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A Methyl-Branched Lipid -Hydroxylase from Mycobacterium Tuberculosis

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A Methyl-Branched Lipid -Hydroxylase from Mycobacterium Tuberculosis Biochemical and structural characterization of CYP124: A methyl-branched lipid ␻-hydroxylase from Mycobacterium tuberculosis Jonathan B. Johnston, Petrea M. Kells, Larissa M. Podust, and Paul R. Ortiz de Montellano1 Department of Pharmaceutical Chemistry, University of California, San Francisco, CA 94158-2517 Edited by Rodney B. Croteau, Washington State University, Pullman, WA, and approved September 25, 2009 (received for review July 6, 2009) Mycobacterium tuberculosis (Mtb) produces a variety of methyl- enzymes of known function, and their organization within the branched lipids that serve important functions, including modu- Mtb genome provides few clues about their potential biological lating the immune response during pathogenesis and contributing roles (2, 3, 17, 18). to a robust cell wall that is impermeable to many chemical agents. CYP124 is found in pathogenic and nonpathogenic mycobac- Here, we report characterization of Mtb CYP124 (Rv2266) that teria species, actinomycetes, and some proteobacteria, which includes demonstration of preferential oxidation of methyl- suggests that it has an important catalytic activity (2). CYP124 branched lipids. Spectrophotometric titrations and analysis of (Rv2266) is located adjacent to a three-gene operon containing reaction products indicate that CYP124 tightly binds and hydroxy- a sulfotransferase (Sft3, Rv2267c) that catalyzes the PAPS- lates these substrates at the chemically disfavored ␻-position. We dependent sulfation at the ␻-position of menaquinone MK-9 also report X-ray crystal structures of the ligand-free and phytanic DH-2 (19, 20). CYP128 (Rv2268c) is thought to hydroxylate the acid-bound protein at a resolution of 1.5 Å and 2.1 Å, respectively, ␻-position before sulfation (19). The sulfated form of the lipid, which provide structural insights into a cytochrome P450 with termed ‘‘S881,’’ is associated with the outer cell membrane of predominant ␻-hydroxylase activity. The structures of ligand-free Mtb, where it acts as a negative modulator of virulence in the and substrate-bound CYP124 reveal several differences induced by mouse model of infection (20). We postulated that CYP124 substrate binding, including reorganization of the I helix and might have a related substrate, i.e., a lipid with repeating methyl CHEMISTRY closure of the active site by elements of the F, G, and D helices that branching due to its proximity to the Sft3 operon. We describe bind the substrate and exclude solvent from the hydrophobic here the biochemical characterization of CYP124 that includes active site cavity. The observed regiospecific catalytic activity identifying a series of substrates consistent with ␻-hydroxylase suggests roles of CYP124 in the physiological oxidation of relevant activity and, importantly, a marked preference for lipids con- Mtb methyl-branched lipids. The enzymatic specificity and struc- taining methyl branching. We also report high-resolution struc- tures reported here provide a scaffold for the design and testing of tures of the ligand-free and phytanic acid-bound forms of specific inhibitors of CYP124. CYP124, the first structures of a native cytochrome P450 that ␻ BIOCHEMISTRY ͉ ͉ ␻ ͉ primarily oxidizes the chemically disfavored -position of a cytochrome P450 phytanic acid -hydroxylation X-ray structure hydrocarbon chain. ycobacterium tuberculosis (Mtb) is the causative agent of Results Mhuman tubercular infection that, according to the World Spectroscopic Characterization of CYP124. Purified CYP124 (Fig. S1 Health Organization (1), results in more than two million deaths in SI Appendix) is in the ferric low-spin six-coordinated form, as each year. Approximately one-third of the world’s population judged by the UV-visible absorption spectrum that shows a large harbors the bacterium in a latent, noninfective state, and new peak at 421 nm and smaller peaks at 538 and 571 nm corre- infections are occurring at an alarming rate. The emergence of sponding to the ␤- and ␣-bands, respectively (Fig. 1). Ferric drug-resistant and multidrug-resistant Mtb strains has made the CYP124 underwent facile reduction to the ferrous form by frontline antituberculosis drugs (isoniazid, streptomycin, rifam- treatment with sodium dithionite, which generated a spectrum picin, ethambutol, and pyrazinamide) less effective. Yet, no new with peaks at 415 and 544 nm. Ferrous CYP124 in complex with effective antitubercular drugs have been approved since the CO, however, showed the signature Soret band at 450 nm as well 1960s, and there is an urgent need to identify new drug targets as a smaller peak at 555 nm. Incubating ferric CYP124 with to help fight the spread of Mtb and quell the rising mortality rates various methyl-branched lipid substrates (see below) shifted the associated with infection. heme to the high-spin form, as indicated by the appearance of a Mtb produces a rich array of lipids that, among other things, dominant peak at 395 nm, small peaks at 511 and 544 nm, and allow it to thrive in the harsh environment of the macrophage, a charge-transfer band at 646 nm (Fig. 1). confer resistance to a variety of chemical agents, and stimulate CYP124, like most P450 enzymes (21), binds azole drugs the host immune response during pathogenesis. A large portion through coordination to the heme iron to produce characteristic of the Mtb genome encodes genes involved in lipid biosynthesis Type-II spectra with a peak between 425 and 435 nm and a broad and metabolism, including 20 putative cytochrome P450 en- zymes that are of interest as potential drug targets (2–5). Five of the 20 Mtb P450 enzymes (CYP51, CYP121, CYP125, CYP130, Author contributions: J.B.J., L.M.P. and P.R.O.d.M. designed research; J.B.J., P.M.K., and and CYP142) have been reported in purified form (2, 3, 6–14). L.M.P. performed research; J.B.J., L.M.P., and P.R.O.d.M. analyzed data; and J.B.J., P.M.K., To date, only CYP51 and CYP121 demonstrate a defined L.M.P., and P.R.O.d.M. wrote the paper. catalytic activity (14, 15). More than 10 years elapsed between The authors declare no conflict of interest. the sequencing of the Mtb genome and the association of a This article is a PNAS Direct Submission. catalytic activity with a second orphan P450 enzyme—CYP121 Data deposition: The atomic coordinates and structure factors have been deposited in the (15). Importantly, the recent breakthrough with CYP121 came Protein Data Bank, www.pdb.org (PDB ID codes 2WM4 and 2WM5). in part from knowledge of the function of its flanking gene (15, 1To whom correspondence should be addressed: E-mail: [email protected]. 16). Catalytic functions are difficult to assign to the remaining This article contains supporting information online at www.pnas.org/cgi/content/full/ Mtb P450s because they have diverged significantly from P450 0907398106/DCSupplemental. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0907398106 PNAS ͉ December 8, 2009 ͉ vol. 106 ͉ no. 49 ͉ 20687–20692 Downloaded by guest on September 28, 2021 unsaturation does not contribute significantly to binding. The proximity of the CYP124 gene to the Sft3 operon led us to also test phylloquinone and menaquinone as ligands of CYP124, but we were unable to detect binding. CYP124 Catalyzes ␻-Hydroxylation of Methyl-Branched Lipids. Based on the Type-I spin shifts and high-affinity binding toward methyl-branched lipids, CYP124 was incubated with spinach ferredoxin, spinach ferredoxin-NADPϩ-reductase, various lip- ids, and NADPH, and the reaction products were compared by GC-MS with those obtained in control reactions in which either CYP124 or NADPH was omitted. New signals appeared in the GC chromatograms that depended on the presence of both NADPH and CYP124 in the reaction mixture (Fig. 1 and Fig. S4 in SI Appendix). In each case, the trimethylsilylated (TMS) metabolites eluted from the GC at a higher temperature than the respective substrates, which is consistent with the presence of a TMS-protected alcohol in each metabolite. The reaction prod- ucts, identified by mass spectrometry using characteristic mo- lecular ion and fragmentation patterns, confirm that CYP124 oxidation occurs primarily at the ␻-position (Fig. S4 in SI Appendix). CYP124 converted phytanic (Fig. 1) and 15-methyl palmitic acid (Fig. S4 in SI Appendix) each into a single product. and their molecular ions at m/z ϭ 472 and 430, respectively, confirm the presence of an additional TMS-protected alcohol in each. The fragment ion at m/z ϭ 103 corresponds to the loss of -CH2OSi(CH3)3 from the ␻-position of a saturated branched- Fig. 1. Biochemical characterization of CYP124. (A) UV-visible absorbance lipid with a TMS-protected hydroxyl group (23), and we ob- spectra of CYP124 in the ferric (black), ferrous (green), ferric high-spin (blue) served such fragments with the new phytanic and 15-methyl and ferrous-CO (red) forms. (B) Overlaid GC chromatograms showing the total palmitic acid metabolites (Fig. S4 in SI Appendix). ion current (TIC) versus retention time for reactions containing Fdx, Fdr, Isoprenoids (farnesol, farnesyl diphosphate, geranylgeraniol) NADPH and phytanic acid incubated for 10 min in the presence (dotted line) were also efficiently oxidized by CYP124 into the respective or absence (solid line) of CYP124. The reaction scheme depicts the CYP124- and ␻-hydroxylated products (Fig. S4 in SI Appendix). These assign- NADPH-dependent conversion of phytanic acid into ␻-hydroxy-phytanic acid. ments were based on comparisons with the product formed in Reactions were extracted, and these compounds were derivatized with BSTFA incubations of farnesol with CYP2E1, which catalyzes ␻- before GC analysis, yielding peaks at 18.2 min (S) and 20.7 min (P) correspond- hydroxylation of farnesol (24). Both the retention times and mass ing to the trimethylsilyl-ester of phytanic acid and TMS-␻-hydroxy phytanic TMS-ester, respectively. spectra of the CYP124 and CYP2E1 metabolites matched in our assays. The product from farnesyl diphosphate was treated with alkaline phosphatase before GC-MS analysis to release the trough at 390–410 nm that reflect azole coordination to the farnesol structure, which then matched the mass and retention heme iron through a nitrogen atom (22).
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