A highly conserved mycobacterial cholesterol catabolic pathway

Degradation of the cholesterol side‐chain in Mycobacterium tuberculosis is initiated by two cytochromes P450, CYP125A1 and CYP142A1, that sequentially oxidize C26 to the alcohol, aldehyde and acid metabolites. Here we report characterization of the homologous enzymes CYP125A3 and CYP142A2 from Mycobacterium smegmatis mc² 155. Heterologously expressed, purified CYP125A3 and CYP142A2 bound cholesterol, 4‐cholesten‐3‐one, and antifungal azole drugs. CYP125A3 or CYP142A2 reconstituted with spinach ferredoxin and ferredoxin reductase efficiently hydroxylated 4‐cholesten‐3‐one to the C‐26 alcohol and subsequently to the acid. The X‐ray structures of both substrate‐free CYP125A3 and CYP142A2 and of cholest‐4‐en‐3‐one‐bound CYP142A2 reveal significant differences in the substrate binding sites compared with the homologous M. tuberculosis proteins. Deletion only of cyp125A3 causes a reduction of both the alcohol and acid metabolites and a strong induction of cyp142 at the mRNA and protein levels, indicating that CYP142A2 serves as a functionally redundant back up enzyme for CYP125A3. In contrast to M. tuberculosis, the M. smegmatis Δcyp125Δcyp142 double mutant retains its ability to grow on cholesterol albeit with a diminished capacity, indicating an additional level of redundancy within its genome.     

Structural and biochemical characterization of Mycobacterium tuberculosis CYP142: evidence for multiple cholesterol 27-hydroxylase activities in a human pathogen

The Mycobacterium tuberculosis cytochrome P450 enzyme CYP142 is encoded in a large gene cluster involved in metabolism of host cholesterol. CYP142 was expressed and purified as a soluble, low spin P450 hemoprotein. CYP142 binds tightly to cholesterol and its oxidized derivative cholest-4-en-3-one, with extensive shift of the heme iron to the high spin state. High affinity for azole antibiotics was demonstrated, highlighting their therapeutic potential. CYP142 catalyzes either 27-hydroxylation of cholesterol/cholest-4-en-3-one or generates 5-cholestenoic acid/cholest-4-en-3-one-27-oic acid from these substrates by successive sterol oxidations, with the catalytic outcome dependent on the redox partner system used. The CYP142 crystal structure was solved to 1.6 Å, revealing a similar active site organization to the cholesterol-metabolizing M. tuberculosis CYP125, but having a near-identical organization of distal pocket residues to the branched fatty acid oxidizing M. tuberculosis CYP124. The cholesterol oxidizing activity of CYP142 provides an explanation for previous findings that ΔCYP125 strains of Mycobacterium bovis and M. bovis BCG cannot grow on cholesterol, because these strains have a defective CYP142 gene. CYP142 is revealed as a cholesterol 27-oxidase with likely roles in host response modulation and cholesterol metabolism.     

Mycobacterial Cytochrome P450 125 (Cyp125) Catalyzes the Terminal Hydroxylation of C27 Steroids

Cyp125 (Rv3545c), a cytochrome P450, is encoded as part of the cholesterol degradation gene cluster conserved among members of the Mycobacterium tuberculosis complex. This enzyme has been implicated in mycobacterial pathogenesis, and a homologue initiates cholesterol catabolism in the soil actinomycete Rhodococcus jostii RHA1. In Mycobacterium bovis BCG, cyp125 was up-regulated 7.1-fold with growth on cholesterol. A cyp125 deletion mutant of BCG did not grow on cholesterol and accumulated 4-cholesten-3-one when incubated in the presence of cholesterol. Wild-type BCG grew on this metabolite. By contrast, a parallel cyp125 deletion mutation of M. tuberculosis H37Rv did not affect growth on cholesterol. Purified Cyp125 from M. tuberculosis, heterologously produced in R. jostii RHA1, bound cholesterol and 4-cholesten-3-one with apparent dissociation constants of 0.20 ± 0.02 μm and 0.27 ± 0.05 μm, respectively. When reconstituted with KshB, the cognate reductase of the ketosteroid 9α-hydroxylase, Cyp125 catalyzed the hydroxylation of these steroids. MS and NMR analyses revealed that hydroxylation occurred at carbon 26 of the steroid side chain, allowing unambiguous classification of Cyp125 as a steroid C26-hydroxylase. This study establishes the catalytic function of Cyp125 and, in identifying an important difference in the catabolic potential of M. bovis and M. tuberculosis, suggests that Cyp125 may have an additional function in pathogenesis.     

Mycobacterium tuberculosis CYP125A1, a steroid C27 monooxygenase that detoxifies intracellularly generated cholest‐4‐en‐3‐one

The infectivity and persistence of Mycobacterium tuberculosis requires the utilization of host cell cholesterol. We have examined the specific role of cytochrome P450 CYP125A1 in the cholesterol degradation pathway using genetic, biochemical and high‐resolution mass spectrometric approaches. The analysis of lipid profiles from cells grown on cholesterol revealed that CYP125A1 is required to incorporate the cholesterol side‐chain carbon atoms into cellular lipids, as evidenced by an increase in the mass of the methyl‐branched phthiocerol dimycocerosates. We observed that cholesterol‐exposed cells lacking CYP125A1 accumulate cholest‐4‐en‐3‐one, suggesting that this is a physiological substrate for this enzyme. Reconstitution of enzymatic activity with spinach ferredoxin and ferredoxin reductase revealed that recombinant CYP125A1 indeed binds both cholest‐4‐en‐3‐one and cholesterol, efficiently hydroxylates both of them at C‐27, and then further oxidizes 27‐hydroxycholest‐4‐en‐3‐one to cholest‐4‐en‐3‐one‐27‐oic acid. We determined the X‐ray structure of cholest‐4‐en‐3‐one‐bound CYP125A1 at a resolution of 1.58 Å. CYP125A1 is essential for growth of CDC1551 in media containing cholesterol or cholest‐4‐en‐3‐one. In its absence, the latter compound is toxic for both CDC1551 and H37Rv when added with glycerol as a second carbon source. CYP125A1 is a key enzyme in cholesterol metabolism and plays a crucial role in circumventing the deleterious effect of cholest‐4‐en‐3‐one.     

Genomic diversity among Beijing and non-Beijing Mycobacterium tuberculosis isolates from Myanmar

Background

The Beijing family of Mycobacterium tuberculosis is dominant in countries in East Asia. Genomic polymorphisms are a source of diversity within the M. tuberculosis genome and may account for the variation of virulence among M. tuberculosis isolates. Till date there are no studies that have examined the genomic composition of M. tuberculosis isolates from the high TB-burden country, Myanmar.

Methodology/Principle Findings

Twenty-two M. tuberculosis isolates from Myanmar were screened on whole-genome arrays containing genes from M. tuberculosis H37Rv, M. tuberculosis CDC1551 and M. bovis AF22197. Screening identified 198 deletions or extra regions in the clinical isolates compared to H37Rv. Twenty-two regions differentiated between Beijing and non-Beijing isolates and were verified by PCR on an additional 40 isolates. Six regions (Rv0071-0074 [RD105], Rv1572-1576c [RD149], Rv1585c-1587c [RD149], MT1798-Rv1755c [RD152], Rv1761c [RD152] and Rv0279c) were deleted in Beijing isolates, of which 4 (Rv1572-1576c, Rv1585c-1587c, MT1798-Rv1755c and Rv1761c) were variably deleted among ST42 isolates, indicating a closer relationship between the Beijing and ST42 lineages. The TbD1 region, Mb1582-Mb1583 was deleted in Beijing and ST42 isolates. One M. bovis gene of unknown function, Mb3184c was present in all isolates, except 11 of 13 ST42 isolates. The CDC1551 gene, MT1360 coding for a putative adenylate cyclase, was present in all Beijing and ST42 isolates (except 1). The pks15/1 gene, coding for a putative virulence factor, was intact in all Beijing and non-Beijing isolates, except in ST42 and ST53 isolates.

Conclusion

This study describes previously unreported deletions/extra regions in Beijing and non-Beijing M. tuberculosis isolates. The modern and highly frequent ST42 lineage showed a closer relationship to the hypervirulent Beijing lineage than to the ancient non-Beijing lineages. The pks15/1 gene was disrupted only in modern non-Beijing isolates. This is the first report of an in-depth analysis on the genomic diversity of M. tuberculosis isolates from Myanmar.     

The Role of Lipid Raft Aggregation in the Infection of Type II Pneumocytes by Mycobacterium tuberculosis

Dynamic, cholesterol-dense regions of the plasma membrane, known as lipid rafts (LR), have been observed to develop during and may be directly involved in infection of host cells by various pathogens. This study focuses on LR aggregation induced in alveolar epithelial cells during infection with Mycobacterium tuberculosis (Mtb) bacilli. We report dose- and time-dependent increases in LR aggregation after infection with three different strains at multiplicities of infection of 1, 10 and 100 from 2–24 hr post infection (hpi). Specific strain-dependent variations were noted among H37Rv, HN878 and CDC1551 with H37Rv producing the most significant increase from 15 aggregates per cell (APC) to 27 APC at MOI 100 during the 24 hour infection period. Treatment of epithelial cells with Culture Filtrate Protein, Total Lipids and gamma-irradiated whole cells from each strain failed to induce the level of LR aggregation observed during infection with any of the live strains. However, filtered supernatants from infected epithelial cells did produce comparable LR aggregation, suggesting a secreted mycobacterial product produced during infection of host cells is responsible for LR aggregation. Disruption of lipid raft formation prior to infection indicates that Mtb bacilli utilize LR aggregates for internalization and survival in epithelial cells. Treatment of host cells with the LR-disruption agent Filipin III produced a nearly 22% reduction in viable bacteria for strains H37Rv and HN878, and a 7% reduction for strain CDC1551 after 6 hpi. This study provides evidence for significant mycobacterial-induced changes in the plasma membrane of alveolar epithelial cells and that Mtb strains vary in their ability to facilitate aggregation and utilization of LR.     

Cytochrome P450 125 (CYP125) catalyses C26‐hydroxylation to initiate sterol side‐chain degradation in Rhodococcus jostii RHA1

The cyp125 gene of Rhodococcus jostii RHA1 was previously found to be highly upregulated during growth on cholesterol and the orthologue in Mycobacterium tuberculosis (rv3545c) has been implicated in pathogenesis. Here we show that cyp125 is essential for R. jostii RHA1 to grow on 3‐hydroxysterols such as cholesterol, but not on 3‐oxo sterol derivatives, and that CYP125 performs an obligate first step in cholesterol degradation. The involvement of cyp125 in sterol side‐chain degradation was confirmed by disrupting the homologous gene in Rhodococcus rhodochrous RG32, a strain that selectively degrades the cholesterol side‐chain. The RG32Ωcyp125 mutant failed to transform the side‐chain of cholesterol, but degraded that of 5‐cholestene‐26‐oic acid‐3β‐ol, a cholesterol catabolite. Spectral analysis revealed that while purified ferric CYP125_RHA1 was < 10% in the low‐spin state, cholesterol (K_D^app = 0.20 ± 0.08 μM), 5α‐cholestanol (K_D^app = 0.15 ± 0.03 μM) and 4‐cholestene‐3‐one (K_D^app = 0.20 ± 0.03 μM) further reduced the low spin character of the haem iron consistent with substrate binding. Our data indicate that CYP125 is involved in steroid C26‐carboxylic acid formation, catalysing the oxidation of C26 either to the corresponding carboxylic acid or to an intermediate state.     

Differential expression of NF-κB in mycobacteria infected THP-1 affects apoptosis

The present study was conducted to see the role of NF-κB in virulent (Mycobacterium tuberculosis H37Rv) and avirulent (M. tuberculosis H37Ra) mycobacterial infection in THP-1 cells. To inactivate NF-κB, pCMV-IκBαM dn containing THP-1 cell line was generated which showed marked increase in apoptosis with M. tuberculosis H37Rv and M. tuberculosis H37Ra. Infected THP-1-IκBαM dn cells showed decrease in mitochondrial membrane potential, cytochrome c release, activation of caspase-3 and enhanced TNF-α production. Increase in apoptosis of infected THP-1-IκBαM dn cells resulted in inhibition of intracellular mycobacterial growth. Differential NF-κB activation potential was observed with M. tuberculosis H37Rv and M. tuberculosis H37Ra. Both the strains activated NF-κB after 4 h in THP-1 cells however after 48 h only M. tuberculosis H37Rv activated NF-κB which lead to up-regulation of bcl-2 family anti-apoptotic member, bfl-1/A1. Our results indicated that NF-κB activation may be a determinant factor for the success of virulent mycobacteria within macrophages.     

Comparative analysis of bacterial genomes: identification of divergent regions in mycobacterial strains using an anchor-based approach

Comparative genomic approaches are useful in identifying molecular differences between organisms. Currently available methods fail to identify small changes in genomes, such as expansion of short repetitive motifs and to analyse divergent sequences. In this report, we describe an anchor-based whole genome comparison (ABWGC) method. ABWGC is based on random sampling of anchor sequences from one genome, followed by analysis of sampled and homologous regions from the target genome. The method was applied to compare two strains of Mycobacterium tuberculosis CDC1551 and H37Rv. ABWGC was able to identify a total of 104 indels including 20 expansion of short repetitive sequences and five recombination events. It included 18 new unidentified genomic differences. ABWGC also identified 188 SNPs including eight new ones. The method was also used to compare M. tuberculosis H37Rv and M. avium genomes. ABWGC was able to correctly pick 1002 additional indels (size>100nt) between the two organisms in contrast to MUMmer, a popular tool for comparative genomics. ABWGC was able to identify correctly repeat expansion and indels in a set of simulated sequences. The study also revealed important role of small repeat expansion in the evolution of M. tuberculosis strains.     

Mycobacterium tuberculosis CDC1551 induces a more vigorous host response in vivo and in vitro, but is not more virulent than other clinical isolates.

Mycobacterium tuberculosis CDC1551, a clinical isolate reported to be hypervirulent and to grow faster than other isolates, was compared with two other clinical isolates (HN60 and HN878) and two laboratory strains (H37Rv and Erdman). The initial (1-14 days) growth of CDC1551, HN60, HN878, and H37Rv was similar in the lungs of aerosol-infected mice, but growth of Erdman was slower. Thereafter, the growth rate of CDC1551 decreased relative to the other strains which continued to grow at comparable rates up to day 21. In the lungs of CDC1551-infected mice, small well-organized granulomas with high levels of TNF-alpha, IL-6, IL-10, IL-12, and IFN-gamma mRNA were apparent sooner than in lungs of mice infected with the other strains. CDC1551-infected mice survived significantly longer. These findings were confirmed in vitro. The growth rates of H37Rv and CDC1551 in human monocytes were the same, but higher levels of TNF-alpha, IL-10, IL-6, and IL-12 were induced in monocytes after infection with CDC1551 or by exposure of monocytes to lipid fractions from CDC1551. CD14 expression on the surface of the monocytes was up-regulated to a greater extent by exposure to the lipids of CDC1551. Thus, CDC1551 is not more virulent than other M. tuberculosis isolates in terms of growth in vivo and in vitro, but it induces a more rapid and robust host response.     

Peptides derived from Mycobacterium tuberculosis Rv2301 protein are involved in invasion to human epithelial cells and macrophages

The specific function of putative cut2 protein (or CFP25), encoded by the Rv2301 gene from Mycobacterium tuberculosis H37Rv, has not been identified yet. The aim of this study was to assess some of CFP25 characteristics and its possible biological role in Mycobacterium tuberculosis H37Rv invasion process to target cells. Molecular assays indicated that the gene encoding Rv2301 is present and transcribed in M. tuberculosis complex strains. The presence of Rv2301 protein over the bacilli surface was confirmed by Western blot and immunoelectron microscopy analyses, using goats sera inoculated with synthetic peptides derived from Rv2301 protein. Receptor–ligand binding assays with carcinomic human alveolar basal epithelial cells (A549) and macrophages derived from human histolytic lymphoma monocytes (U937) allowed us to identify five high activity binding peptides (HABPs) in both cell lines, and two additional HABPs only in A549 cells. U937 HABPs binding interactions were characterized by saturation assays, finding dissociation constants (K _d) within the nanomolar range and positive cooperativity (n _H?>?1). Inhibition assays were performed to assess the possible biological role of Rv2301 identified HABPs, finding that some of them were able to inhibit invasion at a 5?μM concentration, compared with the cytochalasin control. On the other hand, HABPs, and especially HABP 36507 located at the N-terminus of the protein, facilitated the internalization of fluorescent latex beads into A549 cells. These findings are of vital importance for the rational selection of Rv2301 HABPs, to be included as components of an antituberculosis vaccine.     

Functional characterization of Mycobacterium tuberculosis Rv2969c membrane protein

Identifying Mycobacterium tuberculosis membrane proteins involved in binding to and invasion of host cells is important in designing subunit-based anti-tuberculosis vaccines. The Rv2969c gene sequence was identified by PCR in M. tuberculosis complex strains, being transcribed in M. tuberculosis H37Rv, M. tuberculosis H37Ra, and M. bovis BCG. Rabbits immunized with synthetic peptides from highly specific conserved regions of this protein produced antibodies recognizing 27 and 29 kDa bands in M. tuberculosis lysate, which is consistent with the molecular weight of the Rv2969c gene product in M. tuberculosis H37Rv. Immunoelectron microscopy revealed the protein was localized on the bacillus surface. Four and three specific high activity binding peptides (HABPs) to the A549 alveolar epithelial and U937 monocyte cell lines were found, respectively. Two of the HABPs found inhibited M. tuberculosis invasion of A549 cells, suggesting that these peptides might be good candidates to be included in a multiepitopic, subunit-based anti-tuberculosis vaccine.     

Cholesterol Ester Oxidation by Mycobacterial Cytochrome P450

Mycobacteria share a common cholesterol degradation pathway initiated by oxidation of the alkyl side chain by enzymes of cytochrome P450 (CYP) families 125 and 142. Structural and sequence comparisons of the two enzyme families revealed two insertions into the N-terminal region of the CYP125 family (residues 58–67 and 100–109 in the CYP125A1 sequence) that could potentially sterically block the oxidation of the longer cholesterol ester molecules. Catalytic assays revealed that only CYP142 enzymes are able to oxidize cholesteryl propionate, and although CYP125 enzymes could oxidize cholesteryl sulfate, they were much less efficient at doing so than the CYP142 enzymes. The crystal structure of CYP142A2 in complex with cholesteryl sulfate revealed a substrate tightly fit into a smaller active site than was previously observed for the complex of CYP125A1 with 4-cholesten-3-one. We propose that the larger CYP125 active site allows for multiple binding modes of cholesteryl sulfate, the majority of which trigger the P450 catalytic cycle, but in an uncoupled mode rather than one that oxidizes the sterol. In contrast, the more unhindered and compact CYP142 structure enables enzymes of this family to readily oxidize cholesteryl esters, thus providing an additional source of carbon for mycobacterial growth.     

Reverse type I inhibitor of Mycobacteriumtuberculosis CYP125A1

Cytochrome P450 CYP125A1 of Mycobacteriumtuberculosis, a potential therapeutic target for tuberculosis in humans, initiates degradation of the aliphatic chain of host cholesterol and is essential for establishing M. tuberculosis infection in a mouse model of disease. We explored the interactions of CYP125A1 with a reverse type I inhibitor by X-ray structure analysis and UV-vis spectroscopy. Compound LP10 (α-[(4-methylcyclohexyl)carbonyl amino]-N-4-pyridinyl-1H-indole-3-propanamide), previously identified as a potent type II inhibitor of Trypanosomacruzi CYP51, shifts CYP125A1 to a water-coordinated low-spin state upon binding with low micromolar affinity. When LP10 is present in the active site, the crystal structure and spectral characteristics both demonstrate changes in lipophilic and electronic properties favoring coordination of the iron axial water ligand. These results provide an insight into the structural requirements for developing selective CYP125A1 inhibitors.     

Spectrum of heart diseases in a new cardiac service in Nigeria: An echocardiographic study of 1441 subjects in Abeokuta

Background

The recent determination of the complete nucleotide sequence of several Mycobacterium tuberculosis (MTB) genomes allows the use of comparative genomics as a tool for dissecting the nature and consequence of genetic variability within this species. The multiple alignment of the genomes of clinical strains (CDC1551, F11, Haarlem and C), along with the genomes of laboratory strains (H37Rv and H37Ra), provides new insights on the mechanisms of adaptation of this bacterium to the human host.

Findings

The genetic variation found in six M. tuberculosis strains does not involve significant genomic rearrangements. Most of the variation results from deletion and transposition events preferentially associated with insertion sequences and genes of the PE/PPE family but not with genes implicated in virulence. Using a Perl-based software islandsanalyser, which creates a representation of the genetic variation in the genome, we identified differences in the patterns of distribution and frequency of the polymorphisms across the genome. The identification of genes displaying strain-specific polymorphisms and the extrapolation of the number of strain-specific polymorphisms to an unlimited number of genomes indicates that the different strains contain a limited number of unique polymorphisms.

Conclusion

The comparison of multiple genomes demonstrates that the M. tuberculosis genome is currently undergoing an active process of gene decay, analogous to the adaptation process of obligate bacterial symbionts. This observation opens new perspectives into the evolution and the understanding of the pathogenesis of this bacterium.     

MycoProtease-DB: Useful resource for Mycobacterium tuberculosis complex and nontuberculous mycobacterial proteases

MycoProtease-DB is an online MS SQL and CGI-PERL driven relational database that domiciles protease information of Mycobacterium tuberculosis (MTB) complex and Nontuberculous Mycobacteria (NTM), whose complete genome sequence is available. Our effort is to provide comprehensive information on proteases of 5 strains of Mycobacterium tuberculosis (H₃₇Rv, H₃₇Ra, CDC1551, F11 and KZN 1435), 3 strains of Mycobacterium bovis (AF2122/97, BCG Pasteur 1173P2 and BCG Tokyo 172) and 4 strains of NTM (Mycobacterium avium 104, Mycobacterium smegmatis MC2 155, Mycobacterium avium paratuberculosis K-10 and Nocardia farcinica IFM 10152) at gene, protein and structural level. MycoProtease-DB currently hosts 1324 proteases, which include 906 proteases from MTB complex with 237distinct proteases & 418 from NTM with 404 distinct proteases. Flexible database design and easy expandability & retrieval of information are the main features of MycoProtease-DB. All the data were validated with various online resources and published literatures for reliable serving as comprehensive resources of various Mycobacterial proteases.

Availability

The Database is publicly available at http://www.bicjbtdrc-mgims.in/MycoProtease-DB/     

The synthesis and antituberculosis activity of 5′-nor carbocyclic uracil derivatives

A series of new carbocyclic uracil derivatives were synthesized and evaluated as potential antituberculosis agents. Racemic 1-[4′-hydroxy-2′-cyclopenten-1′-yl]-5-tetradecynyluracil completely inhibited the growth of Mycobacterium tuberculosis H37Rv in vitro at a concentration of 10 μg/ml. Individual (+) and (?) isomers of the above uracil derivative were isolated and showed the same level of activity against two strains of Mycobacterium tuberculosis: laboratory sensitive (H37Rv) and clinical resistant to five top antituberculosis drugs (MS-115).     

Stereoselective Formation and Metabolism of 4-Hydroxy-Retinoic Acid Enantiomers by Cytochrome P450 Enzymes

All-trans-retinoic acid (atRA), the major active metabolite of vitamin A, plays a role in many biological processes, including maintenance of epithelia, immunity, and fertility and regulation of apoptosis and cell differentiation. atRA is metabolized mainly by CYP26A1, but other P450 enzymes such as CYP2C8 and CYP3As also contribute to atRA 4-hydroxylation. Although the primary metabolite of atRA, 4-OH-RA, possesses a chiral center, the stereochemical course of atRA 4-hydroxylation has not been studied previously. (4S)- and (4R)-OH-RA enantiomers were synthesized and separated by chiral column HPLC. CYP26A1 was found to form predominantly (4S)-OH-RA. This stereoselectivity was rationalized via docking of atRA in the active site of a CYP26A1 homology model. The docked structure showed a well defined niche for atRA within the active site and a specific orientation of the β-ionone ring above the plane of the heme consistent with stereoselective abstraction of the hydrogen atom from the pro-(S)-position. In contrast to CYP26A1, CYP3A4 formed the 4-OH-RA enantiomers in a 1:1 ratio and CYP3A5 preferentially formed (4R)-OH-RA. Interestingly, CYP3A7 and CYP2C8 preferentially formed (4S)-OH-RA from atRA. Both (4S)- and (4R)-OH-RA were substrates of CYP26A1 but (4S)-OH-RA was cleared 3-fold faster than (4R)-OH-RA. In addition, 4-oxo-RA was formed from (4R)-OH-RA but not from (4S)-OH-RA by CYP26A1. Overall, these findings show that (4S)-OH-RA is preferred over (4R)-OH-RA by the enzymes regulating atRA homeostasis. The stereoselectivity observed in CYP26A1 function will aid in better understanding of the active site features of the enzyme and the disposition of biologically active retinoids.