scholarly journals Residues Distal to the Active Site Contribute to Enhanced Catalytic Activity of Variant and Hybrid β-Lactamases Derived from CTX-M-14 and CTX-M-15

2015 ◽  
Vol 59 (10) ◽  
pp. 5976-5983 ◽  
Author(s):  
Dandan He ◽  
Jiachi Chiou ◽  
Zhenling Zeng ◽  
Lanping Liu ◽  
Xiaojie Chen ◽  
...  
Keyword(s):  
Amino Acids ◽  
Catalytic Activity ◽  
Active Site ◽  
Hydrophobic Interactions ◽  
Catalytic Efficiency ◽  
Structure Stability ◽  
Hydrolytic Activities ◽  
C And N ◽  
Key Residues ◽  
M 14

ABSTRACTA variety of CTX-M-type extended-spectrum β-lactamases (ESBLs), including hybrid ones, have been reported in China that are uncommon elsewhere. To better characterize the substrate profiles and enzymatic mechanisms of these enzymes, we performed comparative kinetic analyses of both parental and hybrid CTX-M enzymes, including CTX-M-15, -132, -123, -64, -14 and -55, that are known to confer variable levels of β-lactam resistance in the host strains. All tested enzymes were susceptible to serine β-lactamase inhibitors, with sulbactam exhibiting the weakest inhibitory effects. CTX-M-55, which differs from CTX-M-15 by one substitution, A77V, displayed enhanced catalytic activity (kcat/Km) against expanded-spectrum cephalosporins (ESCs). CTX-M-55 exhibits higher structure stability, most likely by forming hydrophobic interactions between A77V and various key residues in different helices, thereby stabilizing the core architecture of the helix cluster, and indirectly contributes to a more stable active site conformation, which in turn shows higher catalytic efficiency and is more tolerant to temperature change. Analyses of the hybrids and their parental prototypes showed that evolution from CTX-M-15 to CTX-M-132, CTX-M-123, and CTX-M-64, characterized by gradual enhancement of catalytic activity to ESCs, was attributed to introduction of different substitutions to amino acids distal to the active site of CTX-M-15. Similarly, the increased hydrolytic activities against cephalosporins and sensitivity to β-lactamase inhibitors, clavulanic acid and sulbactam, of CTX-M-64 were partly due to the amino acids that were different from CTX-M-14 and located at both the C and N termini of CTX-M-64. These data indicate that residues distal to the active site of CTX-Ms contributed to their enhanced catalytic activities to ESCs.

scholarly journals 3-D Structure of Serum Paraoxonase 1 Sheds Light on Its Activity, Stability, Solubility and Crystallizability

2007 ◽  
Vol 58 (3) ◽  
pp. 347-353 ◽  
Author(s):  
Michal Harel ◽  
Boris Brumshtein ◽  
Ran Meged ◽  
Hay Dvir ◽  
Raimond Ravelli ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Catalytic Activity ◽  
High Density Lipoprotein ◽  
Active Site ◽  
Density Lipoprotein ◽  
Paraoxonase 1 ◽  
Hydrolytic Activities ◽  
Wide Range ◽  
The Stability ◽  
Serum Paraoxonase

3-D Structure of Serum Paraoxonase 1 Sheds Light on Its Activity, Stability, Solubility and CrystallizabilitySerum paraoxonases (PONs) exhibit a wide range of physiologically important hydrolytic activities, including drug metabolism and detoxification of nerve gases. PON1 and PON3 reside on high-density lipoprotein (HDL) (the "good cholesterol"), and are involved in the alleviation of atherosclerosis. Members of the PON family have been identified not only in mammals and other vertebrates, but also in invertebrates. We earlier described the first crystal structure of a PON family member, a directly-evolved variant of PON1, at 2.2 Å resolution. PON1 is a 6-bladed beta-propeller with a unique active-site lid which is also involved in binding to HDL. The 3-D structure, taken together with directed evolution studies, permitted analysis of mutations which enhanced the stability, solubility and crystallizability of this PON1 variant. The structure permits a detailed description of PON1's active site and suggests possible mechanisms for its catalytic activity on certain substrates.

scholarly journals Set2-Dependent K36 Methylation Is Regulated by Novel Intratail Interactions within H3

2009 ◽  
Vol 29 (24) ◽  
pp. 6413-6426 ◽  
Author(s):  
James N. Psathas ◽  
Suting Zheng ◽  
Song Tan ◽  
Joseph C. Reese
Keyword(s):  
Amino Acids ◽  
Catalytic Activity ◽  
Chromatin Remodeling ◽  
Posttranslational Modifications ◽  
Active Site ◽  
Transcription Initiation ◽  
Gene Activation ◽  
N Terminus

ABSTRACT Posttranslational modifications to histones have been studied extensively, but the requirement for the residues within the tails for different stages of transcription is less clear. Using RNR3 as a model, we found that the residues within the N terminus of H3 are predominantly required for steps after transcription initiation and chromatin remodeling. Specifically, deleting as few as 20 amino acids, or substituting glutamines for lysines in the tail, greatly impaired K36 methylation by Set2. The mutations to the tail described here preserve the residues predicted to fill the active site of Set2, and the deletion mimics the recently described cleavage of the H3 tail that occurs during gene activation. Importantly, maintaining the charge of the unmodified tail by arginine substitutions preserves Set2 function in vivo. The H3 tail is dispensable for Set2 recruitment to genes but is required for the catalytic activity of Set2 in vitro. We propose that Set2 activity is controlled by novel intratail interactions which can be influenced by modifications and changes to the structure of the H3 tail to control the dynamics and localization of methylation during elongation.

scholarly journals Prediction of the Evolution of Ceftazidime Resistance in Extended-Spectrum β-Lactamase CTX-M-9

2006 ◽  
Vol 50 (2) ◽  
pp. 731-738 ◽  
Author(s):  
J. Delmas ◽  
F. Robin ◽  
F. Carvalho ◽  
C. Mongaret ◽  
R. Bonnet
Keyword(s):  
Catalytic Activity ◽  
Amino Acid ◽  
Active Site ◽  
Random Mutagenesis ◽  
Evolutionary Process ◽  
Catalytic Efficiency ◽  
Kinetic Constants ◽  
The Other ◽  
Amino Acid Substitutions ◽  
Evolutionary Potential

ABSTRACT A random mutagenesis technique was used to predict the evolutionary potential of β-lactamase CTX-M-9 toward the acquisition of improved catalytic activity against ceftazidime. Thirty CTX-M mutants were obtained during three rounds of mutagenesis. These mutants conferred 1- to 128-fold-higher MICs of ceftazidime than the parental enzyme CTX-M-9. The CTX-M mutants contained one to six amino acid substitutions. Mutants harbored the substitutions Asp240Gly and Pro167Ser, which were previously observed in clinical CTX-M enzymes. Additional substitutions, notably Arg164His, Asp179Gly, and Arg276Ser, were observed near the active site. The kinetic constants of the three most active mutants revealed two distinct ways of improving catalytic efficiency against ceftazidime. One enzyme had a 17-fold-higher k cat value than CTX-M-9 against ceftazidime. The other two had 75- to 300-fold-lower Km values than CTX-M-9 against ceftazidime. The current emergence of CTX-M β-lactamases with improved activity against ceftazidime may therefore be the beginning of an evolutionary process which might subsequently generate a great diversity of CTX-M-type ceftazidimases.

scholarly journals Comparative Characterization of CTX-M-64 and CTX-M-14 Provides Insights into the Structure and Catalytic Activity of the CTX-M Class of Enzymes

2016 ◽  
Vol 60 (10) ◽  
pp. 6084-6090 ◽  
Author(s):  
Dandan He ◽  
Jiachi Chiou ◽  
Zhenling Zeng ◽  
Edward Wai-Chi Chan ◽  
Jian-Hua Liu ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Amino Acid ◽  
Active Site ◽  
Structural Integrity ◽  
Amino Acid Residues ◽  
Active Site Residues ◽  
Significant Difference ◽  
Function Relationship ◽  
Alignment Analysis ◽  
M 14

ABSTRACTClinical isolates producing hybrid CTX-M β-lactamases, presumably due to recombination between theblaCTX-M-15andblaCTX-M-14elements, have emerged in recent years. Among the hybrid enzymes, CTX-M-64 and CTX-M-14 display the most significant difference in catalytic activity. This study aims to investigate the mechanisms underlying such differential enzymatic activities in order to provide insight into the structure/function relationship of this class of enzymes. Sequence alignment analysis showed that the major differences between the amino acid composition of CTX-M-64 and CTX-M-14 lie at both the N and C termini of the enzymes. Single or multiple amino acid substitutions introduced into CTX-M-64 and CTX-M-14 were found to produce only minor effects on hydrolytic functions; such a finding is consistent with the notion that the discrepancy between the functional activities of the two enzymes is not the result of only a few amino acid changes but is attributable to interactions between a unique set of amino acid residues in each enzyme. This theory is supported by the results of the thermal stability assay, which confirmed that CTX-M-64 is significantly more stable than CTX-M-14. Our data confirmed that, in addition to the important residues located in the active site, residues distal to the active site also contribute to the catalytic activity of the enzyme through stabilizing its structural integrity.

scholarly journals Synergistic effects of functionally distinct substitutions in β-lactamase variants shed light on the evolution of bacterial drug resistance

2018 ◽  
Vol 293 (46) ◽  
pp. 17971-17984 ◽  
Author(s):  
Meha P. Patel ◽  
Liya Hu ◽  
Cameron A. Brown ◽  
Zhizeng Sun ◽  
Carolyn J. Adamski ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Conformational Changes ◽  
Active Site ◽  
Enzyme Stability ◽  
Synergistic Effects ◽  
Catalytic Efficiency ◽  
Bacterial Drug Resistance ◽  
Natural Variant ◽  
Loop Conformation ◽  
Shed Light

The CTX-M β-lactamases have emerged as the most widespread extended-spectrum β-lactamases (ESBLs) in Gram-negative bacteria. These enzymes rapidly hydrolyze cefotaxime, but not the related cephalosporin, ceftazidime. ESBL variants have evolved, however, that provide enhanced ceftazidime resistance. We show here that a natural variant at a nonactive site, i.e. second-shell residue N106S, enhances enzyme stability but reduces catalytic efficiency for cefotaxime and ceftazidime and decreases resistance levels. However, when the N106S variant was combined with an active-site variant, D240G, that enhances enzyme catalytic efficiency, but decreases stability, the resultant double mutant exhibited higher resistance levels than predicted on the basis of the phenotypes of each variant. We found that this epistasis is due to compensatory effects, whereby increased stability provided by N106S overrides its cost of decreased catalytic activity. X-ray structures of the variant enzymes in complex with cefotaxime revealed conformational changes in the active-site loop spanning residues 103–106 that were caused by the N106S substitution and relieve steric strain to stabilize the enzyme, but also alter contacts with cefotaxime and thereby reduce catalytic activity. We noted that the 103–106 loop conformation in the N106S-containing variants is different from that of WT CTX-M but nearly identical to that of the non-ESBL, TEM-1 β-lactamase, having a serine at the 106 position. Therefore, residue 106 may serve as a “switch” that toggles the conformations of the 103–106 loop. When it is serine, the loop is in the non-ESBL, TEM-like conformation, and when it is asparagine, the loop is in a CTX-M–like, cefotaximase-favorable conformation.

scholarly journals Simultaneous Enhancement of Thermostability and Catalytic Activity of a Metagenome-Derived β-Glucosidase Using Directed Evolution for the Biosynthesis of Butyl Glucoside

2019 ◽  
Vol 20 (24) ◽  
pp. 6224 ◽  
Author(s):  
Bangqiao Yin ◽  
Qinyan Hui ◽  
Muhammad Kashif ◽  
Ran Yu ◽  
Si Chen ◽  
...  
Keyword(s):  
Amino Acids ◽  
Catalytic Activity ◽  
Directed Evolution ◽  
Catalytic Efficiency ◽  
Raw Material ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Substitutions ◽  
The Novel ◽  
Wild Type

Butyl glucoside synthesis using bioenzymatic methods at high temperatures has gained increasing interest. Protein engineering using directed evolution of a metagenome-derived β-glucosidase of Bgl1D was performed to identify enzymes with improved activity and thermostability. An interesting mutant Bgl1D187 protein containing five amino acid substitutions (S28T, Y37H, D44E, R91G, and L115N), showed catalytic efficiency (kcat/Km of 561.72 mM−1 s−1) toward ρ-nitrophenyl-β-d-glucopyranoside (ρNPG) that increased by 23-fold, half-life of inactivation by 10-fold, and further retained transglycosidation activity at 50 °C as compared with the wild-type Bgl1D protein. Site-directed mutagenesis also revealed that Asp44 residue was essential to β-glucosidase activity of Bgl1D. This study improved our understanding of the key amino acids of the novel β-glucosidases and presented a raw material with enhanced catalytic activity and thermostability for the synthesis of butyl glucosides.

scholarly journals Four amino acids define the CO2binding pocket of enoyl-CoA carboxylases/reductases

2019 ◽  
Vol 116 (28) ◽  
pp. 13964-13969 ◽  
Author(s):  
Gabriele M. M. Stoffel ◽  
David Adrian Saez ◽  
Hasan DeMirci ◽  
Bastian Vögeli ◽  
Yashas Rao ◽  
...  
Keyword(s):  
Amino Acids ◽  
Active Site ◽  
Catalytic Efficiency ◽  
Catalytic Cycle ◽  
Carboxylating Enzymes ◽  
Order Of Magnitude ◽  
Ordered Water ◽  
And Control ◽  
Atmospheric Concentrations ◽  
Plant Enzyme

Carboxylases are biocatalysts that capture and convert carbon dioxide (CO2) under mild conditions and atmospheric concentrations at a scale of more than 400 Gt annually. However, how these enzymes bind and control the gaseous CO2molecule during catalysis is only poorly understood. One of the most efficient classes of carboxylating enzymes are enoyl-CoA carboxylases/reductases (Ecrs), which outcompete the plant enzyme RuBisCO in catalytic efficiency and fidelity by more than an order of magnitude. Here we investigated the interactions of CO2within the active site of Ecr fromKitasatospora setae. Combining experimental biochemistry, protein crystallography, and advanced computer simulations we show that 4 amino acids, N81, F170, E171, and H365, are required to create a highly efficient CO2-fixing enzyme. Together, these 4 residues anchor and position the CO2molecule for the attack by a reactive enolate created during the catalytic cycle. Notably, a highly ordered water molecule plays an important role in an active site that is otherwise carefully shielded from water, which is detrimental to CO2fixation. Altogether, our study reveals unprecedented molecular details of selective CO2binding and C–C-bond formation during the catalytic cycle of nature’s most efficient CO2-fixing enzyme. This knowledge provides the basis for the future development of catalytic frameworks for the capture and conversion of CO2in biology and chemistry.

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.

scholarly journals A Promiscuous Bacterial P450: The Unparalleled Diversity of BM3 in Pharmaceutical Metabolism

2021 ◽  
Vol 22 (21) ◽  
pp. 11380
Author(s):  
Sian Thistlethwaite ◽  
Laura N. Jeffreys ◽  
Hazel M. Girvan ◽  
Kirsty J. McLean ◽  
Andrew W. Munro
Keyword(s):  
Active Site ◽  
Chemical Properties ◽  
Catalytic Efficiency ◽  
Attractive Alternative ◽  
Wild Type ◽  
Metabolite Production ◽  
The Past ◽  
Valuable Compounds ◽  
Key Residues ◽  
Pharmaceutical Production

CYP102A1 (BM3) is a catalytically self-sufficient flavocytochrome fusion protein isolated from Bacillus megaterium, which displays similar metabolic capabilities to many drug-metabolizing human P450 isoforms. BM3′s high catalytic efficiency, ease of production and malleable active site makes the enzyme a desirable tool in the production of small molecule metabolites, especially for compounds that exhibit drug-like chemical properties. The engineering of select key residues within the BM3 active site vastly expands the catalytic repertoire, generating variants which can perform a range of modifications. This provides an attractive alternative route to the production of valuable compounds that are often laborious to synthesize via traditional organic means. Extensive studies have been conducted with the aim of engineering BM3 to expand metabolite production towards a comprehensive range of drug-like compounds, with many key examples found both in the literature and in the wider industrial bioproduction setting of desirable oxy-metabolite production by both wild-type BM3 and related variants. This review covers the past and current research on the engineering of BM3 to produce drug metabolites and highlights its crucial role in the future of biosynthetic pharmaceutical production.

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