Switching Catalysis from Hydrolysis to Perhydrolysis in <i>Pseudomonas fluorescens</i> Esterase<sup>,</sup>

Yin, Tyler; Bernhardt, Peter; Morley, Krista L.; Jiang, Yun; Cheeseman, Jeremy D.; Purpero, Vincent M.; Schrag, Joseph D.; Kazlauskas, Romas J.

doi:10.1021/bi9021268

Cited by 52 publications

(56 citation statements)

References 55 publications

(81 reference statements)

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“…Perhydrolysis is a promiscuous unnatural reaction, the natural function of these esterases that catalyze perhydrolysis of carboxylic acids may be lactone hydrolysis. 55,56 …”

Section: Ec 1 Oxidoreductasesmentioning

confidence: 99%

How the Same Core Catalytic Machinery Catalyzes 17 Different Reactions: the Serine-Histidine-Aspartate Catalytic Triad of α/β-Hydrolase Fold Enzymes

2015

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Enzymes within a family often catalyze different reactions. In some cases, this variety stems from different catalytic machinery, but in other cases the machinery is identical; nevertheless, the enzymes catalyze different reactions. In this review, we examine the subset of α/β-hydrolase fold enzymes that contain the serine-histidine-aspartate catalytic triad. In spite of having the same protein fold and the same core catalytic machinery, these enzymes catalyze seventeen different reaction mechanisms. The most common reactions are hydrolysis of C–O, C–N and C–C bonds (Enzyme Classification (EC) group 3), but other enzymes are oxidoreductases (EC group 1), acyl transferases (EC group 2), lyases (EC group 4) or isomerases (EC group 5). Hydrolysis reactions often follow the canonical esterase mechanism, but eight variations occur where either the formation or cleavage of the acyl enzyme intermediate differs. The remaining eight mechanisms are lyase-type elimination reactions, which do not have an acyl enzyme intermediate and, in four cases, do not even require the catalytic serine. This diversity of mechanisms from the same catalytic triad stems from the ability of the enzymes to bind different substrates, from the requirements for different chemical steps imposed by these new substrates and, only in about half of the cases, from additional hydrogen bond partners or additional general acids/bases in the active site. This detailed analysis shows that binding differences and non-catalytic residues create new mechanisms and are essential for understanding and designing efficient enzymes.

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“…Perhydrolysis is a promiscuous unnatural reaction, the natural function of these esterases that catalyze perhydrolysis of carboxylic acids may be lactone hydrolysis. 55,56 …”

Section: Ec 1 Oxidoreductasesmentioning

confidence: 99%

How the Same Core Catalytic Machinery Catalyzes 17 Different Reactions: the Serine-Histidine-Aspartate Catalytic Triad of α/β-Hydrolase Fold Enzymes

2015

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“…However, introducing completely new catalytic activities by exploiting native functional groups remains a challenge with these approaches because a starting point with some activity is typically required for directed evolution. Mechanistically related activities have been introduced into enzymes but these approaches require homologs with the desired activity to borrow sequence or structural features from 4–12 .…”

Section: Introductionmentioning

confidence: 99%

Computational redesign of a mononuclear zinc metalloenzyme for organophosphate hydrolysis

Khare

Kipnis

Greisen³

et al. 2012

Nat Chem Biol

200

171

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The ability to redesign enzymes to catalyze non-cognate chemical transformations would have wide-ranging applications. We developed a computational method for repurposing the reactivity of active site functional groups of metalloenzymes to catalyze new reactions. Using this method, we engineered a zinc-containing murine adenosine deaminase to catalyze the hydrolysis of a model organophosphate with a catalytic efficiency kcat/Km ~104 M−1s−1 after directed evolution. In the high-resolution crystal structure of the enzyme, all but one of the designed residues adopt the designed conformation. The designed enzyme efficiently catalyzed the hydrolysis of the RP-isomer of a coumarinyl analog of the nerve agent cyclosarin, and showed striking substrate selectivity for coumarinyl leaving-groups. Computational redesign of native enzyme active sites complements directed evolution methods and offers a general approach for exploring their untapped catalytic potential for new reactivities.

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“…Site-directed mutagenesis of PFE to introduce this proline increased its perhydrolysis activity 43-fold and, as in other perhydrolases, moved the carbonyl oxygen of W28 closer to the active. [15,16] The O–O distance from W28 carbonyl oxygen to the active site S94-Oγ decreased to 5.5 Å. This L29P substitution fully accounted for the difference in perhydrolysis abilities between the esterase PFE and perhydrolases.…”

Section: Introductionmentioning

confidence: 93%

New Structural Motif for Carboxylic Acid Perhydrolases

Yin

Purpero

Fujii

et al. 2013

Chemistry A European J

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Some serine hydrolases also catalyze a promiscuous reaction -reversible perhydrolysis of carboxylic acids to make peroxycarboxylic acids. Five x-ray crystal structures of these carboxylic acid perhydrolases show a proline in the oxyanion loop. Here, we test whether this proline is essential for high perhydrolysis activity using Pseudomonas fluorescens esterase (PFE). The L29P variant of this esterase catalyzes perhydrolysis 43-fold faster (k cat comparison) than wild type. Surprisingly, saturation mutagenesis at the 29 position of PFE identified six other amino acid substitutions that increase perhydrolysis of acetic acid at least fourfold over wild type. The best variant, L29I PFE, catalyzed perhydrolysis 83 times faster (k cat comparison) than wild-type PFE and twice as fast as L29P PFE. Despite the different amino acid in the oxyanion loop, L29I PFE shows a similar selectivity for hydrogen peroxide over water as L29P PFE ( 0 = 170 M −1 vs. 160 M −1 ), and a similar fast formation of acetyl-enzyme (140 U/mg vs. 62 U/mg). X-ray crystal structures of L29I PFE with and without bound acetate show an unusual mixture of two different oxyanion loop conformations. The type II -turn conformation resembles the wild-type structure and is unlikely to increase perhydrolysis, but the type I -turn conformation creates a binding site for a second acetate. Modeling suggests that a previously proposed mechanism for L29P PFE can be extended to include L29I PFE where an acetate accepts a hydrogen bond to promote faster formation of the acetyl enzyme.

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Switching Catalysis from Hydrolysis to Perhydrolysis in Pseudomonas fluorescens Esterase^,

Cited by 52 publications

References 55 publications

How the Same Core Catalytic Machinery Catalyzes 17 Different Reactions: the Serine-Histidine-Aspartate Catalytic Triad of α/β-Hydrolase Fold Enzymes

How the Same Core Catalytic Machinery Catalyzes 17 Different Reactions: the Serine-Histidine-Aspartate Catalytic Triad of α/β-Hydrolase Fold Enzymes

Computational redesign of a mononuclear zinc metalloenzyme for organophosphate hydrolysis

New Structural Motif for Carboxylic Acid Perhydrolases

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Switching Catalysis from Hydrolysis to Perhydrolysis in Pseudomonas fluorescens Esterase,

Cited by 52 publications

References 55 publications

How the Same Core Catalytic Machinery Catalyzes 17 Different Reactions: the Serine-Histidine-Aspartate Catalytic Triad of α/β-Hydrolase Fold Enzymes

How the Same Core Catalytic Machinery Catalyzes 17 Different Reactions: the Serine-Histidine-Aspartate Catalytic Triad of α/β-Hydrolase Fold Enzymes

Computational redesign of a mononuclear zinc metalloenzyme for organophosphate hydrolysis

New Structural Motif for Carboxylic Acid Perhydrolases

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Switching Catalysis from Hydrolysis to Perhydrolysis in Pseudomonas fluorescens Esterase^,