NtellanoPageand androstenedione, which regrettably lacks the important 17-sidechain of cholesterol and
NtellanoPageand androstenedione, which however lacks the vital 17-sidechain of cholesterol and cholest-4-en-3-one. Ultimately, purified CYP125A1 expressed in E. coli was shown to catalyze not simply 26-hydroxylation, but also the subsequent conversion of the 26-alcohol to the 26aldehyde and 26-carboxylic acid (58). Furthermore, the crystal structure of CYP125A1 with cholest-4-en-3-one bound within the active web-site demonstrated that this substrate binds within a tightfitting active web site with the side-chain methyl terminus IL-6, Human (CHO) located close towards the heme iron atom (Fig. 6) (16).Author Manuscript Author Manuscript Author Manuscript Author Tenascin/Tnc Protein Source ManuscriptM. smegmatis, a non-pathogenic mycobacterium, has not one, but two enzymes which are homologous to M. tuberculosis CYP125A1 (20, 68). CYP125A3, like CYP125A1, oxidizes the 26-methyl group of cholesterol and cholest-4-en-3-one to the corresponding aldehyde and carboxylic acid (20). CYP125A4, the second enzyme, also catalyzes the 26hydroxylation of cholesterol, but includes a a lot larger activity for 26-hydroxylation of 7hydroxycholesterol (68). This could be attributed to the presence of a bulky tryptophan (Trp83) in CYP125A3 as opposed to the smaller sized tyrosine in CYP125A4 at a position where it interferes with binding on the 7-hydroxy group. Replacing the tryptophan of CYP125A3 by a tyrosine by site-specific mutagenesis enhances the ability of this enzyme to oxidize 7hydroxycholesterol. The crystal structure on the CYP125A3 W83Y mutant is constant with this interpretation (68).The carbon atoms in the cholesterol side-chain are incorporated into surface lipids of M. tuberculosis. Radiocarbon labeling showed that the label of [26-14C]cholesterol was incorporated in to the virulence elements PDIM (phthiocerol dimycocerosate) (69) and SL-1 (sulfolipid-1) (70). This finding was a lot more precisely demonstrated in the case of PDIM by mass spectrometric evaluation from the PDIM lipid fraction right after development on cholesterol with 25,26,26,26,27,27,27-heptadeuterated cholesterol (16). As expected, the improved mass of PDIM seen when cells are grown in the presence of cholesterol is also noticed when the cells are grown with propionic acid within the medium, supporting the inference that propionoyl CoA released in the degradation of the cholesterol side-chain serves as a feedstock for the synthesis of PDIM and SL-1 (16, 71) Regardless of the tight match of cholest-4-en-3-one in the CYP125A1 active web site observed inside the crystal structure (Fig. six), detailed studies from the oxidation of unlabeled and 25,26,26,26,27,27,27 deuterium labeled cholesterol in the presence of 16O2 versus 18O2 established that sideproducts are formed, at least when the oxidation of cholesterol is performed in vitro (72). Mass spectrometric and chromatographic comparisons allowed detection of your products M1 via M5 (Fig. 7), along with the expected 26-alcohol, 26-aldehyde, and 26-carboxylic acid. Comparison of the goods formed from cholest-4-en-3-one, its 26-alcohol metabolite, and the 26-aldehyde showed that the side-products are formed from alternative reactions of the 26-aldehyde. Some assistance for the mechanisms proposed in Fig. 7 for the formation of these side-products comes in the acquiring that replacement in the cysteine thiolate of CYP125A1 by a seleno cysteine anion shifts product formation within the direction of the alternative pathways, all of which demand nucleophilic attack from the iron dioxy complex on the aldehyde group. The seleno ligand is a greater electron donor.