Molecular Basis of Perhydrolase Activity in Serine Hydrolases

Bernhardt, Peter; Hult, Karl; Kazlauskas, Romas J.

doi:10.1002/anie.200463006

Cited by 69 publications

(56 citation statements)

References 36 publications

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“…However, the Ser-His-Asp catalytic triad of serine hydrolases is not the only determinant for perhydrolase activity because some of these enzymes do not exhibit any perhydrolysis activity. 105 Accordingly, some authors proposed an alternative mechanism where the catalytic serine stabilizes the carboxylic acid substrate with a hydrogen bond instead of forming an acyl-enzyme intermediate. 110 An explanation proposed to elucidate the difference in activities between hydrolases and perhydrolases concerns the electronegative microenvironment of the active site.…”

Section: Ii1 Lipase-catalyzed Perhydrolysismentioning

confidence: 99%

The use of lipases as biocatalysts for the epoxidation of fatty acids and phenolic compounds

et al. 2014

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Lipases are versatile enzymes that can be used for various kinds of biocatalyzed reactions. Owing to their selectivity and their mild reaction conditions, they can be often considered as more interesting than classical chemical catalysts. Besides their application in oil and fat processes, these enzymes have proved to be very attractive for other lipase-catalyzed reactions. This review discusses the latest results where lipases are used for the epoxidation of lipid substrates (namely fatty acids and their derivatives) and phenolic compounds. This chemo-enzymatic process involves a two step synthesis where the biocatalyst acts as a perhydrolase to produce peracids, which then act as catalysts to epoxidize double bonds. Various factors govern the efficiency of the reaction in terms of kinetics, yields and enzyme stability.These parameters are evaluated and discussed herein. (Résumé d'auteur

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Section: Ii1 Lipase-catalyzed Perhydrolysismentioning

confidence: 99%

The use of lipases as biocatalysts for the epoxidation of fatty acids and phenolic compounds

et al. 2014

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“…An esterase from Pseudomonas fluorescens (PFE) has been reengineered to improve the ratio of perhydrolytic to hydrolytic activity up to 2600-fold. 12 Semirational design was used to generate PFE variants (L29P, F57H, F93H, D99E, F125A, F227I) by site-directed mutagenesis. Esterase variants were purified and characterized based on conversion of monochlorodimedone.…”

Section: Introductionmentioning

confidence: 99%

Fluorescent Assay for Directed Evolution of Perhydrolases

et al. 2012

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Directed evolution offers opportunities to improve promiscuous activities of hydrolases in rounds of diversity generation and high-throughput screening. In this article, we developed and validated a screening platform to improve the perhydrolytic activity of proteases and likely other hydrolases (e.g., lipases or esterases). Key was the development of a highly sensitive fluorescent assay (sensitivity in the µM range) based on 3-carboxy-7-hydroxycoumarin (HCC) formation. HCC is released through an hypobromite-mediated oxidation of 7-(4′-aminophenoxy)-3-carboxycoumarin (APCC), which enables for the first time a continuous measurement of peroxycarboxylic acid formation with a standard deviation of 11% in microtiter plates with a wide pH range window (5-9). As example, subtilisin Carlsberg was subjected to site saturation mutagenesis at position G165, yielding a variant T58A/G165L/L216W with 5.4-fold increased k cat for perhydrolytic activity compared with wild type.

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“…In addition, it is interesting to note that 1 and other peracids are produced by a number of bacterial and eukaryotic enzymes. [39][40][41][42][43] This offers the possibility that peracids could be involved in the physiological or pathophysiological regulation of PTPs and other cysteine-dependent enzymes. Overall our work provides new evidence that endogenous and exogenous organic hydroperoxides have the potential to regulate or dysregulate cellular signaling pathways.…”

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confidence: 99%

Oxidative inactivation of protein tyrosine phosphatase 1B by organic hydroperoxides

Bhattacharya

Labutti

Seiner

et al. 2008

Bioorganic & Medicinal Chemistry Letters

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Protein tyrosine phosphatases (PTPs) are cysteine-dependent enzymes that play a central role in cell signaling. Organic hydroperoxides cause thiol-reversible, oxidative inactivation of PTP1B in a manner that mirrors the endogenous signaling agent hydrogen peroxide.Protein tyrosine phosphatases (PTPs) are cysteine-dependent enzymes that catalyze the hydrolytic removal of phosphate groups from tyrosine residues in proteins (Scheme 1). [1][2][3] Thus, PTPs, in concert with protein tyrosine kinases, play a central role in cell signaling by regulating the phosphorylation status and, in turn, the functional properties, of target proteins in various signal transduction pathways. 4,5 The cellular activity of some PTPs is regulated by hydrogen peroxide (H 2 O 2 ) that is produced as a second messenger in response to extracellular stimuli such as insulin, epidermal growth factor, and platelet derived growth factor. 6-9 H 2 O 2 inactivates PTPs via oxidation of the active site cysteine thiol residue to a sulfenic acid. 6,10 In some cases, the cysteine sulfenic acid undergoes subsequent conversion to an active site sulfenyl amide linkage 11-13 or disulfide. 14,15 This type of oxidative inactivation of PTPs is slowly reversed upon reaction of the inactivated enzyme with biological thiols (Scheme 1). [10][11][12][13][14][15] Given the biological importance of PTPs, it may be useful to identify organic reagents that regulate PTP activity through redox mechanisms. [16][17][18][19][20] With this in mind, we examined the ability of several organic hydroperoxides (1-4) to effect thiol-reversible, oxidative inactivation of the archtypal member of the PTP family, PTP1B. 21 We find that peracetic acid (1) is a potent, time-dependent inactivator of the catalytic subunit of PTP1B (a.a. 1-322) (Fig. 1A). The observation of time-dependent inactivation is consistent with a process involving covalent modification of the enzyme. 22 A good linear fit is obtained in a plot of the apparent rates of inactivation versus concentration indicating that the inactivation reaction is a second-order process (Fig. 1B). The slope of the line reveals a rate constant of 2.1 ± 0.2 × 10 4 M −1 s −1 for the reaction of 1 with PTP1B. For comparison, hydrogen peroxide inactivates PTP1B with a rate constant of 10 M −1 s −1 . 10 The inactivation of PTP1B (250 nM) by 1 (1 μM, 3 min) is not reversed upon removal of excess inactivator by gel filtration of the enzyme through G25 Sephadex. The inactivation process is significantly slowed in the presence of phosphate ion, which is an active site-directed inhibitor of the enzyme (K i = 17 mM). Specifically, treatment of the enzyme with 1 (15 μM) for 15 s inactivates 88% of the enzyme under standard conditions, while in the presence of phosphate *Corresponding author. Tel.: +1-573-882-6763; fax: +1-573-882-2754 gatesk@missouri.edu. NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript ion (50 mM) only 49% inactivation is observed. Together, the results suggest that 1 inactiv...

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Molecular Basis of Perhydrolase Activity in Serine Hydrolases

Cited by 69 publications

References 36 publications

The use of lipases as biocatalysts for the epoxidation of fatty acids and phenolic compounds

The use of lipases as biocatalysts for the epoxidation of fatty acids and phenolic compounds

Fluorescent Assay for Directed Evolution of Perhydrolases

Oxidative inactivation of protein tyrosine phosphatase 1B by organic hydroperoxides

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