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

Abstract

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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Rational Engineering of Human Cytochrome P450 2B6 for Enhanced Expression and Stability: Importance of a Leu264→Phe Substitution

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