Peroxidase mutagenesis

Josephy PD. The role of peroxidase-catalyzed activation of aromatic amines in breast cancer. Mutagenesis 1996 11 (1 ) 3—7. 

As part of a subsequent study concerning primarily second-site revertant yeast iso-l-cytochrome c variants, Hazzard et al. evaluated the effect of converting Lys-72 to an aspartyl residue by site-directed mutagenesis on the electron transfer kinetics of the cytochrome c-cytochrome c peroxidase complex [136]. Lys-72 was of interest for this purpose, because it is involved in the hypothetical model for the complex formed by these two proteins that was proposed by Poulos and Kraut on the basis of molecular graphics docking [106]. In these... 

Recent structural studies and electron transfer kinetic experiments focus on structures in which a site-specific covalent crosslink between cytochrome c and cytochrome c peroxidase subunits exists. One of these used site-directed mutagenesis to form a disulfide bond between a V197C mutant CcP and an A81C... 

The mechanisms for the rednction of compounds I and II are less well nnderstood than the mechanism of compound I formation, although a number of suggestions have been presented in the literature (24). It is clear from site-directed mutagenesis studies that Arg38 and His42 are also important in the rednction steps, although their precise role has been difficnlt to define. A detailed mechanism for snbstrate oxidation by plant peroxidases has been proposed, based on data from the crystal... 

Various isoforms of both HO and NOS can be expressed in recombinant systems. As a result, the immediate future will undoubtedly witness a wealth of mutagenesis experiments guided by the crystal structures. It also may be possible to trap in crystalline form the various intermediates of the HO reaction cycle, which will greatly facilitate a deeper understanding of the catalytic mechanism. Conformational dynamics appear to be quite important in HO, and hence, a variety of spectral probes such as NMR and fluorescence should prove especially useful in studying the role of protein dynamics in function. Overall there should be considerable optimism for understanding HO at the level of detail achieved for peroxidases and other well-studied enz5une systems. 

New insights into the peroxide binding and in the catalysis, obtained through site-directed mutagenesis, have led to much-improved understanding of heme peroxidases and their possible applications. New crystal structures of peroxidases have provided much more information on the local heme environments [32],... 

Ruiz-Duenas FJ, Morales M, Perez-Boada M et al (2007) Manganese oxidation site in Pleurotus eryngii versatile peroxidase A site-directed mutagenesis, kinetic and crystallographic study. Biochemistry 46 66-77... 

De Smet L, Sawides SN, Van Horen E et al (2006) Structural and mutagenesis studies on the cytochrome c peroxidase from Rhodobacter capsulatus provide new insights into structure-function relationships of bacterial di-heme peroxidases. J Biol Chem 281 4371 1379... 

Perez-Boada M, Ruiz-Duenas FJ, Pogni R et al (2005) Versatile peroxidase oxidation of high redox potential aromatic compounds Site-directed mutagenesis, spectroscopic and... 

Ruiz-Duenas FJ, Morales M, Mate MJ et al (2008) Site-directed mutagenesis of the catalytic tryptophan environment in Pleurotus eryngii versatile peroxidase. Biochemistry 47 1685-1695... 

The third covalent bond present in MPO is also thought to form by an autocatalytic process, although it has not been clearly demonstrated to do so. However, incubation of an ascorbate peroxidase mutant into which a methionine has been introduced by mutagenesis has been shown to result in covalent attachment of the Met sulfur atom to a heme vinyl group [54]. Although the link is not identical to that in MPO, this finding provides strong circumstantial evidence that the bond in MPO is also formed by an autocatalytic mechanism. 

Doyle WA, Blodig W, Veitch NC et al (1998) Two substrate interaction sites in lignin peroxidase revealed by site-directed mutagenesis. Biochemistry 37 15097-15105... 

Watanabe Y, Ueno T (2003) Introduction of P450, peroxidase, and catalase activities into myoglobin by site-directed mutagenesis diverse reactivities of compound I. Bull Chem Soc Jpn 76 1309-1322... 

Genetic modifications by site-directed and random mutagenesis have been proven with peroxidases, obtaining in both cases interesting results, although in most of the reported cases, these studies are made to understand the relationship between structure and mechanism and not to enhance the catalytic functions for biotechnological applications [115-117],... 

The results illustrate the ability of directed molecular evolution and site-directed mutagenesis technologies to deliver solutions about biotechnological problems of limited performance of peroxidase enzymes, approaching peroxidases as viable industrial biocatalysts. 

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