Site-Directed Mutagenesis of Human Myeloperoxidase: Further Identification of Residues Involved in Catalytic Activity and Heme Interaction

Jacquet, Alain; Garcia‐Quintana, Lida; Deleersnyder, Virginie; Fenna, R.E.; Bollen, A.; Moguilevsky, Nicole

doi:10.1006/bbrc.1994.1895

Cited by 18 publications

(14 citation statements)

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“…To test whether the peroxidase catalytic activity of PXN-2 is important for its developmental functions, we mutated the 'distal histidine' of the peroxidase active site to alanine (H755A). Mutation of the equivalent residue in MPO completely abolishes catalytic activity without affecting protein stability (Jacquet et al, 1994). PXN-2(H755A) transgenes displayed significantly reduced rescue activity compared with wild-type PXN-2 transgenes (Fig.…”

Section: Resultsmentioning

confidence: 95%

TheC. elegansperoxidasin PXN-2 is essential for embryonic morphogenesis and inhibits adult axon regeneration

et al. 2010

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SUMMARYPeroxidasins form a highly conserved family of extracellular peroxidases of unknown cellular function. We identified the C. elegans peroxidasin PXN-2 in screens for mutants defective in embryonic morphogenesis. We find that PXN-2 is essential for specific stages of embryonic morphogenesis and muscle-epidermal attachment, and is also required postembryonically for basement membrane integrity. The peroxidase catalytic activity of PXN-2 is necessary for these developmental roles. pxn-2 mutants display aberrant ultrastructure of the extracellular matrix, suggesting a role in basement membrane consolidation. PXN-2 affects specific axon guidance choice points in the developing nervous system but is dispensable for maintenance of process positions. In adults, loss of pxn-2 function promotes regrowth of axons after injury, providing the first evidence that C. elegans extracellular matrix can play an inhibitory role in axon regeneration. Loss of function in the closely related C. elegans peroxidasin pxn-1 does not cause overt developmental defects. Unexpectedly, pxn-2 mutant phenotypes are suppressed by loss of function in pxn-1 and exacerbated by overexpression of wild-type pxn-1, indicating that PXN-1 and PXN-2 have antagonistic functions. These results demonstrate that peroxidasins play crucial roles in development and reveal a new role for peroxidasins as extracellular inhibitors of axonal regeneration.

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Section: Resultsmentioning

confidence: 95%

TheC. elegansperoxidasin PXN-2 is essential for embryonic morphogenesis and inhibits adult axon regeneration

et al. 2010

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“…In this regard it is noteworthy that a Met 243 3 Gln mutant of recombinant human MPO exhibits a Soret band at 410 -412 nm, considerably blue-shifted from the 428-nm band of the native enzyme in its oxidized form (35,37,38). Similarly, a Glu 242 3 Gln mutant of MPO, presumably lacking one of the heme ester bonds, exhibits a blue-shifted Soret band at 416 -418 nm, and an Asp 94 3 Asn mutant, presumed to lack the other ester bond, also has a blue-shifted Soret band at 414 nm (38,39). These studies of mutants strongly suggest that the covalent linkages to the heme in MPO contribute to the observed red shifts of the Soret band in the visible absorption spectrum.…”

Section: Discussionmentioning

confidence: 99%

X-ray Crystal Structure and Characterization of Halide-binding Sites of Human Myeloperoxidase at 1.8 Å Resolution

Fiedler

Davey²,

Fenna

2000

Journal of Biological Chemistry

342

366

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The x-ray crystal structure of human myeloperoxidase has been extended to 1.8 Å resolution, using x-ray data recorded at ؊180°C (r ‫؍‬ 0.197, free r ‫؍‬ 0.239). Results confirm that the heme is covalently attached to the protein via two ester linkages between the carboxyl groups of Glu 242 and Asp 94 and modified methyl groups on pyrrole rings A and C of the heme as well as a sulfonium ion linkage between the sulfur atom of Met 243 and the ␤-carbon of the vinyl group on pyrrole ring A. In the native enzyme a bound chloride ion has been identified at the amino terminus of the helix containing the proximal His 336 . Determination of the x-ray crystal structure of a myeloperoxidase-bromide complex (r ‫؍‬ 0.243, free r ‫؍‬ 0.296) has shown that this chloride ion can be replaced by bromide. Bromide is also seen to bind, at partial occupancy, in the distal heme cavity, in close proximity to the distal His 95 , where it replaces the water molecule hydrogen bonded to Gln 91 . The bromide-binding site in the distal cavity appears to be the halidebinding site responsible for shifts in the Soret band of the absorption spectrum of myeloperoxidase. It is proposed that halide binding to this site inhibits the enzyme by effectively competing with H 2 O 2 for access to the distal histidine, whereas in compound I, the same site may be the halide substrate-binding site.

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“…The covalent linkage of this residue to heme is responsible for the unusually red-shifted Soret band at 428 nm. MPO with Gln replacing Met409 shows decreased overall peroxidase activity, the loss of its chlorinating activity, and a blue shift of the Soret band from 428 to 411 nm, the latter being typical of heme-containing peroxidases (15,20). However, substituting the corresponding Gln376 of bovine LPO with Met is not sufficient to confer either a red shift of the Soret band or chlorinating activity, underlining the complexity of interactions in the heme environment of mammalian peroxidases (31).…”

Section: Discussionmentioning

confidence: 99%

Microbicidal Activity of Vascular Peroxidase 1 in Human Plasma via Generation of Hypochlorous Acid

Cao

Moore

et al. 2012

Infect Immun

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ABSTRACTMembers of the heme peroxidase family play an important role in host defense. Myeloperoxidase (MPO) is expressed in phagocytes and is the only animal heme peroxidase previously reported to be capable of using chloride ion as a substrate to form the highly microbicidal species hypochlorous acid (HOCl) at neutral pH. Despite the potent bacterial killing activity of HOCl, individuals who fail to express MPO typically show only a modest increase in some fungal infections. This may point to the existence of redundant host defense mechanisms. Vascular peroxidase 1 (VPO1) is newly discovered member of the heme peroxidase family. VPO1 is expressed in cells of the cardiovascular system and is secreted into the bloodstream. In the present study, we investigate whether VPO1 is capable of generating HOCl and its role in host defense. Like MPO, VPO1 in the presence of H2O2and chloride generates HOCl. VPO1-dependent HOCl generation was demonstrated by chlorination of taurine and tyrosine using mass spectrometry. In addition, the VPO1/H2O2/Cl−system can cause the chlorination of monochlorodimedone and the oxidation of 5-thio-2-nitrobenzoic acid. Purified VPO1 and VPO1 in plasma mediate bacterial killing that is dependent on chloride and H2O2; killing is inhibited by peroxidase inhibitors and by the H2O2scavenger catalase. In the presence of erythrocytes, bacterial killing by VPO1 is slightly reduced. Thus, VPO1, in addition to MPO, is the second member of the heme peroxidase family capable of generating HOCl under physiological conditions. VPO1 is likely to participate in host defense, with bactericidal activity mediated through the generation of HOCl.

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Site-Directed Mutagenesis of Human Myeloperoxidase: Further Identification of Residues Involved in Catalytic Activity and Heme Interaction

Cited by 18 publications

References 0 publications

TheC. elegansperoxidasin PXN-2 is essential for embryonic morphogenesis and inhibits adult axon regeneration

TheC. elegansperoxidasin PXN-2 is essential for embryonic morphogenesis and inhibits adult axon regeneration

X-ray Crystal Structure and Characterization of Halide-binding Sites of Human Myeloperoxidase at 1.8 Å Resolution

Microbicidal Activity of Vascular Peroxidase 1 in Human Plasma via Generation of Hypochlorous Acid

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