scholarly journals Lactate monooxygenase. III. Additive contributions of active site residues to catalytic efficiency and stabilization of an anionic transition state.

1994 ◽  
Vol 269 (11) ◽  
pp. 7994-8000
Author(s):  
U. Müh ◽  
C.H. Williams ◽  
V. Massey
Keyword(s):  
Transition State ◽  
Active Site ◽  
Catalytic Efficiency ◽  
Active Site Residues

scholarly journals Structure based protein engineering to confer selectivity of guanine deaminase

2014 ◽  
Vol 70 (a1) ◽  
pp. C437-C437
Author(s):  
Aruna Bitra ◽  
Ruchi Anand
Keyword(s):  
Crystal Structures ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Catalytic Efficiency ◽  
Structural Basis ◽  
Active Site Residues ◽  
X Ray ◽  
Secondary Activity ◽  
Guanine Deaminase ◽  
Differential Selectivity

Guanine deaminases (GDs) are important enzymes involved in both purine metabolism and nucleotide anabolism pathways. Here we present the molecular and catalytic mechanism of NE0047 and use the information obtained to engineer specific enzyme activities. NE0047 from Nitrosomonas europaea was found to be a high fidelity guanine deaminase (catalytic efficiency of 1.2 × 105 M–1 s–1). However; it exhibited secondary activity towards the structurally non-analogous triazine based compound ammeline. The X-ray structure of NE0047 in the presence of the substrate analogue 8-azaguanine help establish that the enzyme exists as a biological dimer and both the proper closure of the C-terminal loop and cross talk via the dimeric interface is crucial for conferring catalytic activity. It was further ascertained that the highly conserved active site residues Glu79 and Glu143 facilitate the deamination reaction by serving as proton shuttles. Moreover, to understand the structural basis of dual substrate specificity, X-ray structures of NE0047 in complex with a series of nucleobase analogs, nucleosides and substrate ammeline were determined. The crystal structures demonstrated that any substitutions in the parent substrates results in the rearrangement of the ligand in a catalytically unfavorable orientation and also impede the closure of catalytically important loop, thereby abrogating activity. However, ammeline was able to adopt a catalytically favorable orientation which, also allowed for proper loop closure. Based on the above knowledge of the crystal structures and the catalytic mechanism, the active site was subsequently engineered to fine-tune NE0047 activity. The mutated versions of the enzyme were designed so that they can function either exclusively as a GD or serve as specific ammeline deaminases. For example, mutations in the active site E143D and N66A confer the enzyme to be an unambiguous GD with no secondary activity towards ammeline. On the other hand, the N66Q mutant of NE0047 only deaminates ammeline. Additionally, a series of crystal structures of the mutant versions were solved that shed light on the structural basis of this differential selectivity.

Structure of a class III engineered cephalosporin acylase: comparisons with class I acylase and implications for differences in substrate specificity and catalytic activity

2013 ◽  
Vol 451 (2) ◽  
pp. 217-226 ◽  
Author(s):  
Emily Golden ◽  
Rachel Paterson ◽  
Wan Jun Tie ◽  
Anandhi Anandan ◽  
Gavin Flematti ◽  
...  
Keyword(s):  
Transition State ◽  
Active Site ◽  
Catalytic Efficiency ◽  
Cephalosporin C ◽  
Class Iii ◽  
Wild Type ◽  
Serine Residue ◽  
Active Site Cavity ◽  
Β Chain ◽  
Enzyme Variant

The crystal structure of the wild-type form of glutaryl-7-ACA (7-aminocephalosporanic acid) acylase from Pseudomonas N176 and a double mutant of the protein (H57βS/H70βS) that displays enhanced catalytic efficiency on cephalosporin C over glutaryl-7-aminocephalosporanic acid has been determined. The structures show a heterodimer made up of an α-chain (229 residues) and a β-chain (543 residues) with a deep cavity, which constitutes the active site. Comparison of the wild-type and mutant structures provides insights into the molecular reasons for the observed enhanced specificity on cephalosporin C over glutaryl-7-aminocephalosporanic acid and offers the basis to evolve a further improved enzyme variant. The nucleophilic catalytic serine residue, Ser1β, is situated at the base of the active site cavity. The electron density reveals a ligand covalently bound to the catalytic serine residue, such that a tetrahedral adduct is formed. This is proposed to mimic the transition state of the enzyme for both the maturation step and the catalysis of the substrates. A view of the transition state configuration of the enzyme provides important insights into the mechanism of substrate binding and catalysis.

scholarly journals Directed evolution induces tributyrin hydrolysis in a virulence factor of Xylella fastidiosa using a duplicated gene as a template

F1000Research ◽  
2014 ◽  
Vol 3 ◽  
pp. 215 ◽  
Author(s):  
Hossein Gouran ◽  
Sandeep Chakraborty ◽  
Basuthkar J. Rao ◽  
Bjarni Asgeirsson ◽  
Abhaya M. Dandekar
Keyword(s):  
Directed Evolution ◽  
Active Site ◽  
Xylella Fastidiosa ◽  
Catalytic Efficiency ◽  
Resistance Mechanisms ◽  
Loss Of Function ◽  
Active Site Residues ◽  
Protein Protein Interaction ◽  
Moonlighting Functions

Duplication of genes is one of the preferred ways for natural selection to add advantageous functionality to the genome without having to reinvent the wheel with respect to catalytic efficiency and protein stability. The duplicated secretory virulence factors ofXylella fastidiosa(LesA, LesB and LesC), implicated in Pierce's disease of grape and citrus variegated chlorosis of citrus species, epitomizes the positive selection pressures exerted on advantageous genes in such pathogens. A deeper insight into the evolution of these lipases/esterases is essential to develop resistance mechanisms in transgenic plants. Directed evolution, an attempt to accelerate the evolutionary steps in the laboratory, is inherently simple when targeted for loss of function. A bigger challenge is to specify mutations that endow a new function, such as a lost functionality in a duplicated gene. Previously, we have proposed a method for enumerating candidates for mutations intended to transfer the functionality of one protein into another related protein based on the spatial and electrostatic properties of the active site residues (DECAAF). In the current work, we presentin vivovalidation of DECAAF by inducing tributyrin hydrolysis in LesB based on the active site similarity to LesA. The structures of these proteins have been modeled using RaptorX based on the closely related LipA protein fromXanthomonas oryzae. These mutations replicate the spatial and electrostatic conformation of LesA in the modeled structure of the mutant LesB as well, providingin silicovalidation before proceeding to the laboriousin vivowork. Such focused mutations allows one to dissect the relevance of the duplicated genes in finer detail as compared to gene knockouts, since they do not interfere with other moonlighting functions, protein expression levels or protein-protein interaction.

scholarly journals Role of Non-Active-Site Residue Trp-93 in the Function and Stability of New Delhi Metallo-β-Lactamase 1

2015 ◽  
Vol 60 (1) ◽  
pp. 356-360 ◽  
Author(s):  
Asad U. Khan ◽  
M. Tabish Rehman
Keyword(s):  
Active Site ◽  
Catalytic Efficiency ◽  
Structure And Function ◽  
Clinical Strain ◽  
New Delhi ◽  
Active Site Residue ◽  
Active Site Residues ◽  
Content Type ◽  
And Function

ABSTRACTNew Delhi metallo-β-lactamase-1 (NDM-1) is expressed by various members ofEnterobacteriaceaeas a defense mechanism to hydrolyze β-lactam antibiotics. Despite various studies showing the significance of active-site residues in the catalytic mechanism, there is a paucity of reports addressing the role of non-active-site residues in the structure and function of NDM-1. In this study, we investigated the significance of non-active-site residue Trp-93 in the structure and function of NDM-1. We clonedblaNDM-1from anEnterobacter cloacaeclinical strain (EC-15) and introduced the mutation of Trp-93 to Ala (yielding the Trp93Ala mutant) by PCR-based site-directed mutagenesis. Proteins were expressed and purified to homogeneity by affinity chromatography. The MICs of the Trp93Ala mutant were reduced 4- to 8-fold for ampicillin, cefotaxime, ceftazidime, cefoxitin, imipenem, and meropenem. The poor hydrolytic activity of the Trp93Ala mutant was also reflected by its reduced catalytic efficiency. The overall catalytic efficiency of the Trp93Ala mutant was reduced by 40 to 55% (theKmwas reduced, while thekcatwas similar to that of wild-type NDM-1 [wtNDM-1]). Heat-induced denaturation showed that the ΔGDoandTmof Trp93Ala mutant were reduced by 1.8 kcal/mol and 4.8°C, respectively. Far-UV circular dichroism (CD) analysis showed that the α-helical content of the Trp93Ala mutant was reduced by 2.9%. The decrease in stability and catalytic efficiency of the Trp93Ala mutant was due to the loss of two hydrogen bonds with Ser-63 and Val-73 and hydrophobic interactions with Leu-65, Val-73, Gln-123, and Asp-124. The study provided insight into the role of non-active-site amino acid residues in the hydrolytic mechanism of NDM-1.

scholarly journals Structural Basis for Stereoselective Dehydration and Hydrogen-Bonding Catalysis by the SAM-Dependent Pericyclase LepI

2018 ◽  
Author(s):  
Yujuan Cai ◽  
Yang Hai ◽  
Masao Ohashi ◽  
Cooper S. Jamieson ◽  
Marc Garcia-Borras ◽  
...  
Keyword(s):  
Transition State ◽  
Active Site ◽  
Claisen Rearrangement ◽  
Biochemical Characterization ◽  
Mutational Analysis ◽  
Diels Alder ◽  
Quinone Methide ◽  
Structural Basis ◽  
Active Site Residues ◽  
Transition State Analog

ABSTRACTLepI is an S-adenosylmethionine (SAM)-dependent pericyclase that catalyzes the formation of 2-pyridone natural product leporin C. Biochemical characterization showed LepI can catalyze the stereoselective dehydration to yield a reactive (E)-quinone methide which can undergo a bifurcating intramolecular Diels-Alder (IMDA) and hetero-Diels-Alder (HDA) cyclization from an ambimodal transition state, and a [3,3]-retro-Claisen rearrangement to recycle the IMDA product into leporin C. Here we solved the X-ray crystal structures of SAM-bound LepI, and in complex with a substrate analog, the product leporin C, and a retro-Claisen reaction transition-state analog to understand the structural basis for the multitude of reactions. Structural and mutational analysis revealed how Nature evolves a classic methyltransferase active site into one that can serve as a dehydratase and a multifunctional pericyclase. Catalysis of both sets of reactions employ His133 and Arg295, two active site residues that are not found in canonical methyltransferases. An alternative role of SAM, which is not found to be in direct contact of the substrate, is also proposed.

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.

Interconversion of a pair of active-site residues in Escherichia coli cystathionine γ-synthase, E. coli cystathionine β-lyase, and Saccharomyces cerevisiae cystathionine γ-lyase and development of tools for the investigation of their mechanisms and reaction specificity

2009 ◽  
Vol 87 (2) ◽  
pp. 445-457 ◽  
Author(s):  
Ali Farsi ◽  
Pratik H. Lodha ◽  
Jennifer E. Skanes ◽  
Heidi Los ◽  
Navya Kalidindi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Active Site ◽  
Affinity Purification ◽  
Catalytic Efficiency ◽  
Active Site Residues ◽  
E Coli ◽  
Transsulfuration Pathway ◽  
Reaction Specificity

Cystathionine γ-synthase (CGS) and cystathionine β-lyase (CBL), which comprise the transsulfuration pathway of bacteria and plants, and cystathionine γ-lyase (CGL), the second enzyme of the fungal and animal reverse transsulfuration pathway, share ∼30% sequence identity and are almost indistinguishable in overall structure. One difference between the active site of Escherichia coli CBL and those of E. coli CGS and Saccharomyces cerevisiae CGL is the replacement of a pair of aromatic residues, F55 and Y338, of the former by acidic residues in CGS (D45 and E325) and CGL (E48 and E333). A series of interconverting, site-directed mutants of these 2 residues was constructed in CBL (F55D, Y338E, F55D/Y338E), CGS (D45F, E325Y and D45F/E325Y) and CGL (E48A,D and E333A,D,Y) to probe the role of these residues as determinants of reaction specificity. Mutation of either position results in a reduction in catalytic efficiency, as exemplified by the 160-fold reduction in the kcat/Kml-Cys of eCGS-D45F and the 2850- and 30-fold reductions in the kcat/Kml-Cth of the eCBL-Y338E and the yCGL-E333A,Y mutants, respectively. However, the in vivo reaction specificity of the mutants was not altered, compared with the corresponding wild-type enzymes. The ΔmetB and ΔmetC strains, the optimized CBL and CGL assay conditions, and the efficient expression and affinity purification systems described provide the necessary tools to enable the continued exploration of the determinants of reaction specificity in the enzymes of the transsulfuration pathways.

scholarly journals Structural Comparisons of Cefotaximase (CTX-M-ase) Sub Family 1

2021 ◽  
Vol 12 ◽  
Author(s):  
Ben A. Shurina ◽  
Richard C. Page
Keyword(s):  
Transition State ◽  
Active Site ◽  
Boronic Acid ◽  
Structural Changes ◽  
Surface Patch ◽  
Active Site Residues ◽  
Transition State Analog ◽  
Drug Candidates ◽  
Existing Structures ◽  
New Antibiotics

The cefotaximase or CTX-M, family of serine-β-lactamases represents a significant clinical concern due to the ability for these enzymes to confer resistance to a broad array of β-lactam antibiotics an inhibitors. This behavior lends CTX-M-ases to be classified as extended spectrum β-lactamases (ESBL). Across the family of CTX-M-ases most closely related to CTX-M-1, the structures of CTX-M-15 with a library of different ligands have been solved and serve as the basis of comparison within this review. Herein we focus on the structural changes apparent in structures of CTX-M-15 in complex with diazabicyclooctane (DABCO) and boronic acid transition state analog inhibitors. Interactions between a positive surface patch near the active site and complementary functional groups of the bound inhibitor play key roles in the dictating the conformations of active site residues. The insights provided by analyzing structures of CTX-M-15 in complex with DABCO and boronic acid transition state analog inhibitors and analyzing existing structures of CTX-M-64 offer opportunities to move closer to making predictions as to how CTX-M-ases may interact with potential drug candidates, setting the stage for the further development of new antibiotics and β-lactamase inhibitors.

Sign in / Sign up

Export Citation Format

Share Document