Processive kinetics in the three-step lanosterol 14α-demethylation reaction catalyzed by human cytochrome P450 51A1.

Cytochrome P450 (P450, CYP) family 51 enzymes catalyze the 14α-demethylation of sterols, leading to critical products used for membranes and the production of steroids, as well as signaling molecules. In mammals, P450 51 catalyzes the 3-step, 6-electron oxidation of lanosterol to form (4ß,5α)-4,4-dimethyl-cholestra-8,14,24-trien-3-ol (FF-MAS). P450 51A1 can also use 24,25-dihydrolanosterol (a natural substrate in the Kandutsch-Russell cholesterol pathway). 24,25-Dihydrolanosterol and the corresponding P450 51A1 reaction intermediates, the 14α-alcohol and -aldehyde derivatives of dihydrolanosterol, were synthesized to study the kinetic processivity of the overall 14α-demethylation reaction of human P450 51A1. A combination of steady-state kinetic parameters, steady-state binding constants, dissociation rates of P450-sterol complexes, and kinetic modeling of the time course of oxidation of a P450-dihydrolanosterol complex showed that the overall reaction is highly processive, with koff rates of P450 51A1-dihydrolanosterol and the 14α-alcohol and 14α-aldehyde complexes being 1 to 2 orders of magnitude less than the forward rates of competing oxidations. epi-Dihydrolanosterol (the 3α-hydroxy analog) was as efficient as the common 3ß-hydroxy isomer in the binding and formation of dihydro FF-MAS. The common lanosterol contaminant dihydroagnosterol was found to be a substrate of human P450 51A1, with roughly one-half the activity of dihydrolanosterol. Steady-state experiments with 14α-methyl deuterated dihydrolanosterol showed no kinetic isotope effect, indicating that C-14α C-H bond breaking is not rate-limiting in any of the individual steps. The high processivity of this reaction generates higher efficiency and also renders the reaction less sensitive to inhibitors.

Induced expression of Ganoderma boninense Lanosterol 14α-Demethylase (ERG11) during interaction with oil palm.

BACKGROUND: The basidiomycete fungus, Ganoderma boninense is the main contributor to oil palm Basal Stem Rot (BSR) in Malaysia and Indonesia. Lanosterol 14α-Demethylase (ERG11) is a key enzyme involved in biosynthesis of ergosterol, which is an important component in the fungal cell membrane. The Azole group fungicides are effective against pathogenic fungi including G. boninense by inhibiting the ERG11 activity. However, the work on molecular characterization of G. boninense ERG11 is still unavailable today. METHODS AND RESULTS: This study aimed to isolate and characterize the full-length cDNA encoding ERG11 from G. boninense. The G. boninense ERG11 gene expression during interaction with oil palm was also studied. A full-length 1860 bp cDNA encoding ERG11 was successfully isolated from G. boninense. The G. boninense ERG11 shared 91% similarity to ERG11 from other basidiomycete fungi. The protein structure homology modeling of GbERG11 was analyzed using the SWISS-MODEL workspace. Southern blot and genome data analyses showed that there is only a single copy of ERG11 gene in the G. boninense genome. Based on the in-vitro inoculation study, the ERG11 gene expression in G. boninense has shown almost 2-fold upregulation with the presence of oil palm. CONCLUSION: This study provided molecular information and characterization study on the G. boninense ERG11 and this knowledge could be used to design effective control measures to tackle the BSR disease of oil palm.

Metabolic and pathologic profiles of human LSS deficiency recapitulated in mice.

Skin lesions, cataracts, and congenital anomalies have been frequently associated with inherited deficiencies in enzymes that synthesize cholesterol. Lanosterol synthase (LSS) converts (S)-2,3-epoxysqualene to lanosterol in the cholesterol biosynthesis pathway. Biallelic mutations in LSS have been reported in families with congenital cataracts and, very recently, have been reported in cases of hypotrichosis. However, it remains to be clarified whether these phenotypes are caused by LSS enzymatic deficiencies in each tissue, and disruption of LSS enzymatic activity in vivo has not yet been validated. We identified two patients with novel biallelic LSS mutations who exhibited congenital hypotrichosis and midline anomalies but did not have cataracts. We showed that the blockade of the LSS enzyme reaction occurred in the patients by measuring the (S)-2,3-epoxysqualene/lanosterol ratio in the forehead sebum, which would be a good biomarker for the diagnosis of LSS deficiency. Epidermis-specific Lss knockout mice showed neonatal lethality due to dehydration, indicating that LSS could be involved in skin barrier integrity. Tamoxifen-induced knockout of Lss in the epidermis caused hypotrichosis in adult mice. Lens-specific Lss knockout mice had cataracts. These results confirmed that LSS deficiency causes hypotrichosis and cataracts due to loss-of-function mutations in LSS in each tissue. These mouse models will lead to the elucidation of the pathophysiological mechanisms associated with disrupted LSS and to the development of therapeutic treatments for LSS deficiency.

Synthesis of obtusifoliol and analogues as CYP51 substrates.

Sterol 14α-demethylases (CYP51s) are a ubiquitous superfamily of cytochrome P450 enzymes that play an essential role in sterol biosynthesis. As fungal CYP51s are the target of azole-based antifungal agents, which are facing the problem of increasing resistance, the substrate specificity of this enzyme subclass has recently garnered significant attention. Herein we report the first chemical synthesis of the final endogenous substrate of this enzyme class, obtusifoliol, in 1.3% yield across ten steps from a commercially available lanosterol mixture. Intermediates along this pathway provide a basis for further derivatisation of the sterol skeleton and future investigation into CYP51 inhibition to overcome pathogens' azole resistance.

Mechanism of enhanced production of triterpenoids in algal-fungal consortium.

Chlorella pyrenoidosa-Ganoderma lucidum symbiotic systems were constructed. The mechanism of enhanced production of triterpenoids in algal-fungal consortium by comparing the contents of triterpenoids in individual fungal systems and algal-fungal consortium systems was investigated. The production of triterpenoids in C. pyrenoidosa-G. lucidum consortium increased significantly (P < 0.05). The categories and relative abundances of metabolites in the individual systems and algal-fungal systems were measured and analyzed by metabonomic tests. There were 57 significant different metabolites (VIP > 1 and P < 0.05) including 12 downregulated metabolites and 45 upregulated metabolites were obtained. The significant enriched metabolic pathways (VIP > 1 and P < 0.05) of citrate cycle (TCA cycle), tyrosine metabolism, glycolysis, and terpenoid backbone biosynthesis in algal-fungal consortium were obtained. The relative abundances of important precursors of triterpenoids including mevalonic acid, lanosterol, and hydroquinone were 1.4 times, 1.7 times, and 2 times, respectively, in algal-fungal consortium than that in the individual fungal systems. The presence of C. pyrenoidosa in algal-fungal consortium promoted the biosynthesis of triterpenoids in G. lucidum.

Serum 4ß-hydroxycholesterol increases during fluconazole treatment.

PURPOSE: The antifungal drugs ketoconazole and itraconazole reduce serum concentrations of 4ß-hydroxycholesterol, which is a validated marker for hepatic cytochrome P450 (CYP) 3A4 activity. We tested the effect of another antifungal triazole agent, fluconazole, on serum concentrations of different sterols and oxysterols within the cholesterol metabolism to see if this inhibitory reaction is a general side effect of azole antifungal agents. METHODS: In a prospective, double-blind, placebo-controlled, two-way crossover design, we studied 17 healthy subjects (nine men, eight women) who received 400 mg fluconazole or placebo daily for 8 days. On day 1 before treatment and on day 8 after the last dose, fasting blood samples were collected. Serum cholesterol precursors and oxysterols were measured by gas chromatography-mass spectrometry-selected ion monitoring and expressed as the ratio to cholesterol (R_sterol). RESULTS: Under fluconazole treatment, serum R_lanosterol and R_24,25-dihydrolanosterol increased significantly without affecting serum cholesterol or metabolic downstream markers of hepatic cholesterol synthesis. Serum R_4ß-, R_24S-, and R_27-hydroxycholesterol increased significantly. CONCLUSION: Fluconazole inhibits the 14α-demethylation of lanosterol and 24,25-dihydrolanosterol, regulated by CYP51A1, without reduction of total cholesterol synthesis. The increased serum level of R_4ß-hydroxycholesterol under fluconazole treatment is in contrast to the reductions observed under ketoconazole and itraconazole treatments. The question, whether this increase is caused by induction of CYP3A4 or by inhibition of the catabolism of 4ß-hydroxycholesterol, must be answered by mechanistic in vitro and in vivo studies comparing effects of various azole antifungal agents on hepatic CYP3A4 activity.

Production of a new triterpenoid disaccharide saponin from sequential glycosylation of ganoderic acid A by 2 Bacillus glycosyltransferases.

Ganoderic acid A (GAA) is a lanostane-type triterpenoid, isolated from medicinal fungus Ganoderma lucidum, and possesses multiple bioactivities. In the present study, GAA was sequentially biotransformed by 2 recently discovered Bacillus glycosyltransferases (GT), BtGT_16345 and BsGT110, and the final product was purified and identified as a new compound, GAA-15,26-O-ß-diglucoside, which showed 1024-fold aqueous solubility than GAA.

Luciferase-based HMG-CoA reductase degradation assay for activity and selectivity profiling of oxy(lano)sterols.

HMG-CoA reductase (HMGCR) is the rate-limiting enzyme in the cholesterol biosynthetic pathway, and is the target of cholesterol-lowering drugs, statins. Previous studies have demonstrated that the enzyme activity is regulated by sterol-induced degradation in addition to transcriptional regulation through sterol-regulatory-element-binding proteins (SREBPs). While 25-hydroxycholesterol induces both HMGCR degradation and SREBP inhibition in a nonselective manner, lanosterol selectively induces HMGCR degradation. Here, to clarify the structural determinants of selectivity for the two activities, we established a luciferase-based assay monitoring HMGCR degradation and used it to profile the structure-activity/selectivity relationships of oxysterols and (oxy)lanosterols. We identified several sterols that selectively induce HMGCR degradation and one sterol, 25-hydroxycholest-4-en-3-one, that selectively inhibits the SREBP pathway. These results should be helpful in designing more potent and selective HMGCR degraders.

Endogenous sterol intermediates of the mevalonate pathway regulate HMGCR degradation and SREBP-2 processing.

Sterol-regulated HMG-CoA reductase (HMGCR) degradation and SREBP-2 cleavage are two major feedback regulatory mechanisms governing cholesterol biosynthesis. Reportedly, lanosterol selectively stimulates HMGCR degradation, and cholesterol is a specific regulator of SREBP-2 cleavage. However, it is unclear whether other endogenously generated sterols regulate these events. Here, we investigated the sterol intermediates from the mevalonate pathway of cholesterol biosynthesis using a CRISPR/Cas9-mediated genetic engineering approach. With a constructed HeLa cell line expressing the mevalonate transporter, we individually deleted genes encoding major enzymes in the mevalonate pathway, used lipidomics to measure sterol intermediates, and examined HMGCR and SREBP-2 statuses. We found that the C4-dimethylated sterol intermediates, including lanosterol, 24,25-dihydrolanosterol, follicular fluid meiosis activating sterol, testis meiosis activating sterol, and dihydro-testis meiosis activating sterol, were significantly upregulated upon mevalonate loading. These intermediates augmented both degradation of HMGCR and inhibition of SREBP-2 cleavage. The accumulated lanosterol induced rapid degradation of HMGCR, but did not inhibit SREBP-2 cleavage. The newly synthesized cholesterol from the mevalonate pathway is dispensable for inhibiting SREBP-2 cleavage. Together, these results suggest that lanosterol is a bona fide endogenous regulator that specifically promotes HMGCR degradation, and that other C4-dimethylated sterol intermediates may regulate both HMGCR degradation and SREBP-2 cleavage.

Biallelic pathogenic variants in the lanosterol synthase gene LSS involved in the cholesterol biosynthesis cause alopecia with intellectual disability, a rare recessive neuroectodermal syndrome.

PURPOSE: Lanosterol synthase (LSS) gene was initially described in families with extensive congenital cataracts. Recently, a study has highlighted LSS associated with hypotrichosis simplex. We expanded the phenotypic spectrum of LSS to a recessive neuroectodermal syndrome formerly named alopecia with mental retardation (APMR) syndrome. It is a rare autosomal recessive condition characterized by hypotrichosis and intellectual disability (ID) or developmental delay (DD), frequently associated with early-onset epilepsy and other dermatological features. METHODS: Through a multicenter international collaborative study, we identified LSS pathogenic variants in APMR individuals either by exome sequencing or LSS Sanger sequencing. Splicing defects were assessed by transcript analysis and minigene assay. RESULTS: We reported ten APMR individuals from six unrelated families with biallelic variants in LSS. We additionally identified one affected individual with a single rare variant in LSS and an allelic imbalance suggesting a second event. Among the identified variants, two were truncating, seven were missense, and two were splicing variants. Quantification of cholesterol and its precursors did not reveal noticeable imbalance. CONCLUSION: In the cholesterol biosynthesis pathway, lanosterol synthase leads to the cyclization of (S)-2,3-oxidosqualene into lanosterol. Our data suggest LSS as a major gene causing a rare recessive neuroectodermal syndrome.

A Genome-Centric Approach Reveals a Novel Glycosyltransferase from the GA A07 Strain of Bacillus thuringiensis Responsible for Catalyzing 15-O-Glycosylation of Ganoderic Acid A.

Strain GA A07 was identified as an intestinal Bacillus bacterium of zebrafish, which has high efficiency to biotransform the triterpenoid, ganoderic acid A (GAA), into GAA-15-O-ß-glucoside. To date, only two known enzymes (BsUGT398 and BsUGT489) of Bacillus subtilis ATCC 6633 strain can biotransform GAA. It is thus worthwhile to identify the responsible genes of strain GA A07 by whole genome sequencing. A complete genome of strain GA A07 was successfully assembled. A phylogenomic analysis revealed the species of the GA A07 strain to be Bacillus thuringiensis. Forty glycosyltransferase (GT) family genes were identified from the complete genome, among which three genes (FQZ25_16345, FQZ25_19840, and FQZ25_19010) were closely related to BsUGT398 and BsUGT489. Two of the three candidate genes, FQZ25_16345 and FQZ25_19010, were successfully cloned and expressed in a soluble form in Escherichia coli, and the corresponding proteins, BtGT_16345 and BtGT_19010, were purified for a biotransformation activity assay. An ultra-performance liquid chromatographic analysis further confirmed that only the purified BtGT_16345 had the key biotransformation activity of catalyzing GAA into GAA-15-O-ß-glucoside. The suitable conditions for this enzyme activity were pH 7.5, 10 mM of magnesium ions, and 30 °C. In addition, BtGT_16345 showed glycosylation activity toward seven flavonoids (apigenein, quercetein, naringenein, resveratrol, genistein, daidzein, and 8-hydroxydaidzein) and two triterpenoids (GAA and antcin K). A kinetic study showed that the catalytic efficiency (kcat/KM) of BtGT_16345 was not significantly different compared with either BsUGT398 or BsUGT489. In short, this study identified BtGT_16345 from B. thuringiensis GA A07 is the catalytic enzyme responsible for the 15-O-glycosylation of GAA and it was also regioselective toward triterpenoid substrates.

A New Triterpenoid Glucoside from a Novel Acidic Glycosylation of Ganoderic Acid A via Recombinant Glycosyltransferase of Bacillus subtilis.

Ganoderic acid A (GAA) is a bioactive triterpenoid isolated from the medicinal fungus Ganoderma lucidum. Our previous study showed that the Bacillus subtilis ATCC (American type culture collection) 6633 strain could biotransform GAA into compound (1), GAA-15-O-ß-glucoside, and compound (2). Even though we identified two glycosyltransferases (GT) to catalyze the synthesis of GAA-15-O-ß-glucoside, the chemical structure of compound (2) and its corresponding enzyme remain elusive. In the present study, we identified BsGT110, a GT from the same B. subtilis strain, for the biotransformation of GAA into compound (2) through acidic glycosylation. BsGT110 showed an optimal glycosylation activity toward GAA at pH 6 but lost most of its activity at pH 8. Through a scaled-up production, compound (2) was successfully isolated using preparative high-performance liquid chromatography and identified to be a new triterpenoid glucoside (GAA-26-O-ß-glucoside) by mass and nuclear magnetic resonance spectroscopy. The results of kinetic experiments showed that the turnover number (kcat) of BsGT110 toward GAA at pH 6 (kcat = 11.2 min-1) was 3-fold higher than that at pH 7 (kcat = 3.8 min-1), indicating that the glycosylation activity of BsGT110 toward GAA was more active at acidic pH 6. In short, we determined that BsGT110 is a unique GT that plays a role in the glycosylation of triterpenoid at the C-26 position under acidic conditions, but loses most of this activity under alkaline ones, suggesting that acidic solutions may enhance the catalytic activity of this and similar types of GTs toward triterpenoids.

Heterologous Expression of Full-Length Lanosterol 14α-Demethylases of Prominent Fungal Pathogens Candida albicans and Candida glabrata Provides Tools for Antifungal Discovery.

Targeting lanosterol 14α-demethylase (LDM) with azole drugs provides prophylaxis and treatments for superficial and disseminated fungal infections, but cure rates are modest for immunocompromised patients and individuals with comorbidities. The efficacy of azole drugs has also been reduced due to the emergence of drug-resistant fungal pathogens. We have addressed these problems by expressing in Saccharomyces cerevisiae functional, hexahistidine-tagged, full-length Candida albicans LDM (CaLDM6×His) and Candida glabrata LDM (CgLDM6×His) for drug discovery purposes and determining their X-ray crystal structures. Compared with S. cerevisiae overexpressing LDM6×His (ScLDM6×His), the reduced susceptibility of CgLDM6×His to all azole drugs tested correlated with its level of overexpression. In contrast, the reduced susceptibility to short-tailed (fluconazole and voriconazole) but not medium-tailed (VT-1161) or long-tailed azoles (itraconazole and posaconazole) indicates CaLDM6×His works best when coexpressed with its cognate NADPH-cytochrome P450 reductase (CaNcp1A) rather than the host reductase (ScNcp1). Overexpression of LDM or Ncp1 modified the ergosterol content of yeast and affected growth inhibition by the polyene antibiotic amphotericin B. Affinity-purified recombinant Candida LDMs bind carbon monoxide and show tight type II binding of a range of azole drugs, including itraconazole, posaconazole, fluconazole, and voriconazole. This study provides a practical basis for the phenotype-, biochemistry-, and structure-directed discovery of novel antifungals that target LDMs of fungal pathogens.

Crystal Structures of Full-Length Lanosterol 14α-Demethylases of Prominent Fungal Pathogens Candida albicans and Candida glabrata Provide Tools for Antifungal Discovery.

Targeting lanosterol 14α-demethylase (LDM) with azole drugs provides prophylaxis and treatments for superficial and disseminated fungal infections, but cure rates are not optimal for immunocompromised patients and individuals with comorbidities. The efficacy of azole drugs has also been reduced due to the emergence of drug-resistant fungal pathogens. We have addressed the need to improve the potency, spectrum, and specificity for azoles by expressing in Saccharomyces cerevisiae functional, recombinant, hexahistidine-tagged, full-length Candida albicans LDM (CaLDM6×His) and Candida glabrata LDM (CgLDM6×His) and determining their X-ray crystal structures. The crystal structures of CaLDM6×His, CgLDM6×His, and ScLDM6×His have the same fold and bind itraconazole in nearly identical conformations. The catalytic domains of the full-length LDMs have the same fold as the CaLDM6×His catalytic domain in complex with posaconazole, with minor structural differences within the ligand binding pocket. Our structures give insight into the LDM reaction mechanism and phenotypes of single-site CaLDM mutations. This study provides a practical basis for the structure-directed discovery of novel antifungals that target LDMs of fungal pathogens.

Identification of key amino acid residues determining product specificity of 2,3-oxidosqualene cyclase in Oryza species.

Triterpene synthases, also known as 2,3-oxidosqualene cyclases (OSCs), synthesize diverse triterpene skeletons that form the basis of an array of functionally divergent steroids and triterpenoids. Tetracyclic and pentacyclic triterpene skeletons are synthesized via protosteryl and dammarenyl cations, respectively. The mechanism of conversion between two scaffolds is not well understood. Here, we report a promiscuous OSC from rice (Oryza sativa) (OsOS) that synthesizes a novel pentacyclic triterpene orysatinol as its main product. The OsOS gene is widely distributed in indica subspecies of cultivated rice and in wild rice accessions. Previously, we have characterized a different OSC, OsPS, a tetracyclic parkeol synthase found in japonica subspecies. Phylogenetic and protein structural analyses identified three key amino acid residues (#732, #365, #124) amongst 46 polymorphic sites that determine functional conversion between OsPS and OsOS, specifically, the chair-semi(chair)-chair and chair-boat-chair interconversions. The different orientation of a fourth amino acid residue Y257 was shown to be important for functional conversion The discovery of orysatinol unlocks a new path to triterpene diversity in nature. Our findings also reveal mechanistic insights into the cyclization of oxidosqualene into tetra- and pentacyclic skeletons, and provide a new strategy to identify key residues determining OSC specificity.

Biosynthesis of a ganoderic acid in Saccharomyces cerevisiae by expressing a cytochrome P450 gene from Ganoderma lucidum.

Ganoderic acid (GA), a triterpenoid from the traditional Chinese medicinal higher fungus Ganoderma lucidum, possesses antitumor and other significant pharmacological activities. Owing to the notorious difficulty and immaturity in genetic manipulation of higher fungi as well as their slow growth, biosynthesis of GAs in a heterologous host is an attractive alternative for their efficient bioproduction. In this study, using Saccharomyces cerevisiae as a host, we did a systematic screening of cytochrome P450 monooxygenase (CYP450) gene candidates from G. lucidum, which may be responsible for the GA biosynthesis from lanosterol but have not been functionally characterized. As a result, overexpression of a CYP450 gene cyp5150l8 was firstly found to produce an antitumor GA, 3-hydroxy-lanosta-8, 24-dien-26 oic acid (HLDOA) in S. cerevisiae, as confirmed by HPLC, LC-MS and NMR. A final titer of 14.5 mg/L of HLDOA was obtained at 120 hr of the yeast fermentation. Furthermore, our in vitro enzymatic experiments indicate that CYP5150L8 catalyzes a three-step biotransformation of lanosterol at C-26 to synthesize HLDOA. To our knowledge, this is the first report on the heterologous biosynthesis of GAs. The results will be helpful to the GA biosynthetic pathway elucidation and to future optimization of heterologous cell factories for GA production.

Cloning, expression, purification and spectrophotometric analysis of lanosterol 14-alpha demethylase from Leishmania braziliensis (LbCYP51).

Leishmaniasis, a neglected tropical disease, is a major cause of morbidity and mortality worldwide. Of the three main clinical forms, cutaneous leishmaniasis (CL) is the most common and 40 million people are at risk in the endemic areas. Currently, the available drugs to fight leishmaniasis have high toxicity and poor efficiency. Then, it is very important to search for effective and safe drugs that would target essential enzymes from the parasite, such as lanosterol 14-alpha demethylase (CYP51, EC 1.14.13.70) from Leishmania braziliensis. Because most drug design efforts have been directed for Leishmania non-braziliensis species, there is no structural or kinetic data regarding L. braziliensis CYP51. Herein, we present for the first time molecular biology efforts and purification protocol to obtain the enzyme LbCYP51. These results lay the ground for future investigation of drugs against this target.

Nitric oxide stimulates cellular degradation of human CYP51A1, the highly conserved lanosterol 14α-demethylase.

Nitric oxide (NO) is known to down-regulate drug-metabolizing cytochrome P450 enzymes in an enzyme-selective manner. Ubiquitin-proteasome-dependent and -independent pathways have been reported. Here, we studied the regulation of expression of human CYP51A1, the lanosterol 14α-demethylase required for synthesis of cholesterol and other sterols in mammals, which is found in every kingdom of life. In Huh7 human hepatoma cells, treatment with NO donors caused rapid post-translational down-regulation of CYP51A1 protein. Human NO synthase (NOS)-dependent down-regulation was also observed in cultured human hepatocytes treated with a cytokine mixture and in Huh7 cells expressing human NOS2 under control of a doxycycline-regulated promoter. This down-regulation was partially attenuated by proteasome inhibitors, but only trace levels of ubiquitination could be found. Further studies with inhibitors of other proteolytic pathways suggest a possible role for calpains, especially when the proteasome is inhibited. NO donors also down-regulated CYP51A1 mRNA in Huh7 cells, but to a lesser degree, than the down-regulation of the protein.

Uridine Diphosphate-Dependent Glycosyltransferases from Bacillus subtilis ATCC 6633 Catalyze the 15-O-Glycosylation of Ganoderic Acid A.

Bacillus subtilis ATCC (American type culture collection) 6633 was found to biotransform ganoderic acid A (GAA), which is a major lanostane triterpenoid from the medicinal fungus Ganoderma lucidum. Five glycosyltransferase family 1 (GT1) genes of this bacterium, including two uridine diphosphate-dependent glycosyltransferase (UGT) genes, BsUGT398 and BsUGT489, were cloned and overexpressed in Escherichia coli. Ultra-performance liquid chromatography confirmed the two purified UGT proteins biotransform ganoderic acid A into a metabolite, while the other three purified GT1 proteins cannot biotransform GAA. The optimal enzyme activities of BsUGT398 and BsUGT489 were at pH 8.0 with 10 mM of magnesium or calcium ion. In addition, no candidates showed biotransformation activity toward antcin K, which is a major ergostane triterpenoid from the fruiting bodies of Antrodia cinnamomea. One biotransformed metabolite from each BsUGT enzyme was then isolated with preparative high-performance liquid chromatography. The isolated metabolite from each BsUGT was identified as ganoderic acid A-15-O-ß-glucoside by mass and nuclear magnetic resonance spectroscopy. The two BsUGTs in the present study are the first identified enzymes that catalyze the 15-O-glycosylation of triterpenoids.

Sterol methyltransferase a target for anti-amoeba therapy: towards transition state analog and suicide substrate drug design.

Ergosterol biosynthesis pathways essential to pathogenic protozoa growth and absent from the human host offer new chokepoint targets. Here, we present characterization and cell-based interference of Acanthamoeba spp sterol 24-/28-methylases (SMTs) that catalyze the committed step in C28- and C29-sterol synthesis. Intriguingly, our kinetic analyses suggest that 24-SMT prefers plant cycloartenol whereas 28-SMT prefers 24(28)-methylene lophenol in similar fashion to the substrate preferences of land plant SMT1 and SMT2. Transition state analog-24(R,S),25-epiminolanosterol (EL) and suicide substrate 26,27-dehydrolanosterol (DHL) differentially inhibited trophozoite growth with IC50 values of 7 nM and 6 µM, respectively, and EL yielded 20-fold higher activity than reference drug voriconazole. Against either SMT assayed with native substrate, EL exhibited tight binding ∼Ki 9 nM. Alternatively, DHL is methylated at C26 by 24-SMT that thereby, generates intermediates that complex and inactivate the enzyme, whereas DHL is not productively bound to 28-SMT. Steroidal inhibitors had no effect on human epithelial kidney cell growth or cholesterol biosynthesis at minimum amoebicidal concentrations. We hypothesize the selective inhibition of Acanthamoeba by steroidal inhibitors representing distinct chemotypes may be an efficient strategy for the development of promising compounds to combat amoeba diseases.