Examining Reactivity and Specificity of Cytochrome c Peroxidase by using Combinatorial Mutagenesis

Wilming, Martin; Iffland, André; Tafelmeyer, Petra; Arrivoli, Claudio; Saudan, C; Johnsson, Kai

doi:10.1002/1439-7633(20021104)3:11<1097::aid-cbic1097>3.0.co;2-4

Cited by 15 publications

(13 citation statements)

References 25 publications

(52 reference statements)

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“…Several other groups [23][24][25][26] have also successfully conducted simultaneous or successive targeted randomization of multiple active-site positions based on structural knowledge, as an efficient means to circumvent limitations inherent either to site-directed mutagenesis or to whole-gene random mutagenesis. Recently, Schultz and colleagues [27] combinatorially randomized two activesite residues of an aminoacyl-tRNA synthetase as a step in the generation of an orthogonal synthetase/tRNA pair 380 Protein technologies and commercial enzymes Residues in or near the enzyme active site that were targeted for semi-rational combinatorial mutagenesis.…”

Section: Targeted Randomization Of Defined Residues Based On Structurmentioning

confidence: 99%

Semi-rational approaches to engineering enzyme activity: combining the benefits of directed evolution and rational design

Chica

Doucet

Pelletier

2005

Current Opinion in Biotechnology

358

229

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Section: Targeted Randomization Of Defined Residues Based On Structurmentioning

confidence: 99%

Semi-rational approaches to engineering enzyme activity: combining the benefits of directed evolution and rational design

Chica

Doucet

Pelletier

2005

Current Opinion in Biotechnology

358

229

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“…[23,30] Significant efforts have been undertaken to extend the versatility of heme enzymatic function and to enhance its catalytic activity by site-directed mutagenesis and/or cofactor modification. While the first approach is considered tedious and often impractical for certain enzymes, for example, cytochrome c peroxidase [31] or catalase, [32] the chemical modification of cofactors and its insertion into apo enzymes, that is, enzymes from which the natural cofactor has been removed, offers a variety of possibilities to change the enzymatic function by synthetic methods. For example, Hayashi and others were able to increase the peroxidase activity of myoglobin (Mb) by modifying the propionate side chains of the hemin with artificial functional groups.…”

Section: Introductionmentioning

confidence: 99%

Kinetic Analysis of Semisynthetic Peroxidase Enzymes Containing a Covalent DNA–Heme Adduct as the Cofactor

Fruk

Müller

Niemeyer

2006

Chemistry A European J

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The reconstitution of apo enzymes with DNA oligonucleotide-modified heme (protoporphyrin IX) cofactors has been employed as a tool to produce artificial enzymes that can be specifically immobilized at the solid surfaces. To this end, covalent heme-DNA adducts were synthesized and subsequently used in the reconstitution of apo myoglobin (aMb) and apo horseradish peroxidase (aHRP). The reconstitution produced catalytically active enzymes that contained one or two DNA oligomers coupled to the enzyme in the close proximity to the active site. Kinetic studies of these DNA-enzyme conjugates, carried out with two substrates, ABTS and Amplex Red, showed a remarkable increase in peroxidase activity of the DNA-Mb enzymes while a decrease in enzymatic activity was observed for the DNA-HRP enzymes. All DNA-enzyme conjugates were capable of specific binding to a solid support containing complementary DNA oligomers as capture probes. Kinetic analysis of the enzymes immobilized by the DNA-directed immobilization method revealed that the enzymes remained active after hybridization to the capture oligomers. The programmable binding properties enabled by DNA hybridization make such semisynthetic enzyme conjugates useful for a broad range of applications, particularly in biocatalysis, electrochemical sensing, and as building blocks for biomaterials.

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“…This proposal is backed by substrate specificity studies 15,16,18 and computation. 19,20 In the absence of experimental support for this unusual mechanism, we elected to carry out saturation mutagenesis 21–23 of this residue to give insight into the requirements at position 67. We, therefore, charged these mutants with the task of catalyzing a reaction with both its native 1 and epimerized substrate 2 .…”

mentioning

confidence: 99%

A High-Throughput Screen for the Engineered Production of β-Lactam Antibiotics

2012

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High-throughput screens and selections have had profound impact on our ability to engineer proteins possessing new, desired properties. These methods are especially useful when applied to the modification of existing enzymes to create natural and unnatural products. In an advance upon existing methods we developed a high-throughput, genetically-regulated screen for the in vivo production of β-lactam antibiotics using a green fluorescent protein (gfp) reporter. This assay proved reliable, sensitive and presents a dynamic range under which a wide array of β-lactam architectural sub-classes can be detected. Moreover, the graded response elicited in this assay can be used to rank mutant activity. The utility of this development was demonstrated in vivo and then applied to the first experimental investigation of a putative catalytic residue in carbapenem synthase (CarC). Information gained about the mutability of this residue defines one parameter for enzymatic activity and sets boundaries for future mechanistic and engineering efforts.

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Examining Reactivity and Specificity of Cytochrome c Peroxidase by using Combinatorial Mutagenesis

Cited by 15 publications

References 25 publications

Semi-rational approaches to engineering enzyme activity: combining the benefits of directed evolution and rational design

Semi-rational approaches to engineering enzyme activity: combining the benefits of directed evolution and rational design

Kinetic Analysis of Semisynthetic Peroxidase Enzymes Containing a Covalent DNA–Heme Adduct as the Cofactor

A High-Throughput Screen for the Engineered Production of β-Lactam Antibiotics

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