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.     

Oxidation of high density lipoproteins: characterization and effects on cholesterol efflux from J774 macrophages

Oxidative modification of high density lipoproteins (HDL) may alter their capacity to mediate cellular cholesterol efflux. We studied the kinetics of copper-mediated oxidation of HDL and cholesterol efflux mediated by unmodified and oxidized HDL (oxHDL). Oxidation was measured by increases in absorbance at 234 nm (ΔA234), production of thiobarbituric acid reactive substances (TBARS) and loss of trinitrobenzene sulfonic acid reactivity. Oxidation was dependent on copper concentration and showed a lag phase and propagation phase. Efflux of cholesterol from J774 macrophages measured by appearance of cellular [³H]cholesterol in the medium was lower by 16% after 4 h and 36% after 24 h with oxHDL compared to HDL. OxHDL-mediated efflux was also lower by 27% to 36% at lipoprotein concentrations of 10 to 200 μg protein/ml. Cholesterol efflux correlated negatively with TBARS production (r= −0.97, P < 0.003) and ΔA234 (r = −0.77, P < 0.080). There was no difference in efflux mediated by apoproteins prepared from HDL and oxHDL. Efflux measured by change in cholesterol mass in medium was 78% lower with oxHDL. Inhibition of oxidation with butylated hydroxytoluene maintained the capacity of HDL to stimulate efflux. These results suggest that oxidation of HDL may impair its protective role against atherosclerosis.     

Differences in the sterol composition of Heliothis zea fed Zea mays versus Medicago sativa

Larvae of Heliothis zea were fed Zea mays or Medicago sativa in order to determine how these plants affected the development and sterol composition of the insect. The larvae fed corn kernels grew more rapidly than those fed alfalfa leaves. The corn kernels contained Δ⁵-24-alkylsterols whereas the leaves as well as the sprouts and flowers of alfalfa contained Δ⁷-24-alkylsterols. The corn-fed larvae dealkylated the dietary Δ⁵-sterols and utilized primarily cholesterol in their tissues. In contrast, although the larvae fed alfalfa dealkylated the Δ⁷-sterols present in the leaves, they were unable to metabolize the B-ring of these sterols and so both the prepupae and adults utilized large quantities of lathosterol in their tissues. Interestingly, H. zea, a generalized feeder, shared its inability to metabolize Δ⁷-sterols to Δ⁵-sterols with Hypera postica, a specialized feeder on alfalfa. This coleopteran also dealkylated the Δ⁷-24-alkylsterols and contained lathosterol, but not cholesterol, in its tissues. Therefore, the ability of H. zea to utilize either Δ⁵- or Δ⁷-sterols in its tissues may help to explain its ability to complete its development on a variety of different plants, although it may prefer a host, such as corn, that produces Δ⁵-sterols and supports a more-rapid rate of growth.     

Biodistribution and tumor uptake of [67Ga]chlorogallium-tetraoctadecyloxy phthalocyanine and its sulfonation products in tumor bearing C3H mice

To evaluate the biodistribution and tumor uptake of chlorogallium tetraoctadecyloxy phthalocyanine, a potential new drug for the photodynamic therapy of cancer, we prepared the radioactive ⁶⁷Ga-labeled complex and its water soluble sulfonated derivative. The non-sulfonated dye was obtained by condensation of octadecyloxyphthalonitrile in the presence of a mixture of ⁶⁷Ga and ⁶⁹Ga chloride. The sulfonated derivative was obtained by treatment of the condensation product with oleum. As singlet molecular oxygen has been implicated in the photodynamic action of phthalocyanines (Pcs), the quantum yield of singlet oxygen (φ_Δ) was evaluated for chlorogallium tetraoctadecyloxy Pc, and also its zinc, aluminum and metal free analogues. After intraperitoneal administration of the non-sulfonated dye into RIF tumor bearing C3H mice, a very high ⁶⁷Ga-uptake was observed in the spleen, while tumor radioactivity remained low during the 3 day study. The in vivo stability of the ⁶⁷Ga-phthalocyanine complex was confirmed by comparing the distribution pattern with that of ⁶⁷Ga-citrate, which proved to be significantly different. Intravenous injection of the sulfonated dye resulted in an overall lowering of ⁶⁷Ga-uptake by most tissues, particularly in the spleen, while tumor radioactivities were slightly higher. These data suggest that amphiphilic photosensitizers, containing both polar sulfonate groups and long aliphatic substituents, exhibit the best distribution pattern for both imaging and photodynamic therapy of tumors.     

Cover Image,Volume 88, Issue 6

Hsp16.3, a molecular chaperone, plays a vital role in the growth and survival of Mycobacterium tuberculosis inside the host. We previously reported that deletion of three amino acid residues (¹⁴²STN¹⁴⁴) from C-terminal extension (CTE) of Hsp16.3 triggers its structural perturbation and increases its chaperone activity, which reaches its apex upon the deletion of its entire CTE (¹⁴¹RSTN¹⁴⁴). Thus, we hypothesized that Arg141 (R141) and Ser142 (S142) in the CTE of Hsp16.3 possibly hold the key in maintaining its native-like structure and chaperone activity. To test this hypothesis, we generated two deletion mutants in which R141 and S142 were deleted individually (Hsp16.3ΔR141 and Hsp16.3ΔS142) and three substitution mutants in which R141 was replaced by lysine (Hsp16.3R141K), alanine (Hsp16.3R141A), and glutamic acid (Hsp16.3R141E), respectively. Hsp16.3ΔS142 or Hsp16.3R141K mutant has native-like structure and chaperone activity. Deletion of R141 from the CTE (Hsp16.3ΔR141) perturbs the secondary and tertiary structure, lowers the subunit exchange dynamics and decreases the chaperone activity of Hsp16.3. But, the substitution of R141 with alanine (Hsp16.3R141A) or glutamic acid (Hsp16.3R141E) perturbs its secondary and tertiary structure. Surprisingly, such charge tampering of R141 enhances the subunit exchange dynamics and chaperone activity of Hsp16.3. Interestingly, neither the deletion of R141/S142 nor the substitution of R141 with lysine, alanine and glutamic acid affects the oligomeric mass/size of Hsp16.3. Overall, our study suggests that R141 (especially the positive charge on R141) plays a crucial role in maintaining the native-like structure as well as in regulating subunit exchange dynamics and chaperone activity of Hsp16.3.     

Effect of triarimol on cholesterol biosynthesis in rat-liver subcellular fractions

Cholesterol biosynthesis was studied in rat-liver subcellular fractions incubated with DL-[2-¹⁴C]mevalonic acid in the presence and absence of triarimol (α-(2,4-dichlorophenyl)-α-phenyl-5-pyrimidine methanol). Triarimol strongly inhibits incorporation of radioactivity into cholesterol and this results in a large accumulation of radioactive lanosterol and 24,25-dihydro-lanosterol. The inhibition of lanosterol 14α-demethylase by triarimol was confirmed by assay of the enzyme in rat-liver microsomal fraction in the presence and absence of the inhibitor. Apart from a slight inhibition of Δ⁷-sterol-Δ⁵-dehydrogenase, triarimol did not affect the activity of any other enzyme involved in cholesterol biosynthesis from acetate.     

The effects of the structure of sterols on the development of Heliothis zea

Heliothis zea was reared on artificial diets which lacked supplementation with plant materials but were supplemented with different sterols in order to determine how certain structural features of a sterol molecule affect the development of this insect. We found that sitosterol and cholesterol supported a more rapid rate of growth than did campesterol. Larvae did not moult when they ingested 5-pregnen-3β-ol. Larvae utilized spinasterol more efficiently than lathosterol. Such a pronounced effect was not observed in the Δ⁵-series. The substitution of a Δ⁷-bond (spinasterol, dihydrospinasterol, lathosterol) for the Δ⁵-bond (stigmasterol, sitosterol, cholesterol) in the 24-ethyl- and desalkylsterols reduced the rate of growth of the larvae. Although larvae developed normally on cholesterol, the addition of a Δ⁷-bond to give the Δ^5,7-diene system apparently altered the functionality of the molecule because 7-dehydrocholesterol did not support larval development. The growth of larvae was also inhibited, although not prevented, when they consumed diets which contained ergosterol. In contrast, the larvae completed their development more rapidly on brassicasterol which lacked the Δ⁷-bond. Cholestanol supported the complete development of the insect. H. zea is unusual among investigated insects because it develops both on cholestanol and lathosterol but does not utilize ergosterol efficiently and fails to grow on 7-dehydrocholesterol.     

The addition of hypobromous acid to 3β,17β-diacetoxy-4-estrene and an equilibration study involving neighboring group participation by the a-ring acetoxy group

The reaction of 3β,17β-diacetoxy-4-estrene with N-bromoacetamide in a two phase ether/water solvent mixture gave 5-bromo-4β,17β-diacetoxy-5α-estran-3β-ol as the major product (75%). Four minor products were also isolated and identified. These were: 4α-bromo-3β, 17β-diacetoxy-5α-estran-5-ol (5%), 5-bromo-3g,17β-diacetoxy-5α-estran-4β-ol (6%), 5-bromo-4α,17β-diacetoxy-5αt-estran-3β-ol (3%), and 4β-bromo-3β,17β-diacetoxy-5α-estran-5-ol (4%). The 5-bromo-4β, 17β-diacetoxy-5α-estran-3β-ol was equilibrated by heating with oxalic acid in refluxing benzene for $\text{ca}$. 16h to give a mixture of it and 5-broino-3β,17β-diacetoxy-5α-estran-4β-ol in the ratio of 16:84 respectively. A similar equilibration mixture (14:86) was obtained under identical conditions when 5-bromo-3β,17β-diacetoxy-5α-estran-4β-ol was the starting material.     

Determination of methylputrescine oxidase by high performance liquid chromatography

N-Methyl-Δ¹-pyrrolinium chloride, the product of the title enzyme, was synthesized by methylation of aminobutyraldehyde diethylacetal followed by acidic cleavage. After purification to homogeneity, it was characterized by NMR and UV spectroscopy. The compound had an absorption maximum at 210 nm; previous data indicating a maximum at 267 nm were shown to arise from an impurity. An HPLC method for the assay of N-methylputrescine oxidase from plant material was developed based on the separation of N-methyl-Δ¹-pyrrolinium chloride on a cation exchange column and direct detection at 210 nm. The enzyme activity was measured in the protein fraction extracted from plant roots and treated by gel filtration on disposable PD 10 columns. A K_m value of 1.9 mM was determined for methylputrescine and the enzyme from tobacco roots. The enzyme activities from N. tabacum and Datura stramonium were compared.     

LDL receptor related protein 1 requires the I3 domain of discs-large homolog 1/DLG1 for interaction with the kinesin motor protein KIF13B

KIF13B, a kinesin-3 family motor, was originally identified as GAKIN due to its biochemical interaction with human homolog of Drosophila discs-large tumor suppressor (hDLG1). Unlike its homolog KIF13A, KIF13B contains a carboxyl-terminal CAP-Gly domain. To investigate the function of the CAP-Gly domain, we developed a mouse model that expresses a truncated form of KIF13B protein lacking its CAP-Gly domain (KIF13BΔCG), whereas a second mouse model lacks the full-length KIF13A. Here we show that the KIF13BΔCG mice exhibit relatively higher serum cholesterol consistent with the reduced uptake of [³H]CO-LDL in KIF13BΔCG mouse embryo fibroblasts. The plasma level of factor VIII was not significantly elevated in the KIF13BΔCG mice, suggesting that the CAP-Gly domain region of KIF13B selectively regulates LRP1-mediated lipoprotein endocytosis. No elevation of either serum cholesterol or plasma factor VIII was observed in the full length KIF13A null mouse model. The deletion of the CAP-Gly domain region caused subcellular mislocalization of truncated KIF13B concomitant with the mislocalization of LRP1. Mechanistically, the cytoplasmic domain of LRP1 interacts specifically with the alternatively spliced I₃ domain of DLG1, which complexes with KIF13B via their GUK-MBS domains, respectively. Importantly, double mutant mice generated by crossing KIF13A null and KIF13BΔCG mice suffer from perinatal lethality showing potential craniofacial defects. Together, this study provides first evidence that the carboxyl-terminal region of KIF13B containing the CAP-Gly domain is important for the LRP1-DLG1-KIF13B complex formation with implications in the regulation of metabolism, cell polarity, and development.     

INTRACRANIAL CONVERSION OF LINOLEIC ACID TO ARACHIDONIC ACID: EVIDENCE FOR LACK OF Δ8 DESATURASE IN THE BRAIN

Eleven-day old rats were given intracranial injection of [1-¹⁴C]linoleic acid (all cis 9,12 octadecadienoic acid) and sacrificed after 8 h. Analysis of brain fatty acids showed that 16:0, 18:2, 20:2,20:3 and 20:4 were labeled. Separation by AgN03:Si02 TLC plates followed by reductive ozonolysis characterized thc polyunsaturated fatty acids as 18:2 (Δ9,12), 20:2 (Δ11,14), 20:3 (Δ8,11,14) and 20:4 (Δ5,8,11,14). A smaller amount of 18:3 (Δ6,9,12) was also identified. This initially suggested 20:2 (A1 1,14) as an intermediate in the optional pathway of biosynthesis of arachidonate. However, when [l-14C]eicosadienoic acid (Δ1 1,141 itself was injected in the brain it was converted to 20:3 (Δ5,11,14) (a non-methylene interrupted double bond system) rather than the expected 20:3 (Δ8,11,14). Only a small amount of arachidonate was formed from 20:2 (Δ11,14). Thus it was concluded that 20:2 (Δ11,14) was not an intermediate in the pathways of arachidonate biosynthesis due to lack of Δ⁵ desaturase in thc brain which agrees with the findings of SPKECRER & LEE (1975) in rat liver.     

Effects of transient anaerobic stress on fatty acid desaturation and electron-transport system in Tetrahymena microsomes

A transient anaerobic stress of Tetrahymena pyriformis NT-1 for 1 h resulted in slight decreases of unsaturated fatty acids in overall lipid composition. The rates of incorporation of [³H]palmitic acid plus sodium [¹⁴C]acetate into unsaturated fatty acids (palmitoleate, 16:1Δ⁹; oleate, 18:1Δ⁹; linoleate, 18:2Δ^9,12; γ-linolenate, 18:3Δ^6,9,12) were drastically reduced by exposing cells to anaerobic conditions for 1 h (anaerobic cells), and thereafter restored to the original levels at 4 h after subsequent reaeration. Activities of Δ⁹- and Δ¹²-desaturases (palmitoyl-CoA, stearoyl-CoA, oleoyl-CoA) and the terminal component of the desaturation system (cyanide-sensitive factor) decreased to 25–30% in microsomes from anaerobic cells, compared to those from control and reaerobic cells. Furthermore, changes induced by the anaerobic shock in the activities of microsomal reductases and the content of cytochrome b₅ were observed to behave comparably to changes in activities of fatty acyl-CoA desaturases. During the anaerobic state, the half-lives of microsomal enzyme activities (desaturases, cyanide-sensitive factor, reductases) and cytochrome b₅ content were diminished to 30–50% of those of control microsomes. But the K_m values of these enzymes did not differ between microsomes from either anaerobic or control cells. These results suggest that the decreased activities of desaturases during anaerobic stress result principally from the reduction of cyanide-sensitive factor content, which would be caused by its instability and repressed synthesis.     

SILICON METABOLISM IN DIATOMS. VI. SILICIC ACID UPTAKE BY A COLORLESS MARINE DIATOM,NITZSCHIA ALBA LEWIN AND LEWIN12

Cells of the colorless, heterotrophic diatom Nitzschia alba, after removal from the culture medium, were able to absorb silicic acid from a salt solution lacking carbon, nitrogen, and phosphorus. Silicic acid uptake continued for approximately 12 hr. At low cell densities (ca. 2.5×10⁵ cells/ml), the cell number doubled under these conditions. At high cell densities (ca. 2 ×10⁶ cells/ml) no cell division resulted. When such cells were washed with a salt solution, their ability to absorb silicic acid was somewhat impaired. The degree of impairment became progressively more pronounced after subsequent washing treatments. A heat-stable factor washed from the cells and present in the first “wash water” was able to restore completely the ability of washed cells to absorb silicic acid. The factor was not identified. Aspartic acid (5 ± 10^-4 M) or glutamine (5 ±10^-4 M) when added to the saline solution similarly promoted complete recovery. A t such concentrations, these substances had only a slight effect on unwashed cells. A solution of 5 ±10^-4 M Na glutamate (or aspartic acid plus glutamine) had an men more pronounced effect, and in addition promoted cell division or growth" of unwashed cells. Several other amino acids and other compounds tested were apparently without effect.     

Methoxylation of methyl 3α,7α-dihydroxychol-4-en-24-oate and its 3β-epimer. —a contribution to chenodeoxycholic acid biogenesis

By the conventional methods of gas liquid chromatography (GLC) as well as mass spectrometry, 3β,7α-dihydroxychol-5-en-24-oic acid (Δ⁵-acid), a key intermediate of chenodeoxycholic acid biogenesis and its metabolic by-product, 3α,7α-dihydroxychol-4-en-24-oic acid (Δ⁴-acid) have not yet been identified as such probably due to thermal decomposition. However, taking advantage of the observation that they are readily methoxylated in methanoi containing a trace of acids, their individual methoxy-compounds were easily prepared and proved to be useful for their identification, even though they are present in minimal amounts as was the case with the human or hen bile. The present paper reported physical as well as spectral properties of the methoxy-compounds derived from methyl 3α,7α-dihydroxychol-4-en-24-oate, compared with those of its 3β-epimer     

Mycobacterium smegmatis is a suitable cell factory for the production of steroidic synthons

A number of pharmaceutical steroid synthons are currently produced through the microbial side‐chain cleavage of natural sterols as an alternative to multi‐step chemical synthesis. Industrially, these synthons have been usually produced through fermentative processes using environmental isolated microorganisms or their conventional mutants. Mycobacterium smegmatis mc²155 is a model organism for tuberculosis studies which uses cholesterol as the sole carbon and energy source for growth, as other mycobacterial strains. Nevertheless, this property has not been exploited for the industrial production of steroidic synthons. Taking advantage of our knowledge on the cholesterol degradation pathway of M. smegmatis mc²155 we have demonstrated that the MSMEG_6039 (kshB1) and MSMEG_5941 (kstD1) genes encoding a reductase component of the 3‐ketosteroid 9α‐hydroxylase (KshAB) and a ketosteroid Δ¹‐dehydrogenase (KstD), respectively, are indispensable enzymes for the central metabolism of cholesterol. Therefore, we have constructed a MSMEG_6039 (kshB1) gene deletion mutant of M. smegmatis MS6039 that transforms efficiently natural sterols (e.g. cholesterol and phytosterols) into 1,4‐androstadiene‐3,17‐dione. In addition, we have demonstrated that a double deletion mutant M. smegmatis MS6039‐5941 [ΔMSMEG_6039 (ΔkshB1) and ΔMSMEG_5941 (ΔkstD1)] transforms natural sterols into 4‐androstene‐3,17‐dione with high yields. These findings suggest that the catabolism of cholesterol in M. smegmatis mc²155 is easy to handle and equally efficient for sterol transformation than other industrial strains, paving the way for valuating this strain as a suitable industrial cell factory to develop à la carte metabolic engineering strategies for the industrial production of pharmaceutical steroids.     

Characterization of glucuronic acid containing glycolipid in Aspergillus fumigatus mycelium

A glucuronic acid containing glycerolipid was isolated from the filamentous fungi Aspergillus fumigatus. This acidic glycolipid was extracted from the membrane of mycelium and purified by two successive chromatographic steps on DEAE-Sephadex and Silica columns. Chemical structural analysis was performed using methylation, gas-chromatography, gas-chromatography-mass spectrometry, nano-electrospray mass spectrometry and ¹H/¹³C NMR spectra. The corresponding structure is a 3-(O-α-glucuronyl)-1,2-diacyl-sn-glycerol, where acyl chains are mainly C_16:0, C_18:0, C_18:1, and C_18:2. This α-GlcA-diacylglycerol is not present in fungal conidia. This acidic glycerolipid is described here for the first time in a fungal species. Two homologs of UDP-glucose dehydrogenase that convert UDP-glucose into UDP-glucuronic acid, are present in A. fumigatus genome, UGD1 and UGD2. Gene deletion showed that only UGD1 is essential for the biosynthesis of GlcA-DG. However, no particular phenotype has been observed in the Ugd1Δ mutant. Biological function of this acidic glycolipid remains unknown in A. fumigatus.