SummaryThe evolutionary pressures that shaped the specificity and catalytic efficiency of enzymes can only be speculated. While directed evolution experiments show that new functions can be acquired under positive selection with few mutations, the role of negative selection in eliminating undesired activities and achieving high specificity remains unclear. Here we examine intermediates along the 'lineage' from a naturally-occurring C 12 -C 20 fatty acid hydroxylase (P450 BM3 ) to a laboratory-evolved P450 propane monooxygenase (P450 PMO ) having 20 heme domain substitutions compared to P450 BM3 . Biochemical, crystallographic and computational analyses show that a minimal perturbation of the P450 BM3 fold and substrate binding pocket accompanies a significant broadening of enzyme substrate range and the emergence of propane activity. In contrast, refinement of the enzyme catalytic efficiency for propane oxidation (~9,000-fold increase in k cat /K m ) involves profound reshaping and partitioning of the substrate access pathway. Remodeling of the substrate recognition mechanisms ultimately results in remarkable narrowing of the substrate profile around propane and enables the acquisition of a basal iodomethane dehalogenase activity as yet unknown in natural alkane monooxygenases. A highly destabilizing L188P substitution in a region of the enzyme that undergoes a large conformational change during catalysis plays an important role in adaptation to the gaseous alkane. This work demonstrates that positive selection alone is sufficient to completely re-specialize the cytochrome P450 for function on a non-native substrate.

The ferryl (Fe(IV)O) intermediate is important in many heme enzymes and thus the precise nature of the Fe(IV)-O bond is critical in understanding enzymatic mechanisms. The 1.40 Å crystal structure of cytochrome c peroxidase Compound I has been solved as a function of x-ray dose while monitoring the visible spectrum. The Fe-O bond increases linearly from 1.73 Å in the low x-ray dose structure to 1.90 Å in the high dose structure. The low dose structure correlates well with a Fe(IV)=O bond while we postulate that the high dose structure is the cryo-trapped Fe(III)-OH species previously thought to be Fe(IV)-OH.

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Fig. 1. Peroxidase mechanism. (A) Traditional "water-modified" mechanism of CmpI formation. In this mechanism, peroxide first coordinates with the heme iron, followed by proton transfer to the distal peroxide O atom via an ordered water molecule and the distal His. The protonation state of the distal His depends on the His pK a . Our computational experiments indicate that the pK a substantially increases in CmpI. (B) Modified mechanism based on the observation that His52 in CCP CmpI is protonated in the neutron diffraction structure (8). Going from the initial peroxide complex to CmpI proceeds via two possible routes, one of which involves the Fe(IV)-OH intermediate. (C) Mechanism of CmpII reduction, which includes a protoncoupled electron transfer event resulting in a net transfer of a proton from the distal His to the ferryl O atom.

The parasitic protozoa Leishmania major produces a peroxidase (L. major peroxidase; LmP) that exhibits activities characteristic of both yeast cytochrome c peroxidase (CCP) and plant cytosolic ascorbate peroxidase (APX

DevS is one of the two sensing kinases responsible for DevR activation and the subsequent entry of Mycobacterium tuberculosis into dormancy. Full length wild-type DevS forms a stable oxy-ferrous complex. The DevS autooxidation rates are extremely low (half-lives > 24 h) in the presence of cations such as K + , Na + , Mg 2+ , and Ca 2+ . At relatively high concentrations (100 µM), Fe 3+ mildly increases the autooxidation rate (six-fold increase) while Cu 2+ accelerates autooxidation more than 1500-fold. Contrary to expectations, removal of the key hydrogen bond between the iron-coordinated oxygen and Tyr171 in the Y171F mutant provides a protein of comparable stability to autooxidation and similar oxygen dissociation rate. This correlates with our earlier finding that the Y171F mutant and wild-type kinase activities are similarly regulated by the binding of oxygen: namely, the ferrous 5c complex is active whereas the oxy ferrous 6c species is inactive. Our results indicate that DevS is a gas sensor in vivo rather than a redox sensor and that the stability of its ferrous-oxy complex is enhanced by inter-domain interactions.Tuberculosis remains a health concern in the 21st century, as it is a leading cause of mortality among infectious diseases and results in the death of 2-3 million people each year (1). Active tuberculosis cases are recruited from an immense reservoir of about 2 billion people latently infected with the bacillus worldwide (1,2). Despite the magnitude of this health problem, no new drug has been introduced for tuberculosis therapy during the past 30 years. The current tuberculosis treatment requires at least six months, which makes it costly and reduces patient compliance. The available medications can only be used with limited success in infections caused by the rapidly emerging resistant tuberculosis strains, particularly multi-drug resistant and extensively drug resistant tuberculosis (3). Latent tuberculosis is itself very difficult to treat, since dormant Mycobacterium tuberculosis displays a diminished susceptibility to drugs.The mechanism of Mycobacterium tuberculosis entrance into the dormant phase still needs to be unraveled (4); this is a key step in the development of new and more effective approaches to the therapy of this disease. When Mycobacterium tuberculosis enters dormancy, it alters its metabolism in response to unfavorable environmental stimuli and undergoes striking morphological changes (5,6). Dormant Mycobacterium tuberculosis bacilli have a thicker modified cell wall (7) that has lost its acid-fastedness and is Ziehl-Neelsen negative (8,9). † This work was supported by grants AI074824 (P.R.O.M.) and GM42614 (T.L.P.) from the National Institutes of Health.*To whom editorial correspondence should be addressed: Dr. Paul Ortiz de Montellano University of California, Genentech Hall GH-N572D, 600 16 th Street, Box 2280 San Francisco, CA 94158-2517 476-2903 FAX: (415) 502-4728, e-mail: ortiz@cgl.ucsf So far, DevS and DosT appear to be redundant in their function, sugge...

Cytochrome P450cin catalyzes the monooxygenation of 1,8-cineole, which is structurally very similar to d-camphor, the substrate for the most thoroughly investigated cytochrome P450, cytochrome P450cam. Both 1,8-cineole and d-camphor are C(10) monoterpenes containing a single oxygen atom with very similar molecular volumes. The cytochrome P450cin-substrate complex crystal structure has been solved to 1.7 A resolution and compared with that of cytochrome P450cam. Despite the similarity in substrates, the active site of cytochrome P450cin is substantially different from that of cytochrome P450cam in that the B' helix, essential for substrate binding in many cytochrome P450s including cytochrome P450cam, is replaced by an ordered loop that results in substantial changes in active site topography. In addition, cytochrome P450cin does not have the conserved threonine, Thr252 in cytochrome P450cam, which is generally considered as an integral part of the proton shuttle machinery required for oxygen activation. Instead, the analogous residue in cytochrome P450cin is Asn242, which provides the only direct protein H-bonding interaction with the substrate. Cytochrome P450cin uses a flavodoxin-like redox partner to reduce the heme iron rather than the more traditional ferredoxin-like Fe(2)S(2) redox partner used by cytochrome P450cam and many other bacterial P450s. It thus might be expected that the redox partner docking site of cytochrome P450cin would resemble that of cytochrome P450BM3, which also uses a flavodoxin-like redox partner. Nevertheless, the putative docking site topography more closely resembles cytochrome P450cam than cytochrome P450BM3.

Cytochrome P450cin (CYP176A1) is a bacterial P450 isolated from Citrobacter braakii that catalyzes the hydroxylation of cineole to (S)-6␤-hydroxycineole. This initiates the biodegradation of cineole, enabling C. braakii to live on cineole as its sole source of carbon and energy. P450cin lacks the almost universally conserved threonine residue believed to be involved in dioxygen activation and instead contains an asparagine at this position (Asn-242). To investigate the role of Asn-242 in P450cin catalysis, it was converted to alanine, and the resultant mutant was characterized. The characteristic CO-bound spectrum and spectrally determined K D for substrate binding were unchanged in the mutant. The x-ray crystal structures of the substrate-free and -bound N242A mutant were determined and show that the only significant change is in a reorientation of the substrate such that (R)-6␣-hydroxycineole should be a major product. Molecular dynamics simulations of both wild type and mutant are consistent with the change in regio-and stereoselectivity predicted from the crystal structure. The mutation has only a modest effect on enzyme activity and on the diversion of the NADPHreducing equivalent toward unproductive peroxide formation. Product profile analysis shows that (R)-6␣-hydroxycineole is the main product, which is consistent with the crystal structure. These results demonstrate that Asn-242 is not a functional replacement for the conserved threonine in other P450s but, rather, is critical in controlling regioselective substrate oxidation.