NtellanoPageand androstenedione, which regrettably lacks the important 17-sidechain of cholesterol and cholest-4-en-3-one. Ultimately, purified CYP125A1 expressed in E. coli was shown to catalyze not only 26-hydroxylation, but in addition the subsequent conversion on the 26-alcohol to the 26aldehyde and 26-carboxylic acid (58). In addition, the crystal structure of CYP125A1 with cholest-4-en-3-one bound inside the active web page demonstrated that this substrate binds within a tightfitting active site using the side-chain methyl terminus positioned close towards the heme iron atom (Fig. 6) (16).Author Manuscript Author Manuscript Author Manuscript Author ManuscriptM. smegmatis, a non-pathogenic mycobacterium, has not one, but two enzymes that are homologous to M. tuberculosis CYP125A1 (20, 68). CYP125A3, like CYP125A1, oxidizes the 26-methyl group of cholesterol and cholest-4-en-3-one towards the corresponding aldehyde and carboxylic acid (20). CYP125A4, the second enzyme, also catalyzes the 26hydroxylation of cholesterol, but has a a great deal larger activity for 26-hydroxylation of 7hydroxycholesterol (68). This can be attributed towards the presence of a bulky tryptophan (Trp83) in CYP125A3 as opposed to the smaller sized tyrosine in CYP125A4 at a position exactly where it interferes with binding in the 7-hydroxy group.THBS1 Protein Storage & Stability Replacing the tryptophan of CYP125A3 by a tyrosine by site-specific mutagenesis enhances the potential of this enzyme to oxidize 7hydroxycholesterol.P4HB, Human (His) The crystal structure in the CYP125A3 W83Y mutant is consistent with this interpretation (68).The carbon atoms of the cholesterol side-chain are incorporated into surface lipids of M.PMID:23551549 tuberculosis. Radiocarbon labeling showed that the label of [26-14C]cholesterol was incorporated into the virulence components PDIM (phthiocerol dimycocerosate) (69) and SL-1 (sulfolipid-1) (70). This acquiring was extra precisely demonstrated in the case of PDIM by mass spectrometric evaluation of your PDIM lipid fraction after growth on cholesterol with 25,26,26,26,27,27,27-heptadeuterated cholesterol (16). As anticipated, the elevated mass of PDIM noticed when cells are grown in the presence of cholesterol is also seen when the cells are grown with propionic acid within the medium, supporting the inference that propionoyl CoA released inside the degradation from the cholesterol side-chain serves as a feedstock for the synthesis of PDIM and SL-1 (16, 71) In spite of the tight match of cholest-4-en-3-one inside the CYP125A1 active internet site observed in the crystal structure (Fig. 6), detailed studies on the oxidation of unlabeled and 25,26,26,26,27,27,27 deuterium labeled cholesterol inside the presence of 16O2 versus 18O2 established that sideproducts are formed, no less than when the oxidation of cholesterol is performed in vitro (72). Mass spectrometric and chromatographic comparisons allowed detection of the merchandise M1 by way of M5 (Fig. 7), as well as the anticipated 26-alcohol, 26-aldehyde, and 26-carboxylic acid. Comparison of the items formed from cholest-4-en-3-one, its 26-alcohol metabolite, and the 26-aldehyde showed that the side-products are formed from option reactions on the 26-aldehyde. Some assistance for the mechanisms proposed in Fig. 7 for the formation of these side-products comes from the finding that replacement with the cysteine thiolate of CYP125A1 by a seleno cysteine anion shifts item formation inside the path on the option pathways, all of which require nucleophilic attack from the iron dioxy complicated on the aldehyde group. The seleno ligand is really a better electron donor.
