College, 204 George Street, Glasgow, GI 1XL. Phenylalanine-393 (F393) ... P450 BM3 is within van der Waals distance of the heme ligand, cysteine-400 (C400).
Biochemical SocietyTransactions (200 I ) Volume 29, Part I
52 azole resistance mechanisms in clinical isolates of Candida
54 Kinetic characterisation of the catalase-peroxidase enzyme
albicans and Cryptococcus neoformans from intensive care patients D. Greetham, D. Lamb, D Kelly and S Kelly Institute of Biological Sciences, Edward Llwyd Building, University of Wales, Aberystwyth, Ceredigion, SY23 3TA
from Mycobacterium tuberculosis Nigel A.I. Eadp, Judit M. Nagy, Anthony E.G. Cass and Katherine A. Brown Department of Biochemistry, Imperial College of Science, Technology and Medicine, Exhibition Road, London, SW7 2AI: UK
Candida albicansand Cryptococcus neoformans are pathogenic yeast species, which cause fungaemia, superficial infections and other systemic infections in humans. Azole antifungal compounds inhibit P-450 sterol 14a-demethylase (Ergllp, a key enzyme in the ergosterol biosynthetic pathway of fungi, resulting in the accumulation of 14a methylated sterols and a decrease in ergosterol levels, leading to cell growth arrest. The widespread use of these antifungals with increasing prominence in AIDS patients has led to appearance of resistant yeast species and other fungi in general. Resistance appears due to three possible mechanisms; a disruption in the ERG3 gene, a lack of sensitivity to Ergllp to fluconazole or via drug-efflux mechanisms of which CDRl and BENR have been identified in Candida. We are investigating thirty clinical isolates of C.albicans and 21 strains ofC.neoformans,will present data on the alteration in sterol biosynthesis with or without treatment as well as measurement of the intracellular concentration of azole drug.
A37
In 1993 the World Health Organisation declared tuberculosis a global emergency and it has been estimated that 70 million people could be infected with the bacteria by 2020. Since 1952 isoniazid has been the core compound used in clinical treatment of the disease and it is believed that a catalase-peroxidase enzyme (CP), encoded by the katG gene, confers sensitivity to this drug. C P is an 80 kDa dual functional enzyme demonstrating both catalase and peroxidase activities. The amino-terminal domain of C P shows good sequence homology with yeast cytochrome c peroxidase with the haem binding motif and catalytic residues in the active site being preserved. However, the carboxy-terminal domain, despite some sequence homology with yeast cytochrome c peroxidase, does not appear to have residues associated with haem binding. To probe the functional properties of C P comparative steadystate parameters have been obtained for the M. tuberculosis C P enzyme. The effect of various inhibitors upon these activities, with complementary spectroscopic data, will also be presented.
53 Phenylalanine-393: Controlling the electronic properties of the heme in Flavocytochrome P450 BM3 T.W.B. Ost, C.S. Miles, A.W. Munro, G.A. Reid, S.K. Chapman Department of Chemistry, University of Edinburgh, Kings Buildings, West Mains Road, Edinburgh, EH9 3JJ, The Institute of Cell and Molecular Biology, University of Edinburgh, Kings Buildings, Mayfield Road, Edinburgh, EH9 317; department of Pure and Appled Chemistry, University of Strathclyde, The Royal College, 204 George Street, Glasgow, GI 1XL Phenylalanine-393 (F393) in flavocytochrome P450 BM3 is within van der Waals distance of the heme ligand, cysteine-400 (C400). Sequence comparisons with other bacterial P450s indicate this residue to be highly conserved within the P450 superfamily. In this study F393 was substituted in order to elucidate its role. The successful overexpression of mutant holoenzymes F393A, F393H and F393Y immediately negated previous assumptions that the residue was essential for heme binding - all proteins had their full complement of heme. Perturbations in the soret absorption of the reduced-CO adduct of mutants F393A (444nm) and F393H (445nm) suggest significant electronic deviations from wild-type (449nm). This theory is supported by the substratefree heme reduction potentials of the mutant enzymes (WT = -427mV, F393H = -331mV, F393A = -312mV, p H 7.0). This suggests that the nature of the residue at position 393 has an important influence on the electronic properties of the heme.
55 Probing the stability of the class I1 peroxidase from Coprinus cinereus N.C.Brissett', L.Jergensenb, P.Schneiderb and A.T.Smith' School of Biological Sciences, University of Sussex, Brighton, BNl 9QG, U.K." Novo Nordisk A/S, 1 Novo Alle, Bagsvaerd, Denmark 2880b The use of peroxidase from Coprinus cinereus (CIP) in industrial processes is well documented. As CIP is the only bulk produced peroxidase involved in an industrial process, studies into the stability of this enzyme are deemed to have high importance. In this study, chaotrope-induced denaturation of C I P m and a mutant (CIP[QUAD]) was carried out. Rapid phase changes in absorption spectra, fluorescence and circular dichroism were recorded using Stopped-flow reaction analysers supplied by Applied Photophysics. The loss of Ca2+was monitored using a fluorescent dye, Calcium Green-1 (Molecular Probes). Results showed that we can accurately estimate the amount of Ca2+per molecule of protein. The experiments also conclusively show the [QUAD] mutant has one molecule of Ca2+less than [wr] per molecule of protein. The difference in rates of Ca2+loss can be directly attributed to the destabilisation of the [QUAD] mutant protein structure. By comparing and contrasting the results, a scheme for chaotrope-induced C I P unfolding is proposed.
0 2001 Biochemical Society
