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1.
Foti RS Honaker M Nath A Pearson JT Buttrick B Isoherranen N Atkins WM 《Biochemistry》2011,50(13):2387-2393
Catalytically promiscuous enzymes are intermediates in the evolution of new function from an existing pool of protein scaffolds. However, promiscuity will only confer an evolutionary advantage if other useful properties are not compromised or if there is no "negative trade-off" induced by the mutations that yield promiscuity. Therefore, identification and characterization of negative trade-offs incurred during the emergence of promiscuity are required to further develop the evolutionary models and to optimize in vitro evolution. One potential negative trade-off of catalytic promiscuity is increased susceptibility to inhibition, or inhibitory promiscuity. Here we exploit cytochrome P450s (CYPs) as a model protein scaffold that spans a vast range of catalytic promiscuity and apply a quantitative index to determine the relationship between promiscuity of catalysis and promiscuity of inhibition for a series of homologues. The aim of these studies is to begin to identify properties that, in general, correlate with catalytic promiscuity, hypothetically such as inhibitory promiscuity. Interestingly, the data indicate that the potential negative trade-off of inhibitory promiscuity is nearly insignificant because even highly substrate specific CYPs have high inhibitory promiscuity, with little incremental increase in susceptibility to inhibitory interactions as the substrate promiscuity increases across the series of enzymes. In the context of evolution, inhibitory promiscuity is not an obligate negative trade-off for catalytic promiscuity. 相似文献
2.
《Journal of structural biology》2022,214(1):107835
Enzyme promiscuity is the ability of (some) enzymes to perform alternate reactions or catalyze non-cognate substrate(s). The latter is referred to as substrate promiscuity, widely studied for its biotechnological applications and understanding enzyme evolution. Insights into the structural basis of substrate promiscuity would greatly benefit the design and engineering of enzymes. Previous studies on some enzymes have suggested that flexibility, hydrophobicity, and active site protonation state could play an important role in enzyme promiscuity. However, it is not known yet whether substrate promiscuous enzymes have distinctive structural characteristics compared to specialist enzymes, which are specific for a substrate. In pursuit to address this, we have systematically compared substrate/catalytic binding site structural features of substrate promiscuous with those of specialist enzymes. For this, we have carefully constructed dataset of substrate promiscuous and specialist enzymes. On careful analysis, surprisingly, we found that substrate promiscuous and specialist enzymes are similar in various binding/catalytic site structural features such as flexibility, surface area, hydrophobicity, depth, and secondary structures. Recent studies have also alluded that promiscuity is widespread among enzymes. Based on these observations, we propose that substrate promiscuity could be defined as a continuum feature that varies from narrow (specialist) to broad range of substrate preferences. Moreover, diversity of conformational states of an enzyme accessible for ligand binding may possibly regulate its substrate preferences. 相似文献
3.
Introductory courses in biochemistry teach that enzymes are specific for their substrates and the reactions they catalyze. Enzymes diverging from this statement are sometimes called promiscuous. It has been suggested that relaxed substrate and reaction specificities can have an important role in enzyme evolution; however, enzyme promiscuity also has an applied aspect. Enzyme condition promiscuity has, for a long time, been used to run reactions under conditions of low water activity that favor ester synthesis instead of hydrolysis. Together with enzyme substrate promiscuity, it is exploited in numerous synthetic applications, from the laboratory to industrial scale. Furthermore, enzyme catalytic promiscuity, where enzymes catalyze accidental or induced new reactions, has begun to be recognized as a valuable research and synthesis tool. Exploiting enzyme catalytic promiscuity might lead to improvements in existing catalysts and provide novel synthesis pathways that are currently not available. 相似文献
4.
Lipases are the most widely used class of enzymes in organic synthesis. Availability of large number of commercial preparations, their broad specificity and relatively better stability (as compared to other enzymes) in media containing organic solvents have all been contributing factors for this. This review has a sharp focus on their specificity. The recent results with catalytic promiscuity have shown that lipases are even more versatile than thought so far. These results have also prompted workers to rationalize the classification of specificity in terms of substrate promiscuity, condition promiscuity and catalytic promiscuity. The review also attempts to recast the known information on specificity of lipases in the context of enzyme promiscuity. Lipases can exhibit regiospecificity, specificity in terms of fatty acids, nature of the alcohol, and stereospecificity (distinction between sn-1 and sn-3 position on the triglyceride). Lipases show varied stability toward presence of organic solvents, extreme pH conditions and ionic liquids. In low water media, condition promiscuity in terms of esterification, transesterification and interesterification has been extensively studied. The catalytic promiscuity is being increasingly observed for CC bond formation reactions. Finally, the beneficial consequences of this promiscuous behavior in biotechnology sectors are also discussed. 相似文献
5.
Matthew T. Honaker Mauro Acchione Wei Zhang Bengt Mannervik William M. Atkins 《The Journal of biological chemistry》2013,288(25):18599-18611
The role of conformational ensembles in enzymatic reactions remains unclear. Discussion concerning “induced fit” versus “conformational selection” has, however, ignored detoxication enzymes, which exhibit catalytic promiscuity. These enzymes dominate drug metabolism and determine drug-drug interactions. The detoxication enzyme glutathione transferase A1–1 (GSTA1–1), exploits a molten globule-like active site to achieve remarkable catalytic promiscuity wherein the substrate-free conformational ensemble is broad with barrierless transitions between states. A quantitative index of catalytic promiscuity is used to compare engineered variants of GSTA1–1 and the catalytic promiscuity correlates strongly with characteristics of the thermodynamic partition function, for the substrate-free enzymes. Access to chemically disparate transition states is encoded by the substrate-free conformational ensemble. Pre-steady state catalytic data confirm an extension of the conformational selection model, wherein different substrates select different starting conformations. The kinetic liability of the conformational breadth is minimized by a smooth landscape. We propose that “local” molten globule behavior optimizes detoxication enzymes. 相似文献
6.
Several proteases, especially pepsin, were observed to directly catalyze asymmetric aldol reactions. Pepsin, which displays well-documented proteolytic activity under acidic conditions, exhibited distinct catalytic activity in a crossed aldol reaction between acetone and 4-nitrobenzaldehyde with high yield and moderate enantioselectivity. Fluorescence experiments indicated that under neutral pH conditions, pepsin maintains its native conformation and that the natural structure plays an important role in biocatalytic promiscuity. Moreover, no significant loss of enantioselectivity was found even after four cycles of catalyst recycling, showing the high stability of pepsin under the selected aqueous reaction conditions. This case of biocatalytic promiscuity not only expands the application of proteases to new chemical transformations, but also could be developed into a potentially valuable method for green organic synthesis. 相似文献
7.
《Bioorganic & medicinal chemistry》2020,28(1):115238
Currently available volumes of compounds and biological activity data enable large-scale analyses of compound promiscuity (multi-target activity). To aid in the exploration of structure-promiscuity relationships, promiscuity cliffs (PCs) were introduced previously. In analogy to activity cliffs, PCs were defined as pairs of structurally analogous compounds with large differences in the number of targets they are active against. Hence, PCs reveal small chemical modifications that are implicated in promiscuity. As introduced originally, PCs were identified by applying the matched molecular pair formalism and were thus confined to changes at a single substitution site. Herein, PCs with multiple substitution sites are introduced and a pilot study on a large collection of protein kinase inhibitors is reported, which provide excellent test cases for promiscuity analysis. For dual-site PCs (dsPCs), which dominated the distribution of multi-site PCs, an extended data structure was generated comprising a dsPC and two single-site analogs accounting for individual substitutions. Using a canonical representation, extended dsPCs are intuitive and easy to interpret from a chemical perspective. The analog quartet representing an extended dsPC is rich in structure-promiscuity relationship information and makes it possible to evaluate the potential interplay of chemical modifications implicated in promiscuity. Furthermore, extended dsPCs provide insights into possible experimental causes of apparent differences in analog promiscuity such as varying test frequencies. Hence, the newly introduced PC format should be of interest for exploring origins of compound promiscuity in medicinal chemistry and for formulating experimentally testable target hypotheses for analogs. 相似文献
8.
Promiscuity, the basis for the evolution of new functions through ‘tinkering’ of residues in the vicinity of the catalytic site, is yet to be quantitatively defined. We present a computational method Promiscuity Indices Estimator (PROMISE) - based on signatures derived from the spatial and electrostatic properties of the catalytic residues, to estimate the promiscuity (PromIndex) of proteins with known active site residues and 3D structure. PromIndex reflects the number of different active site signatures that have congruent matches in close proximity of its native catalytic site, the quality of the matches and difference in the enzymatic activity. Promiscuity in proteins is observed to follow a lognormal distribution (μ = 0.28, σ = 1.1 reduced chi-square = 3.0E-5). The PROMISE predicted promiscuous functions in any protein can serve as the starting point for directed evolution experiments. PROMISE ranks carboxypeptidase A and ribonuclease A amongst the more promiscuous proteins. We have also investigated the properties of the residues in the vicinity of the catalytic site that regulates its promiscuity. Linear regression establishes a weak correlation (R2∼0.1) between certain properties of the residues (charge, polar, etc) in the neighborhood of the catalytic residues and PromIndex. A stronger relationship states that most proteins with high promiscuity have high percentages of charged and polar residues within a radius of 3 Å of the catalytic site, which is validated using one-tailed hypothesis tests (P-values∼0.05). Since it is known that these characteristics are key factors in catalysis, their relationship with the promiscuity index cross validates the methodology of PROMISE. 相似文献
9.
Despite many years of intensive study, the natures of turnover-limiting and enantio-determining steps in catalytic asymmetric hydrogenation of prochiral enamides are poorly understood. An intriguing set of studies involving isotopic labeling distributions in catalytic enamide hydrogenation reactions were reported by Brown and Parker (Organometallics, 1 (1982) 950–956) more than a decade ago. In this paper we report the results of studies re-examining the application of isotopic probes to the catalytic hydrogenation enamides. These results provide some insights into the nature of the H2 activation step in enamide hydrogenation. 相似文献
10.
Kazlauskas RJ 《Current opinion in chemical biology》2005,9(2):195-201
Catalytic promiscuity - the ability of a single active site to catalyse more than one chemical transformation - has a natural role in evolution and occasionally in biosynthesis of secondary metabolites. Catalytic promiscuity is more widespread than often recognized. Recent success in adding and enhancing such catalytic activities by protein engineering suggests new potential applications in enzyme-catalyzed organic synthesis. 相似文献
11.
Honaker MT Acchione M Sumida JP Atkins WM 《The Journal of biological chemistry》2011,286(49):42770-42776
Enzymological paradigms have shifted recently to acknowledge the biological importance of catalytic promiscuity. However, catalytic promiscuity is a poorly understood property, and no thermodynamic treatment has described the conformational landscape of promiscuous versus substrate-specific enzymes. Here, two structurally similar glutathione transferase (GST, glutathione S-transferase) isoforms with high specificity or high promiscuity are compared. Differential scanning calorimetry (DSC) indicates a reversible low temperature transition for the promiscuous GSTA1-1 that is not observed with substrate-specific GSTA4-4. This transition is assigned to rearrangement of the C terminus at the active site of GSTA1-1 based on the effects of ligands and mutations. Near-UV and far-UV circular dichroism indicate that this transition is due to repacking of tertiary contacts with the remainder of the subunit, rather than "unfolding" of the C terminus per se. Analysis of the DSC data using a modified Landau theory indicates that the local conformational landscape of the active site of GSTA1-1 is smooth, with barrierless transitions between states. The partition function of the C-terminal states is a broad unimodal distribution at all temperatures within this DSC transition. In contrast, the remainder of the GSTA1-1 subunit and the GSTA4-4 protein exhibit folded and unfolded macrostates with a significant energy barrier separating them. Their partition function includes a sharp unimodal distribution of states only at temperatures that yield either folded or unfolded macrostates. At intermediate temperatures the partition function includes a bimodal distribution. The barrierless rearrangement of the GSTA1-1 active site within a local smooth energy landscape suggests a thermodynamic basis for catalytic promiscuity. 相似文献
12.
A unique "enzyme catalytic promiscuity" has recently been observed, wherein a phosphodiester and a phosphonate ester are hydrolyzed by a dinuclear aminopeptidase and its metal derivatives from Streptomyces griseus (SgAP) [Park, H. I., Ming, L.-J. (1999) Angew. Chem., Int. Ed. Engl. 38, 2914-2916 and Ercan, A., Park, H. I., Ming, L.-J. (2000) Chem. Commun. 2501-2502]. Because tetrahedral phosphocenters often serve as transition-state inhibitors toward the hydrolysis of the peptide, phosphoester hydrolysis by peptidases is thus not expected to occur effectively and must take place through a unique mechanism. Owing to the very different structures and mechanistic requirements between phosphoesters and peptides during hydrolysis, the study of this effective phosphodiester hydrolysis by SgAP may provide further insight into the action of this enzyme that is otherwise not obtainable from regular peptide substrates. We present herein a detailed investigation of both peptide and phosphodiester hydrolyses catalyzed by SgAP. The latter exhibits a first-order rate enhancement of 4 x 10(10)-fold compared to the uncatalyzed reaction at pH 7.0 and 25 degrees C. The results suggest that peptide and phosphodiester hydrolyses by SgAP may share a common reaction mechanism to a certain extent. However, their differences in pH dependence, phosphate and fluoride inhibition patterns, and proton inventory reflect that they must follow different pathways. Mechanisms for the two hydrolyses are drawn on the basis of the results, which provide the foundation for further investigation of the catalytic promiscuity of this enzyme by means of physical and molecular biology methods. The catalytic versatility of SgAP suggests that this enzyme may serve as a unique "natural model system" for further investigation of dinuclear hydrolysis. A better understanding of enzyme catalytic promiscuity is also expected to shed light on the evolution and action of enzymes. 相似文献
13.
A quantitative index of substrate promiscuity 总被引:1,自引:0,他引:1
Catalytic promiscuity is a widespread, but poorly understood, phenomenon among enzymes with particular relevance to the evolution of new functions, drug metabolism, and in vitro biocatalyst engineering. However, there is at present no way to quantitatively measure or compare this important parameter of enzyme function. Here we define a quantitative index of promiscuity (I) that can be calculated from the catalytic efficiencies of an enzyme toward a defined set of substrates. A weighted promiscuity index (J) that accounts for patterns of similarity and dissimilarity among the substrates in the set is also defined. Promiscuity indices were calculated for three different enzyme classes: eight serine and cysteine proteases, two glutathione S-transferase (GST) isoforms, and three cytochrome P450 (CYP) isoforms. The proteases ranged from completely specific (granzyme B, J = 0.00) to highly promiscuous (cruzain, J = 0.83). The four drug-metabolizing enzymes studied (GST A1-1 and the CYP isoforms) were highly promiscuous, with J values between 0.72 and 0.92; GST A4-4, involved in the clearance of lipid peroxidation products, is moderately promiscuous (J = 0.37). Promiscuity indices also allowed for studies of correlation between substrate promiscuity and an enzyme's activity toward its most-favored substrate, for each of the three enzyme classes. 相似文献
14.
Enzyme evolution is often constrained by aspects of catalysis. Sets of homologous proteins that catalyze different overall reactions but share an aspect of catalysis, such as a common partial reaction, are called mechanistically diverse superfamilies. The common mechanistic steps and structural characteristics of several of these superfamilies, including the enolase, Nudix, amidohydrolase, and haloacid dehalogenase superfamilies have been characterized. In addition, studies of mechanistically diverse superfamilies are helping to elucidate mechanisms of functional diversification, such as catalytic promiscuity. Understanding how enzyme superfamilies evolve is vital for accurate genome annotation, predicting protein functions, and protein engineering. 相似文献
15.
Ben-David M Elias M Filippi JJ Duñach E Silman I Sussman JL Tawfik DS 《Journal of molecular biology》2012,418(3-4):181-196
The origins of enzyme specificity are well established. However, the molecular details underlying the ability of a single active site to promiscuously bind different substrates and catalyze different reactions remain largely unknown. To better understand the molecular basis of enzyme promiscuity, we studied the mammalian serum paraoxonase 1 (PON1) whose native substrates are lipophilic lactones. We describe the crystal structures of PON1 at a catalytically relevant pH and of its complex with a lactone analogue. The various PON1 structures and the analysis of active-site mutants guided the generation of docking models of the various substrates and their reaction intermediates. The models suggest that promiscuity is driven by coincidental overlaps between the reactive intermediate for the native lactonase reaction and the ground and/or intermediate states of the promiscuous reactions. This overlap is also enabled by different active-site conformations: the lactonase activity utilizes one active-site conformation whereas the promiscuous phosphotriesterase activity utilizes another. The hydrolysis of phosphotriesters, and of the aromatic lactone dihydrocoumarin, is also driven by an alternative catalytic mode that uses only a subset of the active-site residues utilized for lactone hydrolysis. Indeed, PON1's active site shows a remarkable level of networking and versatility whereby multiple residues share the same task and individual active-site residues perform multiple tasks (e.g., binding the catalytic calcium and activating the hydrolytic water). Overall, the coexistence of multiple conformations and alternative catalytic modes within the same active site underlines PON1's promiscuity and evolutionary potential. 相似文献
16.
Hou L Honaker MT Shireman LM Balogh LM Roberts AG Ng KC Nath A Atkins WM 《The Journal of biological chemistry》2007,282(32):23264-23274
The structurally related glutathione S-transferase isoforms GSTA1-1 and GSTA4-4 differ greatly in their relative catalytic promiscuity. GSTA1-1 is a highly promiscuous detoxification enzyme. In contrast, GSTA4-4 exhibits selectivity for congeners of the lipid peroxidation product 4-hydroxynonenal. The contribution of protein dynamics to promiscuity has not been studied. Therefore, hydrogen/deuterium exchange mass spectrometry (H/DX) and fluorescence lifetime distribution analysis were performed with glutathione S-transferases A1-1 and A4-4. Differences in local dynamics of the C-terminal helix were evident as expected on the basis of previous studies. However, H/DX demonstrated significantly greater solvent accessibility throughout most of the GSTA1-1 sequence compared with GSTA4-4. A Phe-111/Tyr-217 aromatic-aromatic interaction in A4-4, which is not present in A1-1, was hypothesized to increase core packing. "Swap" mutants that eliminate this interaction from A4-4 or incorporate it into A1-1 yield H/DX behavior that is intermediate between the wild type templates. In addition, the single Trp-21 residue of each isoform was exploited to probe the conformational heterogeneity at the intrasubunit domain-domain interface. Excited state fluorescence lifetime distribution analysis indicates that this core residue is more conformationally heterogeneous in GSTA1-1 than in GSTA4-4, and this correlates with greater stability toward urea denaturation for GSTA4-4. The fluorescence distribution and urea sensitivity of the mutant proteins were intermediate between the wild type templates. The results suggest that the differences in protein dynamics of these homologs are global. The results suggest also the possible importance of extensive conformational plasticity to achieve high levels of functional promiscuity, possibly at the cost of stability. 相似文献
17.
Structure and chemistry of the p300/CBP and Rtt109 histone acetyltransferases: implications for histone acetyltransferase evolution and function 总被引:1,自引:0,他引:1
The recent structure and associated biochemical studies of the metazoan-specific p300/CBP and fungal-specific Rtt109 histone acetyltransferases (HATs) have provided new insights into the ancestral relationship between HATs and their functions. These studies point to a common HAT ancester that has evolved around a common structural framework to form HATs with divergent catalytic and substrate-binding properties. These studies also point to the importance of regulatory loops within HATs and autoacetylation in HAT function. Implications for future studies are discussed. 相似文献
18.
A closer look of the HECTic ubiquitin ligases 总被引:1,自引:0,他引:1
Zheng N 《Structure (London, England : 1993)》2003,11(1):5-6
In comparison with previous studies, a new crystal structure of a HECT ubiquitin ligase catalytic domain has revealed the structural basis of the conformational flexibility of the enzyme and provides insights into the mechanism underlying the ubiquitin transfer reaction in the polyubiquitination pathway. 相似文献
19.
Pseudomonas aeruginosa arylsulfatase (PAS) is a bacterial sulfatase capable of hydrolyzing a range of sulfate esters. Recently, it has been demonstrated to also show very high proficiency for phosphate ester hydrolysis. Such proficient catalytic promiscuity is significant, as promiscuity has been suggested to play an important role in enzyme evolution. Additionally, a comparative study of the hydrolyses of the p-nitrophenyl phosphate and sulfate monoesters in aqueous solution has demonstrated that despite superficial similarities, the two reactions proceed through markedly different transition states with very different solvation effects, indicating that the requirements for the efficient catalysis of the two reactions by an enzyme will also be very different (and yet they are both catalyzed by the same active site). This work explores the promiscuous phosphomonoesterase activity of PAS. Specifically, we have investigated the identity of the most likely base for the initial activation of the unusual formylglycine hydrate nucleophile (which is common to many sulfatases), and demonstrate that a concerted substrate-as-base mechanism is fully consistent with the experimentally observed data. This is very similar to other related systems, and suggests that, as far as the phosphomonoesterase activity of PAS is concerned, the sulfatase behaves like a "classical" phosphatase, despite the fact that such a mechanism is unlikely to be available to the native substrate (based on pK(a) considerations and studies of model systems). Understanding such catalytic versatility can be used to design novel artificial enzymes that are far more proficient than the current generation of designer enzymes. 相似文献
20.
Rhodanese catalyzes the sulfur-transfer reaction that transfers sulfur from thiosulfate to cyanide by a double-displacement mechanism, in which an active cysteine residue plays a central role. Previous studies indicated that the phage-shock protein E (PspE) from Escherichia coli is a rhodanese composed of a single active domain and is the only accessible rhodanese among the three single-domain rhodaneses in E. coli. To understand the catalytic mechanism of rhodanese at the molecular level, we determined the solution structures of the sulfur-free and persulfide-intermediate forms of PspE by nuclear magnetic resonance (NMR) spectroscopy and identified the active site by NMR titration experiments. To obtain further insights into the catalytic mechanism, we studied backbone dynamics by NMR relaxation experiments. Our results demonstrated that the overall structures in both sulfur-free and persulfide-intermediate forms are highly similar, suggesting that no significant conformational changes occurred during the catalytic reaction. However, the backbone dynamics revealed that the motional properties of PspE in its sulfur-free form are different from the persulfide-intermediate state. The conformational exchanges are largely enhanced in the persulfide-intermediate form of PspE, especially around the active site. The present structural and biochemical studies in combination with backbone dynamics provide further insights in understanding the catalytic mechanism of rhodanese. 相似文献