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

-generating system has yet been obtained after transfecting Duox2 into non-thyroid cell lines, because it is retained in the endoplasmic reticulum (ER). We investigated the level of maturation of various Duox2 truncated proteins in an attempt to identify the region of Duox2 responsible for its remaining in the ER. Duox2-Q686X mutant, corresponding to the Nterminal ectodomain including the first putative transmembrane domain, was expressed in different cell lines. Carbohydrate content analysis revealed that complex type-specific Golgi apparatus (GA) oligosaccharides were present on pig Duox2-Q686X, whereas human truncated Duox2 carried only high mannose-type sugar chains characteristic of the ER. Further characterization using surface biotinylation and flow cytometry assays indicated that pig Duox2-Q686X was present at the plasma membrane, whereas human Duox2-Q686X remained inside the cell. The replacement of the last 90 residues of the human Duox2-Q686X with the pig equivalent region allowed the chimerical peptide to reach the Golgi apparatus. Pig mutants containing the complete first intracellular loop with or without the second transmembrane domain accumulated in the ER. These findings show that 1) the human Duox2-Q686X region encompassing residues 596 -685 prevents mutant exportation from the ER and 2) there is a pig Duox2 retention domain in the first intracellular loop. In addition, missense mutations of four cysteines (Cys-351, -370, -568, or -582) completely inhibited the emergence of pig Duox2-Q686X from the ER compartment, indicating their importance in Duox2 maturation.

Section: Discussionmentioning

confidence: 99%

Targeting of the Dual Oxidase 2 N-terminal Region to the Plasma Membrane

Morand

Agnandji

Noël-Hudson

et al. 2004

“…1B. This partial sequence alignment with the classical peroxidases demonstrates that both DUOX homologs lack the catalytic distal histidine residue (Ser 108 , hDUOX1; and Tyr 105 , CeDUOX1) and the aspartate residue that in MPO and LPO covalently binds to the heme co-factor through an ester linkage (Leu 107 , hDUOX1; and Ala 104 , CeDUOX1) (25,26). hDUOX1 lacks additional critical peroxidase residues: the glutamate that forms the second covalent bond to the heme (Arg 241 ) and the proximal histidine residue (Ser 331 ).…”

Section: Design and Expression Of Soluble Duox1mentioning

confidence: 99%

Caenorhabditis elegans and Human Dual Oxidase 1 (DUOX1) “Peroxidase” Domains

Meitzler

Montellano

2009

The seven members of the NOX/DUOX family are responsible for generation of the superoxide and H 2 O 2 required for a variety of host defense and cell signaling functions in nonphagocytic cells. Two members, the dual oxidase isozymes DUOX1 and DUOX2, share a structurally unique feature: an N-terminal peroxidase-like domain. Despite sequence similarity to the mammalian peroxidases, the absence of key active site residues makes their binding of heme and their catalytic function uncertain. To explore this domain we have expressed in a baculovirus system and purified the Caenorhabditis elegans (CeDUOX1 1-589 ) and human (hDUOX1 1-593 ) DUOX1 "peroxidase" domains. Evaluation of these proteins demonstrated that the isolated hDUOX1 1-593 does not bind heme and has no intrinsic peroxidase activity. In contrast, CeDUOX1 1-589 binds heme covalently, exhibits a modest peroxidase activity, but does not oxidize bromide ion. Surprisingly, the heme appears to have two covalent links to the protein despite the absence of a second conserved carboxyl group in the active site. Although the N-terminal dual oxidase motif has been proposed to directly convert superoxide to H 2 O 2 , neither DUOX1 domain demonstrated significant superoxide dismutase activity. These results strengthen the in vivo conclusion that the CeDUOX1 protein supports controlled peroxidative polymerization of tyrosine residues and indicate that the hDUOX1 protein either has a unique function or must interact with other protein factors to express its catalytic activity.

“…is the reference enzyme for the mammalian peroxidases because its crystal structure has been determined (1,22). No other mammalian peroxidase structure is available.…”

Section: Structure Superposition and Choice Of Mutations-mpomentioning

confidence: 99%

“…Mammalian peroxidases share a unique feature, the formation of two or three covalent bonds to their prosthetic heme 1 group, not found in the peroxidases of other organisms. The two common bonds in all mammalian peroxidases are ester links between a conserved glutamate and aspartate and the heme 1-methyl and 5-methyl substituents, respectively.…”

mentioning

confidence: 99%

“…The two common bonds in all mammalian peroxidases are ester links between a conserved glutamate and aspartate and the heme 1-methyl and 5-methyl substituents, respectively. In MPO a third bond is forged between a methionine and the ␤ carbon of the 2-vinyl substituent (1). Mutagenesis studies with LPO have demonstrated that at least one of the two ester bonds is required for catalytic activity (2).…”

mentioning

confidence: 99%

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Horseradish Peroxidase Mutants That Autocatalytically Modify Their Prosthetic Heme Group

Colas

Montellano

2004

The mammalian peroxidases, including myeloperoxidase and lactoperoxidase, bind their prosthetic heme covalently through ester bonds to two of the heme methyl groups. These bonds are autocatalytically formed. No other peroxidase is known to form such bonds. To determine whether features other than an appropriately placed carboxylic acid residue are important for covalent heme binding, we have introduced aspartate and/or glutamic acid residues into horseradish peroxidase, a plant enzyme that exhibits essentially no sequence identity with the mammalian peroxidases. Based on superposition of the horseradish peroxidase and myeloperoxidase structures, the mutated residues were Leu 37 , Phe 41 , Gly 69 , and Ser 73 . The F41E mutant was isolated with no covalently bound heme, but the heme was completely covalently bound upon incubation with H 2 O 2 . As predicted, the modified heme released from the protein was 3-hydroxymethylheme. The S73E mutant did not covalently bind its heme but oxidized it to the 8-hydroxymethyl derivative. The hydroxyl group in this modified heme derived from the medium. The other mutations gave unstable proteins. The rate of compound I formation for the F41E mutant was 100 times faster after covalent bond formation, but the reduction of compound I to compound II was similar with and without the covalent bond. The results clearly establish that an appropriately situated carboxylic acid group is sufficient for covalent heme attachment, strengthen the proposed mechanism, and suggest that covalent heme attachment in the mammalian peroxidases relates to peroxidase biology or stability rather than to intrinsic catalytic properties.Mammalian peroxidases share a unique feature, the formation of two or three covalent bonds to their prosthetic heme 1 group, not found in the peroxidases of other organisms. The two common bonds in all mammalian peroxidases are ester links between a conserved glutamate and aspartate and the heme 1-methyl and 5-methyl substituents, respectively. In MPO a third bond is forged between a methionine and the ␤ carbon of the 2-vinyl substituent (1). Mutagenesis studies with LPO have demonstrated that at least one of the two ester bonds is required for catalytic activity (2). Interestingly, a single covalent link between the heme and protein is also found in most members of the CYP4A, -4B, and -4F classes of cytochrome P450 enzymes (3-7). In contrast to LPO, however, mutagenesis studies show that the heme-protein covalent bond is not essential for the catalytic activity of at least some of these P450 enzymes (8, 6). A long known example of heme covalent binding is provided by cytochrome c, in which a cysteine residue is covalently bound to each of the two porphyrin vinyl groups. Although many hypotheses have been formulated concerning the functional advantages of the links in cytochrome c, including bending of the heme, increasing the heme affinity (9), and increasing the stability of the methionyl-Fe(II) coordination (10), the question has yet to be definitively answered.Despite m...