Crystal Structure of a Thermophilic Cytochrome P450 from the Archaeon Sulfolobus solfataricus

Yano, Jason; Koo, Laura S.; Schüller, David; Li, Huiying; Montellano, Paul R. Ortiz de; Poulos, T.L.

doi:10.1074/jbc.m004281200

Cited by 195 publications

(195 citation statements)

References 38 publications

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“…Such an open conformation observed in the substrate-free form might leave the active site relatively exposed and enable the substrate to access the substrate-binding pocket near the heme. Similar open/closed conformations were also reported for CYP119 (14) and P450BM-3 (15). Substrate binding shifts the heme by 1.3 Å toward helix I [ Fig.…”

Section: Resultssupporting

confidence: 61%

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.

105

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: Resultssupporting

confidence: 61%

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.

105

111

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Section: Discussionmentioning

confidence: 99%

“…On the other hand, these azoles are not strong MTCYP51 inhibitors, IC 50 /P450 (M/M) being only of ~9, and 5 for fluconazole and ketoconazole, respectively. Taking into consideration that changes in the P450 structure might be dependent on the identity of the inhibitor bound [45] it is not excluded that conformational rearrangement, detectable by FRET analysis, might follow binding of more potent CYP51 inhibitors.Until recently MTCYP51 was the only P450 in which the BC loop (at least upon crystallization) acquires an open conformation. Because accessibility of the heme to the solvent is not typical for cytochromes P450 (it has now also been observed in CYP8A1 (PGIS) [46]) it is very likely that the opening closes when substrate enters the active site [14].…”

mentioning

confidence: 99%

Conformational dynamics in the F/G segment of CYP51 from Mycobacterium tuberculosis monitored by FRET

Lepesheva

Seliškar

Knutson

et al. 2007

Archives of Biochemistry and Biophysics

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A cysteine was introduced into the FG-loop (P187C) of CYP51 from Mycobacterium tuberculosis (MT) for selective labeling with BODIPY and fluorescence energy transfer (FRET) analysis. Forster radius for the BODIPY-heme pair was calculated assuming that the distance between the heme and Cys187 in solution corresponds to that in the crystal structure of ligand free MTCYP51. Interaction of MTCYP51 with azole inhibitors ketoconazole and fluconazole or the substrate analog estriol did not influence the fluorescence, but titration with the substrate lanosterol quenched BODIPY emission, the effect being proportional to the portion of substrate-bound MTCYP51. The detected changes correspond to ~10 Å decrease in the calculated distance between BODIPY-Cys187 and the heme. The results confirm 1) functional importance of conformational motions in the MTCYP51 F/G segment and 2) applicability of FRET to monitor them in solution. KeywordsCytochrome P450; sterol 14α-demethylase; ligand binding; conformational changes; FRET The cytochrome P450 superfamily currently includes more than 6,300 enzymes (http://drnelson.utmem.edu/CytochromeP450.html) oxidizing a wide variety of xenobiotics (compounds from the environment) and endogenous substrates. Even at less than 16% amino acid identity, all P450s preserve a common overall structural fold (α-helical with orthogonal bundle architecture according to CATH classification [1]) and the set of secondary structural elements that allow the superfamily to metabolize a wide variety of diverse and unrelated structures. By now it has become quite generally accepted that this enormous catalytic versatility arises from the P450 conformational flexibility [2,3]. Ligandinduced conformational changes encompassing small to large movements are seen in the Xray structures of several bacterial and drug-metabolizing mammalian CYPs 1 (e.g. CYPs101 * Corresponding author: Michael R. Waterman, Tel.: 615-343-1373; Fax: 615-322-4349; E-mail: michael.waterman@vanderbilt.edu.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. . On the other hand, site-directed mutagenesis of five amino acid residues in this region (L172, G175, P178, R194 and D195), which are highly conserved in the CYP51 family and exposed into the substrate binding cavity of MTCYP51 has shown that all of them are essential for MTCYP51 catalytic activity at the stage of the interaction with substrate. Moreover, mutation of A197 in the middle of the MTCYP51 G-helix to the helix-breaking glycine causes about one order of magnitude enzyme activation and sharp increase in the substrate bound (high-spin) po...

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“…However, amino acid sequence diversity of enzymes within the superfamily has limited use of the molecular replacement approach for cytochrome P450 structure determination (49). Fortunately, the intrinsic heme-iron atom of cytochromes P450 can be used as an anomalous scatterer to facilitate protein structure determination using the multiple anomalous dispersion technique.…”

Section: Resultsmentioning

confidence: 99%

The 1.92-Å Structure of Streptomyces coelicolor A3(2) CYP154C1

Podust

Kim

Arase

et al. 2003

Journal of Biological Chemistry

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Evolutionary links between cytochrome P450 monooxygenases, a superfamily of extraordinarily divergent heme-thiolate proteins catalyzing a wide array of NADPH/NADH-and O 2 -dependent reactions, are becoming better understood because of availability of an increasing number of fully sequenced genomes. Among other reactions, P450s catalyze the site-specific oxidation of the precursors to macrolide antibiotics in the genus Streptomyces introducing regiochemical diversity into the macrolide ring system, thereby significantly increasing antibiotic activity. Developing effective uses for Streptomyces enzymes in biosynthetic processes and bioremediation requires identification and engineering of additional monooxygenases with activities toward a diverse array of small molecules. To elucidate the molecular basis for substrate specificity of oxidative enzymes toward macrolide antibiotics, the x-ray structure of CYP154C1 from Streptomyces coelicolor A3(2) was determined (Protein Data Bank code 1GWI). Relocation of certain common P450 secondary structure elements, along with a novel structural feature involving an additional ␤-strand transforming the five-stranded ␤-sheet into a six-stranded variant, creates an open cleft-shaped substrate-binding site between the two P450 domains. High sequence similarity to macrolide monooxygenases from other microbial species translates into catalytic activity of CYP154C1 toward both 12-and 14-membered ring macrolactones in vitro.

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Crystal Structure of a Thermophilic Cytochrome P450 from the Archaeon Sulfolobus solfataricus

Cited by 195 publications

References 38 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

Conformational dynamics in the F/G segment of CYP51 from Mycobacterium tuberculosis monitored by FRET

The 1.92-Å Structure of Streptomyces coelicolor A3(2) CYP154C1

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