweaver–Burk plots in the presence of fixed concentrations of Bz-Gly has shown that activation of the hydrolysis of neutral and basic peptides by CPB, as reflected in the values of the extrapolated parameters, Km(app) and keat, occurs by different mechanisms. For Bz-Gly-Gly-Phe, activation occurs because the enzyme–modifier complex has a higher affinity than the free enzyme for the substrate, whereas activation of the hydrolysis of Bz-Gly-Lys derives from an increase in the rate of breakdown of the enzyme–substrate complex to give products.Cyclohexanol differs from Bz-Gly and Bz-Gly-Gly in that it displays no inhibitory mode of binding with any of the substrates examined, activates only the hydrolysis of dipeptides by CPB, and has a greater effect on the hydrolysis of the basic dipeptide than on the neutral dipeptide. Moreover, when Bz-Gly-Lys is the substrate, cyclohexanol activates its hydrolysis by CPB by increasing both the enzyme–substrate binding affinity and the rate of the catalytic step, an effect different from that observed when Bz-Gly is the modifier.The anomalous kinetic behavior of CPB is remarkably similar to that of carboxypeptidase A, and is a good indication that both enzymes have very similar structures in and around their respective active sites. A binding site for activator molecules down the cleft of the active site is proposed for CPB to explain the observed kinetic behavior.

Human cholinesterases

2020 ◽  
pp. 63-120
Author(s):  
Sergey Varfolomeev ◽  
Bella Grigorenko ◽  
Sofya Lushchekina ◽  
Alexander Nemuchin
Keyword(s):  
Active Site ◽  
The Body ◽  
Polymorphic Modification ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Mechanism Of Hydrolysis ◽  
The Central Nervous System ◽  
The Stability ◽  
Hydrolysis Of ◽  
Enzyme Reactivity

The work is devoted to modeling the elementary stages of the hydrolysis reaction in the active site of enzymes belonging to the class of cholinesterases — acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). The study allowed to describe at the molecular level the effect of the polymorphic modification of BChE, causing serious physiolog ical consequences. Cholinesterase plays a crucial role in the human body. AChE is one of the key enzymes of the central nervous system, and BChE performs protective functions in the body. According to the results of calculations using the combined method of quantum and molecular mechanics (KM/MM), the mechanism of the hydrolysis of the native acetylcholine substrate in the AChE active center was detailed. For a series of ester substrates, a method for estimation of dependence of the enzyme reactivity on the structure of the substrate has been developed. The mechanism of hydrolysis of the muscle relaxant of succininylcholine BChE and the effect of the Asp70Gly polymorph on it were studied. Using various computer simulation methods, the stability of the enzyme-substrate complex of two enzyme variants with succinylcholine was studied.

Destabilization is as important as binding

1993 ◽  
Vol 345 (1674) ◽  
pp. 3-10 ◽  
Keyword(s):  
Binding Energy ◽  
Gibbs Energy ◽  
Active Site ◽  
Electrostatic Interactions ◽  
Large Fraction ◽  
Geometric Distortion ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Non Covalent Interactions ◽  
Covalent Interactions

Enzymes make use of non-covalent interactions with their substrates to bring about a large fraction of their catalytic activity. These interactions must destabilize, or increase the Gibbs energy, of the substrate in the active site in order that the transition state can be reached easily. This destabilization may be brought about by utilization of the intrinsic binding energy between the active site and the bound substrate by desolvation of charged groups, geometric distortion, electrostatic interactions and, especially, loss of entropy in the enzyme-substrate complex. These mechanisms are described by interaction energies and require utilization of the intrinsic binding energy that is realized from non-covalent interactions between the enzyme and substrate. Receptors and coupled vectorial processes, such as muscle contraction and active transport, utilize binding energy similarly to avoid large peaks and valleys along the Gibbs energy profile of the reaction under physiological conditions.

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.

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.

Essential Arginine Residues in Lactate Dehydrogenase from Germinating Soybean

1994 ◽  
Vol 59 (2) ◽  
pp. 467-472 ◽  
Author(s):  
Jana Barthová ◽  
Irena Hulová ◽  
Miroslava Birčáková
Keyword(s):  
Enzyme Activity ◽  
Glycine Max ◽  
Lactate Dehydrogenase ◽  
Chemical Modification ◽  
Active Site ◽  
Enzyme Modification ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Arginine Residues ◽  
Blue Sepharose

The lactate dehydrogenase was isolated from soybean (Glycine max. L.) by a procedure that employed biospecific chromatography on a column of Blue-Sepharose CL-6B. The participation of the guanidine group of arginine residues in the mechanism of enzyme action was determined through kinetic and chemical modification studies. The dependence of enzyme activity on pH was followed in the alkaline region (pH 8.6 - 12.8). The pK values found were 12.4 for the enzyme substrate complex and 11.1 for the free enzyme. The enzyme was inactivated by phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione and p-hydroxyphenylglyoxal reagents used in modification experiments. Kinetic analysis of the modification indicated that one arginine residue is modified when inactivation occurs. No effect was observed on the rate of inactivation upon addition of coenzyme. The extent of enzyme modification by p-hydroxyphenylglyoxal was determined. It appears there are at least two arginine residues in the active site of the enzyme.

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).

Three factors that modulate the activity of class D β-lactamases and interfere with the post-translational carboxylation of Lys70

2010 ◽  
Vol 432 (3) ◽  
pp. 495-506 ◽  
Author(s):  
Lionel Vercheval ◽  
Cédric Bauvois ◽  
Alexandre di Paolo ◽  
Franck Borel ◽  
Jean-Luc Ferrer ◽  
...  
Keyword(s):  
Active Site ◽  
Chloride Ions ◽  
Side Chain ◽  
Wild Type ◽  
Post Translational Modification ◽  
Rate Limiting Step ◽  
Class D ◽  
The Individual ◽  
Rate Limiting

The activity of class D β-lactamases is dependent on Lys70 carboxylation in the active site. Structural, kinetic and affinity studies show that this post-translational modification can be affected by the presence of a poor substrate such as moxalactam but also by the V117T substitution. Val117 is a strictly conserved hydrophobic residue located in the active site. In addition, inhibition of class D β-lactamases by chloride ions is due to a competition between the side chain carboxylate of the modified Lys70 and chloride ions. Determination of the individual kinetic constants shows that the deacylation of the acyl–enzyme is the rate-limiting step for the wild-type OXA-10 β-lactamase.

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