The Mycobacterium tuberculosis cytochrome P450 enzyme CYP142 is encoded in a large gene cluster involved in metabolism of host cholesterol. CYP142 was expressed and purified as a soluble, low spin P450 hemoprotein. CYP142 binds tightly to cholesterol and its oxidized derivative cholest-4-en-3-one, with extensive shift of the heme iron to the high spin state. High affinity for azole antibiotics was demonstrated, highlighting their therapeutic potential. CYP142 catalyzes either 27-hydroxylation of cholesterol/cholest-4-en-3-one or generates 5-cholestenoic acid/cholest-4-en-3-one-27-oic acid from these substrates by successive sterol oxidations, with the catalytic outcome dependent on the redox partner system used. The CYP142 crystal structure was solved to 1.6 Å , revealing a similar active site organization to the cholesterol-metabolizing M. tuberculosis CYP125, but having a near-identical organization of distal pocket residues to the branched fatty acid oxidizing M. tuberculosis CYP124. The cholesterol oxidizing activity of CYP142 provides an explanation for previous findings that ⌬CYP125 strains of Mycobacterium bovis and M. bovis BCG cannot grow on cholesterol, because these strains have a defective CYP142 gene. CYP142 is revealed as a cholesterol 27-oxidase with likely roles in host response modulation and cholesterol metabolism. Tuberculosis (TB)3 is a debilitating and frequently fatal disease caused by the bacterium Mycobacterium tuberculosis. The disease usually presents as a pulmonary condition but can also affect other parts of the body (1). M. tuberculosis can also remain in a dormant phase in the host (latent TB), and a proportion of individuals with latent TB will later develop active TB (2). The emergence and proliferation of multidrugresistant and extensively drug-resistant M. tuberculosis strains have provided a major challenge for TB therapeutic development (3). Numerous M. tuberculosis clinical strains are now resistant to one or several front line and second line drugs, including antibiotics such as rifampicin, isoniazid, streptomycin, and ethambutol (4). TB and drug-resistant strains are prevalent in the less developed countries of Asia and Africa, and synergy between M. tuberculosis and HIV leads to high rates of TB infection and deaths in HIV-infected individuals (5). Consequently, there has been recent intensive research on the genetics and biochemistry of M. tuberculosis, as well as work to develop novel therapeutics and to identify new drug targets (6 -8). Recent successes include the development of 1,3-benzothiazin-4-ones that inhibit the decaprenylphosphoryl-␤-D-ribose 2Ј-epimerase enzyme and formation of cell wall arabinans, and clavulanate/␤-lactam combinations inhibiting formation of cell wall peptidoglycan (9, 10).The M. tuberculosis H37Rv and then the M. tuberculosis CDC1551 genome sequences provided key information on the M. tuberculosis proteome, highlighting the complexity of M. tuberculosis lipid metabolism as well as several genes previously found only in eukaryotes, e.g. the a...

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Gd4O2S:Yb:Tm rare-earth upconversion phosphors have been utilised to monitor the redox behaviour of flavin mononucleotide and report on the turnover of a flavo-protein, (pentaerythritol tetranitrate reductase). The presence of two bands separated by over 300 nm in the UCP emission spectra allows ratiometric signalling of these processes with high sensitivity.

The role of transition-state stabilization in enzyme catalysis, as proposed by Pauling, has been clearly demonstrated by extensive studies. In contrast, ground-state destabilization can also contribute to enzyme catalysis, but experimental evidence has been more limited. In recent years, high-resolution X-ray crystal structures of enzyme–substrate complexes have been obtained which show evidence for ground-state strain. We found that Y71F and F448H mutant tyrosine phenol-lyase (TPL) form complexes with 3-fluoro-l-tyrosine, a substrate, which shows a bending of the substrate aromatic ring about 20° out of plane, and we suggested that this was evidence for ground-state destabilization in TPL catalysis. Here, we have now evaluated quantitatively the role of ground-state destabilization in TPL catalysis. Phe-448 and Phe-449 are in close contact with the bound substrate side chain, and by mutating these residues to alanine and leucine, the contribution they play via ground-state destabilization was investigated. F448A, F448L and F449A TPL have activity for elimination of phenol from l-tyrosine reduced by a factor of 104, 103, and 104, respectively, but they have near-normal activity with the alternate substrates S-(o-nitrophenyl)-l-cysteine and S-ethyl-l-cysteine. F448A TPL forms quinonoid intermediates from l-tyrosine and S-ethyl-l-cysteine with rate constants similar to those of wild-type TPL. In addition, F448A TPL can form an aminoacrylate intermediate from S-ethyl-l-cysteine but not l-tyrosine, with a rate constant similar to that of wild-type TPL. Thus, the effect of the mutation is specifically on the elimination of phenol from l-tyrosine. We also examined the effect of hydrostatic pressure on the rates and equilibria of formation of the quinonoid intermediates from F448H and F448A TPL and 3-fluoro-l-tyrosine. Although the fastest phase shows only a small effect of pressure, the three slower phases have significant pressure dependences, suggesting that they may be associated with a conformational change. These results demonstrate that Phe-448 and Phe-449 contribute a total of about 108 to catalysis in TPL, about 50% of the estimated rate acceleration, by introducing ground-state destabilization into the l-tyrosine substrate.

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