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Iron-containing monooxygenases in Mycobacterium tuberculosis cholesterol degradation : biochemical and phylogenetic perspectives

University of British Columbia

Mycobacterium tuberculosis (Mtb) is the human pathogen that causes tuberculosis. A gene cluster encoding a cholesterol degradation pathway plays a role in Mtb virulence. Two iron-containing monooxygenases in this pathway were characterized with respect to their roles in bacterial cholesterol catabolism: the Rieske oxygenase (RO) KshAB, and the cytochrome P450 (P450) Cyp125. These enzymes are predicted to catalyze the first ring-opening step and the first transformation of the steroid side chain, respectively. Cyp125A1 (Mtb) and Cyp125A14P (Rhodococcus jostii RHA1) were expressed in R. jostii RHA1 and characterized in vitro using the Mtb reductase KshB. Both enzymes were purified with the heme iron in a predominantly high spin state and exhibiting thiolate ligation of the heme iron. Both P450s bound cholesterol and 4-cholesten-3-one with apparent submicromolar affinity. Cyp125A1 was demonstrated to catalyze C26-monohydroxylation of both steroids. KshA (a terminal oxygenase) and KshB (an oxygenase reductase) of Mtb were produced in Escherichia coli and characterized in vitro. KshAB had over twenty times the apparent substrate specificity for steroid substrates with isopropionyl-CoA side chains than for the corresponding 17-keto steroids. The apparent KMO₂ with a CoA thioester-bearing steroid was 90 ± 10 μM whereas that for the corresponding 17-keto steroid was in excess of 1.2 mM. These results suggest that the physiological substrate(s) for KshAB is likely a CoA thioester intermediate of cholesterol side chain degradation. A comprehensive phylogenetic analysis was undertaken to consolidate the available RO literature. Six hundred fifty enzymes that are fully representative of the RO terminal oxygenase (RO-O) sequences in the NCBI database were collected and aligned to a structure-based sequence template. The structure-based alignment was also used to objectively define the structurally conserved positions that were included in phylogenetic reconstruction. The resulting analysis revealed a level of RO-O diversity that has been unrecognized in previous literature and that necessitates a different approach to RO-O classification. A classification scheme based on the system currently in use for P450s was proposed. This work provides significant insight into the cholesterol degradation pathway of Mtb and the RO-O protein family and contributes to potential commercial applications in bioremediation, biocatalysis, and Mtb therapeutics.

Text

2012-03-09

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/41309

Biochemistry and Molecular Biology

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/