Identification by ENDOR of Trp
            <sup>191</sup>
            as the Free-Radical Site in Cytochrome c Peroxidase Compound ES

Sivaraja, M.; Goodin, David B.; Smith, Michael B.; Hoffman, Brian M.

doi:10.1126/science.2549632

Cited by 503 publications

(448 citation statements)

References 28 publications

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“…A similar relative orientation between the indole and the heme was demonstrated for in the active site of cytochrome c peroxidase (CcP) (21)(22)(23), which is a heme-containing enzyme that catalyzes H 2 O 2 -dependent oxidation of ferrous cytochrome c (Fig. 4).…”

Section: Resultsmentioning

confidence: 99%

Crystal structures and catalytic mechanism of cytochrome P450 StaP that produces the indolocarbazole skeleton

Makino

Sugimoto

Shiro

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

106

111

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Staurosporine isolated fromThe subsequent oxidative decarboxylation reaction is also discussed based on the crystal structure. Our crystallographic study shows the first crystal structures of enzymes involved in formation of the indolocarbazole core and provides valuable insights into the process of staurosporine biosynthesis, combinatorial biosynthesis of indolocarbazoles, and the diversity of cytochrome P450 chemistry.heme ͉ staurosporine ͉ rebeccamycin ͉ secondary metabolism S taurosporine and rebeccamycin (Fig. 1A) are natural products that have attracted much attention because of their strong inhibitory activity for protein kinase or DNA topoisomerase, which makes them therapeutically important anticancer agents. These natural products are members of a family of indolocarbazole alkaloids which have a similar structure, including an indole[2,3-a]carbazole core with a C-N linkage to a sugar moiety.The staurosporine biosynthetic gene cluster from Streptomyces sp. TP-A0274 and the rebeccamycin biosynthetic gene cluster from Lechevalieria aerocolonigenes (39243; American Type Culture Collection, Manassas, VA) have been cloned and characterized. The staurosporine biosynthetic gene cluster consists of 15 ORFs spanning 22 kb (1), and the rebeccamycin biosynthetic gene cluster consists of 11 genes spanning 17 kb (2, 3). Gene disruption and/or heterologous gene expression experiments revealed that four genes (staO, staD, staP, and staC in Streptomyces sp. TP-A0274 and the homologous genes rebO, rebD, rebP, and rebC in L. aerocolonigenes) are responsible for the biosynthesis of the indolocarbazole skeleton (2, 3). In staurosporine biosynthesis, StaO initiates the synthesis by catalyzing the reaction of tryptophan to the imine form of indole-3-pyruvic acid (IPA imine), and StaD then catalyzes the coupling of two molecules of IPA imine to yield chromopyrrolic acid (CPA). Finally, the key skeleton structure referred to as the indolocarbazole core is constructed through the following two oxidation steps by StaP and StaC (2, 4) (Fig. 1B).StaP (CYP245A1) is a member of the cytochrome P450 family (5), which includes heme enzymes involved in steroid hormone biosynthesis, drug metabolism, and many other physiologically

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Section: Resultsmentioning

confidence: 99%

Crystal structures and catalytic mechanism of cytochrome P450 StaP that produces the indolocarbazole skeleton

Makino

Sugimoto

Shiro

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

106

111

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“…However, intramolecular electron transfer between the oxoferryl porphyrin radical and a protein residue (Tyr or Trp) can occur, e.g. formation of tryptophan radicals in cytochrome c peroxidase (22) and lignin peroxidase (23) or a tyrosyl radical in turnip peroxidase (24). There is a correlation between the standard reduction potential of the ferric/ferrous couple and the stability of high valent porphyrin species (25).…”

Section: Effect On the Chemical Nature Of The Radical Intermediates Imentioning

confidence: 99%

Influence of the Unusual Covalent Adduct on the Kinetics and Formation of Radical Intermediates in Synechocystis Catalase Peroxidase

Jakopitsch¹,

Ivancich

Schmuckenschlager³

et al. 2004

Journal of Biological Chemistry

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Catalase-peroxidases (KatGs) are heme peroxidases with a catalatic activity comparable to monofunctional catalases. They contain an unusual covalent distal side adduct with the side chains of Trp 122 , Tyr 249 , and Met 275 (Synechocysis KatG numbering). The known crystal structures suggest that Tyr 249 and Met 275 could be within hydrogen-bonding distance to Arg 439 . To investigate the role of this peculiar adduct, the variants Y249F, M275I, R439A, and R439N were investigated by electronic absorption, steady-state and transient-state kinetic techniques and EPR spectroscopy combined with deuterium labeling. Exchange of these conserved residues exhibited dramatic consequences on the bifunctional activity of this peroxidase. The turnover numbers of catalase activity of M275I, Y249F, R439A, and R439N are 0.6, 0.17, 4.9, and 3.14% of wild-type activity, respectively. By contrast, the peroxidase activity was unaffected or even enhanced, in particular for the M275I variant. As shown by mass spectrometry and EPR spectra, the KatG typical adduct is intact in both Arg 439 variants, as is the case of the wild-type enzyme, whereas in the M275I variant the covalent link exists only between Tyr 249 and Trp 122 . In the Y249F variant, the link is absent. EPR studies showed that the radical species formed upon reaction of the Y249F and R439A/N variants with peroxoacetic acid are the oxoferrylporphyrin radical, the tryptophanyl and the tyrosyl radicals, as in the wild-type enzyme. The dramatic loss in catalase activity of the Y249F variant allowed the comparison of the radical species formed with hydrogen peroxide and peroxoacetic acid. The EPR data strongly suggest that the sequence of intermediates formed in the absence of a one electron donor substrate, is por ⅐ ؉ 3 Trp ⅐ (or Trp ⅐ ؉ ) 3 Tyr ⅐ . The M275I variant did not form the Trp ⅐ species because of the dramatic changes on the heme distal side, most probably induced by the repositioning of the remaining Trp 122 -Tyr 249 adduct. The results are discussed with respect to the bifunctional activity of catalase-peroxidases.Catalase-peroxidases (KatGs) 1 are present in bacteria and fungi and function primarily as hydrogen peroxide scavengers (1-3). Sequence similarities indicate they are members of the class I of the superfamily of plant, fungal, and bacterial peroxidases (4). In contrast to the other members of this group, such as cytochrome c peroxidase (CcP) and ascorbate peroxidase (APX), they show a high catalase activity comparable with monofunctional catalases. Although the catalase activity is overwhelming, KatGs also show a substantial peroxidase activity with various one electron donors.There are three crystal structures of KatGs available, namely the Archaebacterium Haloarcula marismortui (5), Burkholderia pseudomallei (6), and Synechococcus PCC 7942 (PDB entry 1UB2), the latter being a cyanobacterial KatG with high homology to Synechocystis KatG. The structures show that the arrangement of the active site is similar to CcP and APX, containing the distal triad arg...

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“…Furthermore, the reaction cycle of yeast cytochrome c peroxidase involves the one electron oxidation of an active site byptophan residue to a tryptophan radical (Sivaraja et al, 1989); this implies that the putative mauG gene product, functioning as a peroxidase, may be able to utilise H,O, to initiate a free-radical-mediated cross-linking of the two Lchain tryptophan residues forming the TTQ prosthetic group. prepared from any of the cytochrome-deficient mutants of either organism (Fig.…”

Section: Methylamine-dehydrogenase L-subunit Polypeptides Synthesisedmentioning

confidence: 99%

Mutants of Methylobacterium extorquens and Paracoccus denitrificans deficient in c‐type cytochrome biogenesis synthesise the methylamine‐dehydrogenase polypeptides but cannot assemble the tryptophan‐tryptophylquinone group

Page

Ferguson

1993

European Journal of Biochemistry

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Five mutants of Methylobacterium extorquens and four mutants of Paracoccus denitrificans that have a general defect in c-type cytochrome synthesis also failed to assemble an active methylamine dehydrogenase. In all cases methanol dehydrogenase, another periplasmic enzyme, was fully active. All nine mutant strains accumulated both the heavy and light subunits of methylamine dehydrogenase to essentially wild-type levels. In all nine mutants, the heavy-subunit and light-subunit polypeptides were proteolytically processed, suggesting that translocation to the periplasm had occurred; in the case of the f? denitrificans mutants, a periplasmic location for the heavy and light subunits was confirmed experimentally. While specific quinone staining of the methylamine dehydrogenase light subunit in wild-type M. extorquens and f? denitrificans strains could readily be demonstrated, the light subunit polypeptides accumulated by the mutants did not quinone stain, indicating that the methylamine dehydrogenase prosthetic group, tryptophan tryptophylquinone, is not assembled in the absence of functional c-type cytochromes.Methylamine dehydrogenase, catalysing the oxidation of methylamine to formaldehyde and ammonia, has been identified in several groups of Gram-negative bacteria, the facultative (e.g. Methylobacterium extorquens) and restricted facultative (e.g. Methylophilus spp.) methylotrophs and the facultative autotrophs (e.g. Paracoccus denitrificans) (Eady and Large, 1968;Haywood et al., 1982;Husain and Davidson, 1987). The enzymes have an a& subunit structure with a heavy (H) subunit of 40-45 kDa and light (L) subunit of 14-15 kDa; in all known cases the enzyme is periplasmic (Burton et al., 1983;Husain and Davidson, 1987;Ubbink et al., 1991;Chistoserdov et al., 1992). The methylamine dehydrogenase prosthetic group, recently shown to be a unique quinonoid moiety termed tryptophan tryptophylquinone (TTQ), is formed from two tryptophan residues (positions 55 and 106 in the M . extorquens enzyme; Ishii et al., 1983, Chistoserdov et al., 1990 in the L-subunit polypeptide chain (McIntire et al., 1991). The tryptophan residue that occurs nearer the N-terminal in the amino acid sequence (1.e.

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Identification by ENDOR of Trp ¹⁹¹ as the Free-Radical Site in Cytochrome c Peroxidase Compound ES

Cited by 503 publications

References 28 publications

Crystal structures and catalytic mechanism of cytochrome P450 StaP that produces the indolocarbazole skeleton

Crystal structures and catalytic mechanism of cytochrome P450 StaP that produces the indolocarbazole skeleton

Influence of the Unusual Covalent Adduct on the Kinetics and Formation of Radical Intermediates in Synechocystis Catalase Peroxidase

Mutants of Methylobacterium extorquens and Paracoccus denitrificans deficient in c‐type cytochrome biogenesis synthesise the methylamine‐dehydrogenase polypeptides but cannot assemble the tryptophan‐tryptophylquinone group

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Identification by ENDOR of Trp 191 as the Free-Radical Site in Cytochrome c Peroxidase Compound ES

Cited by 503 publications

References 28 publications

Crystal structures and catalytic mechanism of cytochrome P450 StaP that produces the indolocarbazole skeleton

Crystal structures and catalytic mechanism of cytochrome P450 StaP that produces the indolocarbazole skeleton

Influence of the Unusual Covalent Adduct on the Kinetics and Formation of Radical Intermediates in Synechocystis Catalase Peroxidase

Mutants of Methylobacterium extorquens and Paracoccus denitrificans deficient in c‐type cytochrome biogenesis synthesise the methylamine‐dehydrogenase polypeptides but cannot assemble the tryptophan‐tryptophylquinone group

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Identification by ENDOR of Trp ¹⁹¹ as the Free-Radical Site in Cytochrome c Peroxidase Compound ES