scholarly journals Exploring the challenges of computational enzyme design by rebuilding the active site of a dehalogenase

2018 ◽  
Vol 116 (2) ◽  
pp. 389-394 ◽  
Author(s):  
Garima Jindal ◽  
Katerina Slanska ◽  
Veselin Kolev ◽  
Jiri Damborsky ◽  
Zbynek Prokop ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Active Site ◽  
Test Case ◽  
Wild Type ◽  
Haloalkane Dehalogenase ◽  
Enzyme Design ◽  
Wild Type Enzyme ◽  
Computational Enzyme Design ◽  
Activity Design ◽  
Computational Predictions

Rational enzyme design presents a major challenge that has not been overcome by computational approaches. One of the key challenges is the difficulty in assessing the magnitude of the maximum possible catalytic activity. In an attempt to overcome this challenge, we introduce a strategy that takes an active enzyme (assuming that its activity is close to the maximum possible activity), design mutations that reduce the catalytic activity, and then try to restore that catalysis by mutating other residues. Here we take as a test case the enzyme haloalkane dehalogenase (DhlA), with a 1,2-dichloroethane substrate. We start by demonstrating our ability to reproduce the results of single mutations. Next, we design mutations that reduce the enzyme activity and finally design double mutations that are aimed at restoring the activity. Using the computational predictions as a guide, we conduct an experimental study that confirms our prediction in one case and leads to inconclusive results in another case with 1,2-dichloroethane as substrate. Interestingly, one of our predicted double mutants catalyzes dehalogenation of 1,2-dibromoethane more efficiently than the wild-type enzyme.

scholarly journals Active-site serine mutants of the Streptomyces albus G β-lactamase

1991 ◽  
Vol 277 (3) ◽  
pp. 647-652 ◽  
Author(s):  
F Jacob ◽  
B Joris ◽  
J M Frère
Keyword(s):  
Active Site ◽  
Specific Activity ◽  
Site Directed Mutagenesis ◽  
Beta Lactamase ◽  
Wild Type ◽  
Serine Residue ◽  
Wild Type Enzyme ◽  
Ph Values ◽  
Streptomyces Albus ◽  
Small Production

By using site-directed mutagenesis, the active-site serine residue of the Streptomyces albus G beta-lactamase was substituted by alanine and cysteine. Both mutant enzymes were produced in Streptomyces lividans and purified to homogeneity. The cysteine beta-lactamase exhibited a substrate-specificity profile distinct from that of the wild-type enzyme, and its kcat./Km values at pH 7 were never higher than 0.1% of that of the serine enzyme. Unlike the wild-type enzyme, the activity of the mutant increased at acidic pH values. Surprisingly, the alanine mutant exhibited a weak but specific activity for benzylpenicillin and ampicillin. In addition, a very small production of wild-type enzyme, probably due to mistranslation, was detected, but that activity could be selectively eliminated. Both mutant enzymes were nearly as thermostable as the wild-type.

scholarly journals Enhancing a De Novo Enzyme Activity by Computationally-Focused, Ultra-Low-Throughput Sequence Screening

2020 ◽  
Author(s):  
Valeria A. Risso ◽  
Adrian Romero-Rivera ◽  
Luis I. Gutierrez-Rus ◽  
Mariano Ortega-Muñoz ◽  
Francisco Santoyo-Gonzalez ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Active Site ◽  
De Novo ◽  
Enzymatic Reaction ◽  
Valence Bond ◽  
Computational Procedure ◽  
Enzyme Design ◽  
Computational Enzyme Design ◽  
Stability Ranking ◽  
Random Library

<div> <div> <div> <p>Directed evolution has revolutionized protein engineering. Still, enzyme optimization by random library screening remains a sluggish process, in large part due to futile probing of mutations that are catalytically neutral and/or impair stability and folding. FuncLib (funclib-weizmann.ac.il) is a novel automated computational procedure which uses phylogenetic analysis and Rosetta design to rank enzyme variants with multiple mutations, on the basis of a stability metric. Here, we use it to target the active site region of a minimalist-designed, de novo Kemp eliminase. The similarity between the Michaelis complex and transition state for the enzymatic reaction makes this a particularly challenging system to optimize. Yet, experimental screening of a very small number of active-site, multi-point variants at the top of the predicted stability ranking leads to catalytic efficiencies and turnover numbers (~2·104 M-1 s-1 and ~102 s-1) that compare well with modern natural enzymes, and that approach the catalysis levels for the best Kemp eliminases derived from extensive screening. This result illustrates the promise of FuncLib as a powerful tool with which to speed up directed evolution, by guiding screening to regions of the sequence space that encode stable and catalytically diverse enzymes. Empirical valence bond calculations reproduce the experimental activation energies for the optimized eliminases to within ~2 kcal·mol-1 and indicate that the improvements in activity are linked to better geometric preorganization of the active site. This raises the possibility of further enhancing the stability-guidance of FuncLib by EVB-based computational predictions of catalytic activity, as a generalized approach for computational enzyme design. </p> </div> </div> </div>

scholarly journals Hydration change during the aging of phosphorylated human butyrylcholinesterase: importance of residues aspartate-70 and glutamate-197 in the water network as probed by hydrostatic and osmotic pressures

1999 ◽  
Vol 343 (2) ◽  
pp. 361-369 ◽  
Author(s):  
Patrick MASSON ◽  
Cécile CLÉRY ◽  
Patrice GUERRA ◽  
Arnaud REDSLOB ◽  
Christine ALBARET ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Hydrostatic Pressure ◽  
Transition State ◽  
Osmotic Pressure ◽  
Active Site ◽  
Aging Process ◽  
Wild Type ◽  
Water Network ◽  
Wild Type Enzyme ◽  
Human Butyrylcholinesterase

Wild-type human butyrylcholinesterase (BuChE) and Glu-197 → Asp and Asp-70 → Gly mutants (E197D and D70G respectively) were inhibited by di-isopropyl phosphorofluoridate under standard conditions of pH, temperature and pressure. The effect of hydrostatic and osmotic pressures on the aging process (dealkylation of an isopropyl chain) of phosphorylated enzymes [di-isopropylated (DIP)-BuChE] was investigated. Hydrostatic pressure markedly increased the rate of aging of wild-type enzyme. The average activation volume (δV≠) for the dealkylation reaction was -170 ml/mol for DIP wild-type BuChE. On the other hand, hydrostatic pressure had little effect on the aging of the DIP mutants (δV≠ = -2.6 ml/mol for E197D and -2 ml/mol for D70G), suggesting that the transition state of the aging process was associated with an extended hydration and conformational change in wild-type BuChE, but not in the mutants. The rate of aging of wild-type and mutant enzymes decreased with osmotic pressure, allowing very large positive osmotic activation volumes (δV≠osm) to be estimated, thus probing the participation of water in the aging process. Molecular dynamics simulations performed on the active-site gorge of the wild-type DIP adduct showed that the isopropyl chain involved in aging was highly solvated, supporting the idea that water is important for stabilizing the transition state of the dealkylation reaction. Wild-type BuChE was inhibited by soman (pinacolyl methylphosphonofluoridate). Electrophoresis performed under high pressure [up to 2.5 kbar (1 bar = 105 Pa)] showed that the soman-aged enzyme did not pass through a pressure-induced, molten-globule transition, unlike the native wild-type enzyme. Likewise, this transition was not seen for the native E197D and D70G mutants, indicating that these mutants are resistant to the penetration of water into their structure. The stability energetics of native and soman-aged wild-type BuChE were determined by differential scanning calorimetry. The pH-dependence of the midpoint transition temperature of endotherms indicated that the high difference in stabilization energy between aged and native BuChE (δδG = 23.7 kJ/mol at pH 8.0) is mainly due to the salt bridge between protonated His-438 and PO-, with pKHis-438 = 8.3. A molecular dynamics simulation on the MIP adduct showed that there is no water molecule around the ion pair. The ‘hydrostatic versus osmotic pressure’ approach probed the importance of water in aging, and also revealed that Asp-70 and Glu-197 are the major residues controlling both the dynamics and the structural organization of the water/hydrogen-bond network in the active-site gorge of BuChE. In wild-type BuChE both residues function like valves, whereas in the mutant enzymes the water network is slack, and residues Gly-70 and Asp-197 function like check valves, i.e. forced penetration of water into the gorge is not easily achieved, thereby facilitating the release of water.

scholarly journals Enhancing the α-Cyclodextrin Specificity of Cyclodextrin Glycosyltransferase from Paenibacillus macerans by Mutagenesis Masking Subsite −7

2016 ◽  
Vol 82 (8) ◽  
pp. 2247-2255 ◽  
Author(s):  
Lei Wang ◽  
Xuguo Duan ◽  
Jing Wu
Keyword(s):  
Active Site ◽  
Wild Type ◽  
Cyclodextrin Glycosyltransferase ◽  
Wild Type Enzyme ◽  
Widespread Application ◽  
Content Type ◽  
Hydrogen Bonding Interactions ◽  
Paenibacillus Macerans ◽  
Site Blocking ◽  
Cyclodextrin Production

ABSTRACTCyclodextrin glycosyltransferases (CGTases) (EC 2.4.1.19) catalyze the conversion of starch or starch derivates into mixtures of α-, β-, and γ-cyclodextrins. Because time-consuming and expensive purification procedures hinder the widespread application of single-ingredient cyclodextrins, enzymes with enhanced specificity are needed. In this study, we tested the hypothesis that the α-cyclodextrin selectivity ofPaenibacillus maceransα-CGTase could be augmented by masking subsite −7 of the active site, blocking the formation of larger cyclodextrins, particularly β-cyclodextrin. Five single mutants and three double mutants designed to remove hydrogen-bonding interactions between the enzyme and substrate at subsite −7 were constructed and characterized in detail. Although the rates of α-cyclodextrin formation varied only modestly, the rate of β-cyclodextrin formation decreased dramatically in these mutants. The increase in α-cyclodextrin selectivity was directly proportional to the increase in the ratio of theirkcatvalues for α- and β-cyclodextrin formation. The R146A/D147P and R146P/D147A double mutants exhibited ratios of α-cyclodextrin to total cyclodextrin production of 75.1% and 76.1%, approximately one-fifth greater than that of the wild-type enzyme (63.2%), without loss of thermostability. Thus, these double mutants may be more suitable for the industrial production of α-cyclodextrin than the wild-type enzyme. The production of β-cyclodextrin by these mutants was almost identical to their production of γ-cyclodextrin, which was unaffected by the mutations in subsite −7, suggesting that subsite −7 was effectively blocked by these mutations. Further increases in α-cyclodextrin selectivity will require identification of the mechanism or mechanisms by which these small quantities of larger cyclodextrins are formed.

scholarly journals New properties of Bacillus subtilis succinate dehydrogenase altered at the active site. The apparent active site thiol of succinate oxidoreductases is dispensable for succinate oxidation

1989 ◽  
Vol 260 (2) ◽  
pp. 491-497 ◽  
Author(s):  
L Hederstedt ◽  
L O Hedén
Keyword(s):  
Bacillus Subtilis ◽  
Succinate Dehydrogenase ◽  
Active Site ◽  
Amino Acid Position ◽  
Biochemical Properties ◽  
Cysteine Residue ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Succinate Oxidation ◽  
E Coli

Mammalian and Escherichia coli succinate dehydrogenase (SDH) and E. coli fumarate reductase apparently contain an essential cysteine residue at the active site, as shown by substrate-protectable inactivation with thiol-specific reagents. Bacillus subtilis SDH was found to be resistant to this type of reagent and contains an alanine residue at the amino acid position equivalent to the only invariant cysteine in the flavoprotein subunit of E. coli succinate oxidoreductases. Substitution of this alanine, at position 252 in the flavoprotein subunit of B. subtilis SDH, by cysteine resulted in an enzyme sensitive to thiol-specific reagents and protectable by substrate. Other biochemical properties of the redesigned SDH were similar to those of the wild-type enzyme. It is concluded that the invariant cysteine in the flavoprotein of E. coli succinate oxidoreductases corresponds to the active site thiol. However, this cysteine is most likely not essential for succinate oxidation and seemingly lacks an assignable specific function. An invariant arginine in juxtaposition to Ala-252 in the flavoprotein of B. subtilis SDH, and to the invariant cysteine in the E. coli homologous enzymes, is probably essential for substrate binding.

scholarly journals Probing the catalytic mechanism of Escherichia coli amine oxidase using mutational variants and a reversible inhibitor as a substrate analogue

2002 ◽  
Vol 365 (3) ◽  
pp. 809-816 ◽  
Author(s):  
Colin G. SAYSELL ◽  
Winston S. TAMBYRAJAH ◽  
Jeremy M. MURRAY ◽  
Carrie M. WILMOT ◽  
Simon E.V. PHILLIPS ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Amine Oxidase ◽  
Reaction Pathway ◽  
Strong Support ◽  
Reversible Inhibitor ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Binding Data ◽  
Copper Amine

Copper amine oxidases are homodimeric enzymes containing one Cu2+ ion and one 2,4,5-trihydroxyphenylalanine quinone (TPQ) per monomer. Previous studies with the copper amine oxidase from Escherichia coli (ECAO) have elucidated the structure of the active site and established the importance in catalysis of an active-site base, Asp-383. To explore the early interactions of substrate with enzyme, we have used tranylcypromine (TCP), a fully reversible competitive inhibitor, with wild-type ECAO and with the active-site base variants D383E and D383N. The formation of an adduct, analogous to the substrate Schiff base, between TCP and the TPQ cofactor in the active site of wild-type ECAO and in the D383E and D383N variants has been investigated over the pH range 5.5–9.4. For the wild-type enzyme, the plot of the binding constant for adduct formation (Kb) against pH is bell-shaped, indicating two pKas of 5.8 and ∼8, consistent with the preferred reaction partners being the unprotonated active-site base and the protonated TCP. For the D383N variant, the reaction pathway involving unprotonated base and protonated TCP cannot occur, and binding must follow a less favoured pathway with unprotonated TCP as reactant. Surprisingly, for the D383E variant, the Kb versus pH behaviour is qualitatively similar to that of D383N, supporting a reaction pathway involving unprotonated TCP. The TCP binding data are consistent with substrate binding data for the wild type and the D383E variant using steady-state kinetics. The results provide strong support for a protonated amine being the preferred substrate for the wild-type enzyme, and emphasize the importance of the active-site base, Asp-383, in the primary binding event.

scholarly journals The Structure of the Dizinc Subclass B2 Metallo-β-Lactamase CphA Reveals that the Second Inhibitory Zinc Ion Binds in the Histidine Site

2009 ◽  
Vol 53 (10) ◽  
pp. 4464-4471 ◽  
Author(s):  
Carine Bebrone ◽  
Heinrich Delbrück ◽  
Michaël B. Kupper ◽  
Philipp Schlömer ◽  
Charlotte Willmann ◽  
...  
Keyword(s):  
Active Site ◽  
Aeromonas Hydrophila ◽  
Zinc Ion ◽  
Amide Bond ◽  
Zinc Ions ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
X Ray ◽  
Metal Ligands ◽  
Class B

ABSTRACT Bacteria can defend themselves against β-lactam antibiotics through the expression of class B β-lactamases, which cleave the β-lactam amide bond and render the molecule harmless. There are three subclasses of class B β-lactamases (B1, B2, and B3), all of which require Zn2+ for activity and can bind either one or two zinc ions. Whereas the B1 and B3 metallo-β-lactamases are most active as dizinc enzymes, subclass B2 enzymes, such as Aeromonas hydrophila CphA, are inhibited by the binding of a second zinc ion. We crystallized A. hydrophila CphA in order to determine the binding site of the inhibitory zinc ion. X-ray data from zinc-saturated crystals allowed us to solve the crystal structures of the dizinc forms of the wild-type enzyme and N220G mutant. The first zinc ion binds in the cysteine site, as previously determined for the monozinc form of the enzyme. The second zinc ion occupies a slightly modified histidine site, where the conserved His118 and His196 residues act as metal ligands. This atypical coordination sphere probably explains the rather high dissociation constant for the second zinc ion compared to those observed with enzymes of subclasses B1 and B3. Inhibition by the second zinc ion results from immobilization of the catalytically important His118 and His196 residues, as well as the folding of the Gly232-Asn233 loop into a position that covers the active site.

scholarly journals A large displacement of the SXN motif of Cys115-modified penicillin-binding protein 5 from Escherichia coli

2005 ◽  
Vol 392 (1) ◽  
pp. 55-63 ◽  
Author(s):  
George Nicola ◽  
Alena Fedarovich ◽  
Robert A. Nicholas ◽  
Christopher Davies
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Major Change ◽  
Penicillin Binding Protein ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Penicillin Binding Proteins ◽  
Peptidoglycan Biosynthesis ◽  
Covalent Adduct

Penicillin-binding proteins (PBPs), which are the lethal targets of β-lactam antibiotics, catalyse the final stages of peptidoglycan biosynthesis of the bacterial cell wall. PBP 5 of Escherichia coli is a D-alanine CPase (carboxypeptidase) that has served as a useful model to elucidate the catalytic mechanism of low-molecular-mass PBPs. Previous studies have shown that modification of Cys115 with a variety of reagents results in a loss of CPase activity and a large decrease in the rate of deacylation of the penicilloyl–PBP 5 complex [Tamura, Imae and Strominger (1976) J. Biol. Chem. 251, 414–423; Curtis and Strominger (1978) J. Biol. Chem. 253, 2584–2588]. The crystal structure of wild-type PBP 5 in which Cys115 fortuitously had formed a covalent adduct with 2-mercaptoethanol was solved at 2.0 Å (0.2 nm) resolution, and these results provide a structural rationale for how thiol-directed reagents lower the rate of deacylation. When compared with the structure of the unmodified wild-type enzyme, a major change in the architecture of the active site is observed. The two largest differences are the disordering of a loop comprising residues 74–90 and a shift in residues 106–111, which results in the displacement of Ser110 of the SXN active-site motif. These results support the developing hypothesis that the SXN motif of PBP 5, and especially Ser110, is intimately involved in the catalytic mechanism of deacylation.

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