To pursue structure-function relationships of heme enzymes in the activation of peroxides, we have chosen to use myoglobin as the framework for our molecular engineering studies. Comparison of the crystal structures of myoglobin and peroxidases reveals differences in the arrangement of amino acid residues in heme active sites. On the basis of these structural differences and the reaction mechanisms of peroxidases, we have converted myoglobin into a peroxidase-like enzyme by alternation of the heme distal pocket via site-directed mutagenesis. The replacement of the proximal histidine with cysteine and the exogenous substituted imidazoles slightly accelerates the peroxide O-O bond cleavage due to the electron donor characteristics. However, we have not observed an enhancement in the activation of peroxide by the proximal mutant with tyrosine, the exogenous phenolate, and benzoate. A clear understanding of the absolute role of the proximal ligand remains elusive.

The covalently bound prosthetic group of lactoperoxidase (LPO) has been obtained by hydrolysis of the protein and identified as a dihydroxylated heme. A baculovirus expression system has been developed for LPO and used to obtain protein in which the heme is only partially covalently bound. Reaction of the purified heme⅐apoLPO complex with H 2 O 2 results in both autocatalytic modification of the heme and covalent attachment to the protein. Hydrolytic experiments establish that the autocatalytically incorporated heme is bound normally. Two monohydroxylated heme intermediates have been detected. The peroxidative activity of LPO increases in proportion to the extent of covalently bound heme. The LPO results provide a paradigm for autocatalytic incorporation of heme groups into the mammalian peroxidases, including myeloperoxidase and eosinophil peroxidase, all of which exhibit strong sequence similarity with LPO and have covalentlybound heme groups.The mammalian peroxidases, including lactoperoxidase (LPO), 1 myeloperoxidase (MPO), eosinophil peroxidase, and thyroid peroxidase, utilize H 2 O 2 to catalyze a diverse set of reactions. LPO (1), MPO (2), and eosinophil peroxidase (3) contribute to the nonimmune host defense system by oxidizing chloride ion and the pseudohalide thiocyanate to the potent microbicidal agents hypochlorous and hypothiocyanous acids, respectively. LPO fulfills this function in exocrine secretions, including tears, milk, and saliva, while eosinophil peroxidase and MPO carry out this chemistry in the phagosomes of neutrophils and eosinophils during engulfment of microorganisms. Thyroid peroxidase, which is distinguished from the other peroxidases in that it is an intracellular membrane-bound protein, catalyzes the iodination and coupling of thyroglobulin moieties in the biosynthesis of the thyroid hormones thyroxine and triiodothyronine (4). The mammalian peroxidases also participate in the oxidative metabolism of xenobiotics responsible for hypersensitivity reactions and other toxic sequelae (5, 6).The ease with which the mammalian peroxidases oxidize high potential substrates such as pseudohalides distinguishes them from enzymes such as horseradish peroxidase and yeast cytochrome c peroxidase. The oxidative potency of the mammalian enzymes may be related to one of their distinguishing features: the presence of a modified, covalently bound heme group. The prosthetic group of MPO has been identified by crystallographic and chemical studies as a 1,5-dihydroxymethyl-modified heme b attached by three bonds to the protein (7-9). Two of these are ester bonds that link Asp and Glu residues with the heme hydroxymethyl groups, and the third is a sulfonium ion link obtained by addition of a Met sulfur atom to the 2-vinyl moiety of the heme. The structure of the heme group in the other mammalian peroxidases and the mechanism by which the hemes are incorporated into the mature proteins remain unknown. EXPERIMENTAL PROCEDURESCloning and Expression-The cDNA for bovine LPO (10) was subcloned into pAcGP67B us...

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The heme acquisition system A protein secreted by Pseudomonas aeruginosa (HasA(p)) can capture several synthetic metal complexes other than heme. The crystal structures of HasA(p) harboring synthetic metal complexes revealed only small perturbation of the overall HasA(p) structure. An inhibitory effect upon heme acquisition by HasA(p) bearing synthetic metal complexes was examined by monitoring the growth of Pseudomonas aeruginosa PAO1. HasA(p) bound to iron-phthalocyanine inhibits heme acquisition in the presence of heme-bound HasA(p) as an iron source.

The F43W/H64L myoglobin mutant was previously constructed to investigate the effects of electron-rich tryptophan residue in the heme vicinity on the catalysis, where we found that Trp-43 in the mutant was oxidatively modified in the reaction with m-chloroperbenzoic acid (mCPBA). To identify the exact structure of the modified tryptophan in this study, the mCPBAtreated F43W/H64L mutant has been digested stepwise with Lys-C achromobacter and trypsin to isolate two oxidation products by preparative fast protein liquid chromatography. The close examinations of the 1 H NMR spectra of peptide fragments reveal that two forms of the modified tryptophan must have 2,6-disubstituted indole substructures. The 13 C NMR analysis suggests that one of the modified tryptophan bears a unique hydroxyl group in stead of the NH 2 group at the amino-terminal. The results together with mass spectrometry (MS)/MS analysis (30 Da increase in mass of Trp-43) indicate that oxidation products of Trp-43 are 2,6-dihydro-2,6-dioxoindole and 2,6-dihydro-2-imino-6-oxoindole derivatives. Our finding is the first example of the oxidation of aromatic carbons by the myoglobin mutant system. Myoglobin (Mb), 1 a carrier of molecular oxygen, can perform oxidation reactions in the presence of hydrogen peroxide (H 2 O 2 ), although the activity is not as great as that of peroxidase (1-3). The accumulated biochemical and biophysical data allow us to utilize Mb as a heme enzyme model system, and various myoglobin mutants have been constructed to elucidate structure-function relationship on the activation of peroxides (3-5). For example, F43H/H64L Mb, one of the distal histidine relocation mutants, exhibits the enhanced reactivity with H 2 O 2 and the longer lifetime of an active intermediate, a ferryl porphyrin radical cation (OϭFe IV porphyrin ⅐ ϩ ) (6). Therefore as the results, the F43H/H64L mutant is able to catalyze the sulfoxidation and epoxidation reaction at the rate comparable with the values of peroxidases.On the other hand, cytochromes P-450 (P-450) catalyze the hydroxylation of a wide variety of substrates, including hydrocarbons and polycyclic aromatic molecules (7,8). The variance in reactivity of Mb and P-450 could arise from differences in the active site structure and the arrangement of functional amino acid residues. The crystal structure of P-450cam with d-camphor reveals that the substrate is tightly bound in the hydrophobic heme pocket through hydrogen bonding interaction with the hydroxyl group of Tyr-96 and the carbonyl oxygen of dcamphor (Fig. 1A) (9). The distance between the heme iron and C5 of d-camphor, the hydroxylation site, is 4.2 Å. On the contrary, the active site of myoglobin is exposed to the exterior and does not provide any specific interactions for accommodating a foreign substrate with high affinity (10). Therefore, it will be difficult for a ferryl porphyrin radical cation of Mb to hydroxylate a substrate molecule, which is not bound in an appropriate position nearby the heme. We hypothesize that a ferryl oxygen atom...

Resveratrol was glucosylated to its 3- and 4′-β-glucosides by cultured cells of Phytolacca americana. On the other hand, cultured P. americana cells glucosylated pterostilbene to its 4′-β-glucoside. P. americana cells converted piceatannol into its 4′-β-glucoside. The 3- and 4′-β-glucosides of resveratrol were further glucosylated to 3- and 4′-β-maltosides of resveratrol, 4′-β-maltoside of which is a new compound, by cyclodextrin glucanotransferase. Resveratrol 3-β-glucoside and 3-β-maltoside showed low 2,2-diphenyl-1-picrylhydrazyl free-radical-scavenging activity, whereas other glucosides had no radical-scavenging activity. Piceatannol 4′-β-glucoside showed the strongest inhibitory activity among the stilbene glycosides towards histamine release from rat peritoneal mast cells. Pterostilbene 4′-β-glucoside showed high phosphodiesterase inhibitory activity.

The H64D/V68A and H64D/V68S mutants of Myoglobin are found to oxidize thioanisole with high enantioselectivity and reactivity. These mutants are also capable of enantioselective binding of alpha-methylbenzylamine, which mimics an expected sulfoxidation intermediate. The kinetic study of the amine binding shows that the Fe-O bond cleavage in the intermediate may be the chiral discrimination step of the sulfoxidation.