Discrimination of Esterase and Peptidase Activities of Acylaminoacyl Peptidase from Hyperthermophilic Aeropyrum pernix K1 by a Single Mutation

Wang, Qiuyan; Yang, Guangyu; Yan-li, Liu; Feng, Yan

doi:10.1074/jbc.m601015200

Cited by 32 publications

(32 citation statements)

References 25 publications

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“…Thus, 4-nitrophenyl caprylate is a good substrate for ApAAP (40). Table 2 shows that the values of k cat /K m for the mutant enzyme reactions diminished by 2 orders of magnitude.…”

Section: The Binding Strengths Of the Oligopeptide Are Similar For Thmentioning

confidence: 99%

Structure and Catalysis of Acylaminoacyl Peptidase

Harmat

Domokos

Menyhárd

et al. 2011

Journal of Biological Chemistry

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Acylaminoacyl peptidase from Aeropyrum pernix is a homodimer that belongs to the prolyl oligopeptidase family. The monomer subunit is composed of one hydrolase and one propeller domain. Previous crystal structure determinations revealed that the propeller domain obstructed the access of substrate to the active site of both subunits. Here we investigated the structure and the kinetics of two mutant enzymes in which the aspartic acid of the catalytic triad was changed to alanine or asparagine. Using different substrates, we have determined the pH dependence of specificity rate constants, the rate-limiting step of catalysis, and the binding of substrates and inhibitors. The catalysis considerably depended both on the kind of mutation and on the nature of the substrate. The results were interpreted in terms of alterations in the position of the catalytic histidine side chain as demonstrated with crystal structure determination of the native and two mutant structures (D524N and D524A). Unexpectedly, in the homodimeric structures, only one subunit displayed the closed form of the enzyme. The other subunit exhibited an open gate to the catalytic site, thus revealing the structural basis that controls the oligopeptidase activity. The open form of the native enzyme displayed the catalytic triad in a distorted, inactive state. The mutations affected the closed, active form of the enzyme, disrupting its catalytic triad. We concluded that the two forms are at equilibrium and the substrates bind by the conformational selection mechanism.

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“…Thus, 4-nitrophenyl caprylate is a good substrate for ApAAP (40). Table 2 shows that the values of k cat /K m for the mutant enzyme reactions diminished by 2 orders of magnitude.…”

Section: The Binding Strengths Of the Oligopeptide Are Similar For Thmentioning

confidence: 99%

Structure and Catalysis of Acylaminoacyl Peptidase

Harmat

Domokos

Menyhárd

et al. 2011

Journal of Biological Chemistry

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“…The latter is the catalytic domain, which includes the active site and a Ser445-Asp524-His556 catalytic triad. Moreover, we found a conserved residue in the active site (Arg526) which plays a crucial role in catalysis and substrate discrimination (Wang et al, 2006). Saturation mutagenesis at this position had dramatic effects on esterase and peptidase activities.…”

Section: Introductionmentioning

confidence: 99%

“…Saturation mutagenesis at this position had dramatic effects on esterase and peptidase activities. Whereas the esterase activity of the wild type enzyme for p-nitrophenyl caprylate (pNP-C8) was 7 times higher than its peptidase activity for Ac-Leu-pnitroanilide, the esterase activities of mutants R526V and R526E were about 150-and 785-fold higher than their peptidase activities, respectively, using the same substrates (Wang et al, 2006). Further characterization showed that the mutants possessed hydrolytic activity towards a wide range of p-nitrophenyl alkanoate esters, with optimal acyl chain lengths ranging from C4 to C8.…”

Section: Introductionmentioning

confidence: 99%

“…Variants of apAAP were expressed in E. coli BL21-CodonPlus (DE3)-RIL and purified as described previously (Wang et al, 2006). Esterase, thioesterase, and peptidase activities were determined according to previously described protocols, using pNP-C12, Smethyl thiobutanoate, and Ac-Leu-p-nitroanilide as substrates, respectively (Mandrich et al, 2006;Yang et al, 2009).…”

Section: Protein Expression Purification and Enzymatic Assaysmentioning

confidence: 99%

“…Molecular modeling of the apAAP mutants and substrate docking were performed according to a previously described method (Wang et al, 2006), except that pNP-C12 was used as a ligand instead of pNP-C8. Briefly, mutations were introduced by MODELLER 9v7 (MartiRenom et al, 2000), based on a 1.8 Å crystallographic structure of wild type apAAP (PDB: 1VE6) and scored by PROCHECK (Laskowski et al, 1993) and Profile3D (Luthy et al, 1992) in the Insight II software package (Accelrys Inc., San Diego, CA).…”

Section: Molecular Modeling and Substrate Dockingmentioning

confidence: 99%

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Switch of substrate specificity of hyperthermophilic acylaminoacyl peptidase by combination of protein and solvent engineering

Liu

Yang

et al. 2011

Protein Cell

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The inherent evolvability of promiscuous enzymes endows them with great potential to be artificially evolved for novel functions. Previously, we succeeded in transforming a promiscuous acylaminoacyl peptidase (apAAP) from the hyperthermophilic archaeon Aeropyrum pernix K1 into a specific carboxylesterase by making a single mutation. In order to fulfill the urgent requirement of thermostable lipolytic enzymes, in this paper we describe how the substrate preference of apAAP can be further changed from p-nitrophenyl caprylate (pNP-C8) to p-nitrophenyl laurate (pNP-C12) by protein and solvent engineering. After one round of directed evolution and subsequent saturation mutagenesis at selected residues in the active site, three variants with enhanced activity towards pNP-C12 were identified. Additionally, a combined mutant W474V/F488G/R526V/ T560W was generated, which had the highest catalytic efficiency (k cat /K m ) for pNP-C12, about 71-fold higher than the wild type. Its activity was further increased by solvent engineering, resulting in an activity enhancement of 280-fold compared with the wild type in the presence of 30% DMSO. The structural basis for the improved activity was studied by substrate docking and molecular dynamics simulation. It was revealed that W474V and F488G mutations caused a significant change in the geometry of the active center, which may facilitate binding and subsequent hydrolysis of bulky substrates. In conclusion, the combination of protein and solvent engineering may be an effective approach to improve the activities of promiscuous enzymes and could be used to create naturally rare hyperthermophilic enzymes.

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Recent progress in engineering α/β hydrolase‐fold family members

Qian¹,

Fields²,

Yu³

et al. 2007

Biotechnology Journal

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The members of the alpha/beta hydrolase-fold family represent a functionally versatile group of enzymes with many important applications in biocatalysis. Given the technical significance of alpha/beta hydrolases in processes ranging from the kinetic resolution of enantiomeric precursors for pharmaceutical compounds to bulk products such as laundry detergent, optimizing and tailoring enzymes for these applications presents an ongoing challenge to chemists, biochemists, and engineers alike. A review of the recent literature on alpha/beta hydrolase engineering suggests that the early successes of "random processes" such as directed evolution are now being slowly replaced by more hypothesis-driven, focused library approaches. These developments reflect a better understanding of the enzymes' structure-function relationship and improved computational resources, which allow for more sophisticated search and prediction algorithms, as well as, in a very practical sense, the realization that bigger is not always better.

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Discrimination of Esterase and Peptidase Activities of Acylaminoacyl Peptidase from Hyperthermophilic Aeropyrum pernix K1 by a Single Mutation

Cited by 32 publications

References 25 publications

Structure and Catalysis of Acylaminoacyl Peptidase

Structure and Catalysis of Acylaminoacyl Peptidase

Switch of substrate specificity of hyperthermophilic acylaminoacyl peptidase by combination of protein and solvent engineering

Recent progress in engineering α/β hydrolase‐fold family members

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