Cloning of a potential cytochrome P450 from the Archaeon <i>Sulfolobus solfataricus</i>

Wright, Rhonda L.; Harris, Kathryn A.; Solow, Barbara T.; White, Robert H.; Kennelly, Peter J.

doi:10.1016/0014-5793(96)00322-5

Cited by 63 publications

(48 citation statements)

References 14 publications

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“…In addition, fatty acid hydroxylation could not be detected using H 2 O 2 or bovine adrenodoxin/adrenodoxin reductase as redox partners. 2 A superimposition of the P450BM-3 structure with fatty acid bound (1FAG) on to CYP175A1 provides clues on why we failed to observe fatty acid binding (Fig. 6).…”

Section: Resultsmentioning

confidence: 93%

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Preliminary Characterization and Crystal Structure of a Thermostable Cytochrome P450 from Thermus thermophilus

Yano¹,

Blasco²,

Li³

et al. 2003

Journal of Biological Chemistry

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The second structure of a thermophile cytochrome P450, CYP175A1 from the thermophilic bacterium Thermus thermophilus HB27, has been solved to 1.8-Å resolution. The overall P450 structure remains conserved despite the low sequence identity between the various P450s. The CYP175A1 structure lacks the large aromatic network found in the only other thermostable P450, CYP119, thought to contribute to thermal stability. The primary difference between CYP175A1 and its mesophile counterparts is the investment of charged residues into salt-link networks at the expense of single chargecharge interactions. Additional factors involved in the thermal stability increase are a decrease in the overall size, especially shortening of loops and connecting regions, and a decrease in the number of labile residues such as Asn, Gln, and Cys.Cytochromes P450 (P450s) 1 catalyze the monooxygenation of a vast array of organic compounds in the following reaction.P450s are ubiquitous enzymes essential for steroid biosynthesis, catabolism of drugs, utilization of carbon compounds as an energy source in bacteria, and in the production of various macrolide antibiotics. P450s are found throughout the biosphere and various genome projects have revealed a remarkable and unexpectedly large number of P450s in a variety of organisms. For example, humans have ϳ180, Caenorhabditis elegans 81, Drosophila 90 (83 functional 7 pseudogenes) (1), and Arabidopsis 273 (drnelson.utmem.edu/CytochromeP450. html). Several bacteria also have P450s, but normally bacteria produce only one or at most a few P450s.Another surprising occurrence of a P450 was demonstrated in 1996 when the first P450 from a hyperthermophile, CYP119, was discovered (2). CYP119 was identified in the acidothermophilic archaeon Sulfolobus solfataricus. This was not only the first thermostable P450 to be discovered but also the first demonstration of an archaeon P450. Subsequent cloning, expression, and purification of CYP119 led to the finding that CYP119 exhibits a melting temperature of Ϸ90°C compared with Ϸ55°C for mesophilic bacterial P450cam (3). This was soon followed by our determination of the CYP119 crystal structure (4), followed by an independent structure determination by Park et al. (5). The enhanced thermal stability of CYP119 was attributed to an unusually large clustering of aromatic side chains not found in all other known P450 structures (3-6). The ever increasing data base of known structures of proteins from both mesophiles and their thermophilic counterparts reveals that aromatic clustering does, indeed, correlate with enhanced thermal stability, but that this is only one of several possible mechanisms for enhancing thermal stability (7). Whether or not aromatic clustering is a universal feature of thermostable P450s will require additional crystal structures. Fortunately, the second P450 from a thermophile was recently discovered, CYP175A1 from Thermus thermophilus. The present report describes cloning, expression, purification, crystallization, and structure determinatio...

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Section: Resultsmentioning

confidence: 93%

“…Another surprising occurrence of a P450 was demonstrated in 1996 when the first P450 from a hyperthermophile, CYP119, was discovered (2). CYP119 was identified in the acidothermophilic archaeon Sulfolobus solfataricus.…”

Section: Nad(p)hϩhmentioning

confidence: 99%

Preliminary Characterization and Crystal Structure of a Thermostable Cytochrome P450 from Thermus thermophilus

Yano¹,

Blasco²,

Li³

et al. 2003

Journal of Biological Chemistry

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“…CYP119 is a P450 enzyme from the thermophile Sulfolobus solfataricus (7,(21)(22)(23). The enzyme used in this work was expressed in Escherichia coli and was of high purity as judged from the ratio of the absorbance at ϭ 415 nm ( max for Soret band of the heme group) to that at 280 nm, the R/Z ratio.…”

Section: Resultsmentioning

confidence: 99%

X-ray absorption spectroscopic characterization of a cytochrome P450 compound II derivative

Newcomb

Halgrimson

Horner

et al. 2008

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

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The cytochrome P450 enzyme CYP119, its compound II derivative, and its nitrosyl complex were studied by iron K-edge x-ray absorption spectroscopy. The compound II derivative was prepared by reaction of the resting enzyme with peroxynitrite and had a lifetime of Ϸ10 s at 23°C. The CYP119 nitrosyl complex was prepared by reaction of the enzyme with nitrogen monoxide gas or with a nitrosyl donor and was stable at 23°C for hours. Samples of CYP119 and its derivatives were studied by x-ray absorption spectroscopy at temperatures below 140 (K) at the Advanced Photon Source of Argonne National Laboratory. The x-ray absorption near-edge structure spectra displayed shifts in edge and pre-edge energies consistent with increasing effective positive charge on iron in the series native CYP119 < CYP119 nitrosyl complex < CYP119 compound II derivative. Extended x-ray absorption fine structure spectra were simulated with good fits for k ‫؍‬ 12 Å ؊1 for native CYP119 and k ‫؍‬ 13 Å ؊1 for both the nitrosyl complex and the compound II derivative. The important structural features for the compound II derivative were an iron-oxygen bond length of 1.82 Å and an iron-sulfur bond length of 2.24 Å, both of which indicate an iron-oxygen single bond in a ferryl-hydroxide, Fe IV OH, moiety.EXAFS ͉ ferryl-oxo ͉ XANES

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“…Until now this has not been possible with P450s because the available structures are all from mesophiles. However, quite recently, Wright et al (10) accidentally discovered a P450 while attempting to clone the thymidylate synthase gene from the acidothermophilic archaeon Sulfolobus solfataricus. Based on the accepted P450 nomenclature (5), this P450 has been termed CYP119.…”

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confidence: 99%

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

Yano¹,

Koo²,

Schüller³

et al. 2000

Journal of Biological Chemistry

195

180

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The structure of the first P450 identified in Archaea, CYP119 from Sulfolobus solfataricus, has been solved in two different crystal forms that differ by the ligand (imidazole or 4-phenylimidazole) coordinated to the heme iron. A comparison of the two structures reveals an unprecedented rearrangement of the active site to adapt to the different size and shape of ligands bound to the heme iron. These changes involve unraveling of the F helix C-terminal segment to extend a loop structure connecting the F and G helices, allowing the longer loop to dip down into the active site and interact with the smaller imidazole ligand. A comparison of CYP119 with P450cam and P450eryF indicates an extensive clustering of aromatic residues may provide the structural basis for the enhanced thermal stability of CYP119. An additional feature of the 4-phenylimidazole-bound structure is a zinc ion tetrahedrally bound by symmetryrelated His and Glu residues.

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Cloning of a potential cytochrome P450 from the Archaeon Sulfolobus solfataricus

Cited by 63 publications

References 14 publications

Preliminary Characterization and Crystal Structure of a Thermostable Cytochrome P450 from Thermus thermophilus

Preliminary Characterization and Crystal Structure of a Thermostable Cytochrome P450 from Thermus thermophilus

X-ray absorption spectroscopic characterization of a cytochrome P450 compound II derivative

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

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