An enlarged, adaptable active site in CYP164 family P450 enzymes, the sole P450 in Mycobacterium leprae.

CYP164 family P450 enzymes are found in only a subset of mycobacteria and include CYP164A1, which is the sole P450 found in Mycobacterium leprae, the causative agent of leprosy. This has previously led to interest in this enzyme as a potential drug target. Here we describe the first crystal structure of a CYP164 enzyme, CYP164A2 from Mycobacterium smegmatis. CYP164A2 has a distinctive, enlarged hydrophobic active site that extends above the porphyrin ring toward the access channels. Unusually, we find that CYP164A2 can simultaneously bind two econazole molecules in different regions of the enlarged active site and is accompanied by the rearrangement and ordering of the BC loop. The primary location is through a classic interaction of the azole group with the porphyrin iron. The second econazole molecule is bound to a unique site and is linked to a tetracoordinated metal ion complexed to one of the heme carboxylates and to the side chains of His 105 and His 364. All of these features are preserved in the closely homologous M. leprae CYP164A1. The computational docking of azole compounds to a homology model of CYP164A1 suggests that these compounds will form effective inhibitors and is supported by the correlation of parallel docking with experimental binding studies of CYP164A2. The binding of econazole to CYP164A2 occurs primarily through the high-spin "open" conformation of the enzyme (K(d) [dissociation constant] of 0.1 µM), with binding to the low-spin "closed" form being significantly hindered (K(d) of 338 µM). These studies support previous suggestions that azole derivatives may provide an effective strategy to improve the treatment of leprosy.

A quantitative assay of porphyrins in leprosy patients. A spectrophotometric study.

Quantitative estimations of porphyrin in the blood, urine, and feces of 30 leprosy patients under treatment with dapsone, ten untreated cases, and 100 normal subjects were done by Rimington's method. Dapsone had no adverse effect on porphyrin metabolism because none of the cases of leprosy under study developed statistically significantly raised porphyrin levels in the blood, urine, and stools. Although erythrocyte coproporphyrin levels were significantly higher in leprosy patients than controls and urinary uroporphyrin levels significantly lower, most values fell within the normal range. These differences did not appear to have any clinical significance, and their cause remains unknown.

Biochemical properties of cultivated Mycobacterium lepraemurium.

1. Dehydrogenase activity of whole cell of cultivated M. lepraemurium is accelerated with sodium laurate, but not with the other substrates. 2. Dehydrogenase activity of cell free extract of cultivated M. lepraemurium is accelerated with citrate or malate. 3. There is no acceleration of oxygen consumption corresponding to added substrates in the respiration activity of whole cells of cultivated M. lepraemurium, but endogenous QO2 is 1.7 mul. 4. Cell free extract of cultivated M. lepraemurium shows slight acceleration of oxygen consumption with NADH, but does not with citrate or alpha-ketoglutarate. 5. NADH is not oxidized rapidly with the particle fraction of cultivated M. lepraemurium. 6. Type b1 cytochrome having an absorption peak at a wave length of 561 mmu and type a2 cytochrome having an absorption peak at 625 mmu are detected in an oxidoreductive difference spectrum or particle fraction of cultivated M. lepraemurium, but type c cytochrome having the absorption peak at 550 mmu is not seen. Since the other cultivable acid-fast bacilli always have type c cytochrome, nondetection of type c cytochrome is characteristic for M. lepraemurium. 7. These cytochromes are reduced with NADH. 8. M. lepraemurium produces a red pigment which emits a red fluorescence with ultraviolet light on its 1% Ogawa yolk medium. This phenomenon is a characteristic of M. lepraemurium, M. avium and M. intracellulare.