Functional characterization of CYP81C16 involved in the tanshinone biosynthetic pathway in Salvia miltiorrhiza / 中国天然药物

Danshen, the dried roots and rhizomes of Salvia miltiorrhiza Bunge (S. miltiorrhiza), is widely used in the treatment of cardiovascular and cerebrovascular diseases. Tanshinones, the bioactive compounds from Danshen, exhibit a wide spectrum of pharmacological properties, suggesting their potential for future therapeutic applications. Tanshinone biosynthesis is a complex process involving at least six P450 enzymes that have been identified and characterized, most of which belong to the CYP76 and CYP71 families. In this study, CYP81C16, a member of the CYP71 clan, was identified in S. miltiorrhiza. An in vitro assay revealed that it could catalyze the hydroxylation of four para-quinone-type tanshinones, namely neocryptotanshinone, deoxyneocryptotanshinone, and danshenxinkuns A and B. SmCYP81C16 emerged as a potential broad-spectrum oxidase targeting the C-18 position of para-quinone-type tanshinones with an impressive relative conversion rate exceeding 90%. Kinetic evaluations andin vivo assays underscored its highest affinity towards neocryptotanshinone among the tested substrates. The overexpression of SmCYP81C16 promoted the accumulation of (iso)tanshinone in hairy root lines. The characterization of SmCYP81C16 in this study accentuates its potential as a pivotal tool in the biotechnological production of tanshinones, either through microbial or plant metabolic engineering.

Relationship between serum 25(OH)D and SIRT4 levels and glycolipid metabolism in children with different levels of obesity / 中国基层医药

Objective:To investigate the relationship between serum 25(OH)D and SIRT4 levels and glycolipid metabolism in children with different levels of obesity.Methods:A total of 124 children with different levels of obesity who received treatment in Shaoxing Women's and Children's Health Care Hospital from February 2016 to February 2021 were included in this study. These children were divided into mild/moderate obesity group ( n = 76) and severe obesity group ( n = 48) according to body mass index. An additional 62 healthy children who concurrently received a physical examination were selected for controls. The general data of all children were collected. The relationship between the factors that affect obesity in children and serum 25(OH)D and SIRT4 levels and glycolipid metabolism was analyzed. Results:In the control, mild/moderate obesity, and severe obesity groups, body mass was (26.68 ± 4.98) kg, (33.24 ± 5.48) kg, (37.18 ± 5.88) kg, respectively; waist circumference was (56.12 ± 4.62) cm, (68.45 ± 5.20) cm, (79.34 ± 5.65) cm, respectively; hip circumference was (68.42 ± 5.08) cm, (72.45 ± 6.45) cm, (80.56 ± 6.95) cm, respectively; body mass index (BMI) was (15.90 ± 2.04) kg/m 2, (23.58 ± 2.45) kg/m 2, (25.89 ± 2.35) kg/m 2], respectively; fasting insulin (FINS) level was (26.65 ± 3.68) pmol/L, (34.82 ± 4.15) pmol/L, (48.56 ± 5.49) pmol/l, respectively; homeostasis model assessment of insulin resistance (HOMA-IR) was (1.06 ± 0.24), (2.12 ± 0.35), (3.84 ± 0.52), respectively; total cholesterol (TC) level was (2.21 ± 0.45) mmol/L, (4.14 ± 0.58) mmol/L, (5.96 ± 0.64) mmol/L, respectively; triacylglycerol (TG) level was (0.68 ± 0.16) mmol/L, (1.12 ± 0.24) mmol/L, (1.56 ± 0.35) mmol/L, respectively; low density lipoprotein cholesterol (LDL-C) was (2.68 ± 0.42) mmol/L, (2.10 ± 0.32) mmol/L, (1.41 ± 0.25) mmol/L, respectively; high density lipoprotein cholesterol (HDL-C) was (1.98 ± 0.42) mmol/L, (3.12 ± 0.51) mmol/L, (4.10 ± 0.56) mmol/L, respectively. There were significant differences in body mass, waist circumference, hip circumference, BMI, FINS, HOMA-IR, TC, TG, HDL-C, and LDL-C among the three groups ( F = 53.62, 280.42, 53.33, 303.44, 338.48, 755.71, 618.75, 165.81, 186.89, 251.42, all P < 0.001). Body mass, waist circumference, hip circumference, BMI, FINS, HOMA-IR, TC level, TG level, HDL-C level, and LDL-C level were lower in the control group than in the mild/moderate obesity group ( t = -7.28, -14.56, -4.00, -19.72, -6.49, -21.45, -12.36, 9.20, -14.12, all P < 0.05). Body mass, waist circumference, hip circumference, BMI, FINS, HOMA-IR, TC, TG, HDL-C and LDL-C were lower in the mild/moderate obesity group than in the severe obesity group ( t = -3.79, -10.98, -6.61, -5.19, -15.81, -22.02, -16.34, -8.30, 12.68, -10.03, all P < 0.05). Serum 25(OH)D [(60.52 ± 8.95) nmol/L vs. (49.88 ± 8.12) nmol /L, t = 7.31, P < 0.05] and SIRT4 [(1.98 ± 0.38) mmol/L vs. (1.06 ± 0.30) mmol/L, t = 15.89, P < 0.05] levels were significantly greater in the control group than in the mild/moderate obesity group. Serum 25(OH)D [(49.88 ± 8.12) nmol/L vs. (41.62 ± 7.50) nmol /L, t = 5.68, P < 0.05] and SIRT4 [(1.06 ± 0.30) mmol/L vs. (0.52 ± 0.15) mmol/L, t = 11.57, P < 0.05] levels were significantly greater in the mild/moderate obesity group than in the severe obesity group. Multiple linear regression analysis showed that body mass, waist circumference, hip circumference, FINS, HOMA-IR, TC, TG, and LDL were the positive influential factors of childhood obesity ( B = 0.170, 0.310, 0.403, 1.000, 3.464, 2.080, 2.656, 4.324); HDL, serum 25(OH)D and SIRT4 were the negative influential factors of childhood obesity ( B = -2.096, -0.156, -6.615). Pearson correlation analysis showed that serum 25(OH)D was significantly negatively correlated with FINS, HOMA-IR, TC, TG and LDL ( r = -0.20, -0.46, -0.30, -0.36, all P < 0.01), and significantly positively correlated with FPG and HDL ( r = 0.43, 0.77, both P < 0.01). Serum SIRT4 was negatively correlated with FINS, TC, TG, and LDL ( r = -0.48, -0.74, -0.61, -0.64, all P < 0.01), and positively correlated with FPG and HDL ( r = 0.21, 0.84, both P < 0.01). Conclusion:Serum 25(OH)D and SIRT4 levels decrease with the aggravation of obesity in children and are closely related to glycolipid metabolism. Therefore, early detection of obesity can reflect the degree of obesity and glycolipid metabolism in children.

Analysis of hydroxylation and O-glycosylation on lysine sites in deer-hide gelatin / 中国中药杂志

Nano-LC MS/MS was used to analyze trypsin digested deer-hide gelatin(DHG) samples, hydroxylation and O-glycosylation on lysine sites of DHG were comprehensive identified by using PEAKS Studio software. The sites, sorts and amounts of hydroxylation and O-glycosylation on Type Ⅰ collagen α1 chain(COL1 A1) and α2 chain(COL1 A2) of DHG were revealed. As a result, 5 284 peptides were identified from DHG samples, which were mainly from COL1 A1 and COL1 A2. Among these peptides, there were 449 peptides with hydroxylysine, 442 with galactosyl-hydroxylysine, 449 with glucosyl-galactosyl-hydroxylysine. The major modified sites of hydroxylation and O-glycosylation in DHG were shown as follow: α1-9 N and α2-5 N in N-telopeptides, α1-87, α1-174, α1-930, α2-87, α2-174, α2-933 in triple helix domain, and α1-16 C in C-telopeptides. These hydroxylation and O-glycosylation were correlated with the formation and stability of collagen molecules and collagen fibrils. It is feasible for the collagens and peptides dissolving from deer skin collagen fibrils under high temperature and pressure decocting, high temperature and pressure also might destroy inter-molecular covalent cross-linking and help those glycol-peptides formations. The present study provided ideas and strategies for the in-depth investigation on DHG chemical constituents, and showed good theoretical significance and application value.

Advances in bacterial Rieske non-heme iron ring-hydroxylating dioxygenases that initiate polycyclic aromatic hydrocarbons degradation / 生物工程学报

Polycyclic aromatic hydrocarbons (PAHs) are a class of persistent organic pollutants, which have received widespread attentions due to their carcinogenic and mutagenic toxicity. The microbial degradation of PAHs are usually started from the hydroxylation, followed by dehydrogenation, ring cleavage and step-by-step removal of branched chains, and finally mineralized by the tricarboxylic acid cycle. Rieske type non-heme iron aromatic ring-hydroxylating dioxygenases (RHOs) or cytochrome P450 oxidases are responsible for the conversion of hydrophobic PAHs into hydrophilic derivatives by the ring hydroxylation. The ring hydroxylation is the first step of PAHs degradation and also one of the rate-limiting steps. Here, we review the distribution, substrate specificity, and substrate recognition mechanisms of RHOs, along with some techniques and methods used for the research of RHOs and PAHs.

A case of vitamin D hydroxylation-deficient rickets type 1A caused by 2 novel pathogenic variants in CYP27B1 gene

Vitamin D hydroxylation-deficient rickets type 1A (VDDR1A, OMIM 264700) is a rare autosomal recessive inherited disorder. Pathogenic variants in the CYP27B1 gene lead to loss of 1α-hydroxylase activity. We report the case of a 22-month-old toddler who presented with growth retardation and delayed development. The patient exhibited the typical laboratory findings of VDDR1A, including hypocalcemia (calcium: 5.2 mg/dL), elevated serum level of alkaline phosphatase (2,600 U/L), elevated serum level of intact-parathyroid hormone (238 pg/mL), low 1,25(OH)₂D₃ level (11.2 pg/mL), and normal 25(OH)D₃ level (40.7 ng/mL). His height and weight were 76.5 cm and 9.5 kg, respectively (both <3rd percentile). The Bayley Scales of Infant and Toddler Development II indicated significantly delayed development (mental development index <50, psychomotor development index <50). The patient was a compound heterozygous for two novel pathogenic variants in the CYP27B1 gene: c.57_69del (p.Glu20Profs*2) and c.171dupG (p.Leu58Alafs*275), inherited from his mother and father, respectively. The patient showed remarkable improvement after treatment with calcitriol and calcium carbonate.

Steroids hydroxylation catalyzed by the monooxygenase mutantfromBM3

The search of new substrates with pharmaceutical and industrial potential for biocatalysts including cytochrome P450 enzymes is always challenging. Cytochrome P450 BM3 mutant, a versatile biocatalyst, exhibited hydroxylation activities towards fatty acids and alkanes. However, there were limited reports about its hydroxylation activity towards steroids. Herein, an-based whole-cell extract containing the recombinant 139-3 protein was used as the biocatalyst to screen 13 steroids. Results revealed that 139-3 was able to specifically hydroxylate androstenedione () at 1-position, generating a hydroxylated steroid 1-OH-androstenedione (). To investigate whether C-1hydroxylation catalyzed by BM3 mutantcould be industrially used, an optimization of catalyzing conditions was performed. Accordingly, the BM3 mutant 139-3 enzyme was observed to display maximum activity at 37 °C, under pH 7.0 for 4 h, with 37% transformation rate. Moreover, fourvariants were generated by random mutagenesis with the aim of improving its activity and expanding substrate scope. Surprisingly, these mutants, sharing a common mutated site R379S, lost their activities towards androstenedione (). These data clearly indicated that arginine residue located at site 379 played key role in the hydroxylation activities of 139-3. Overall, these new findings broadened the substrate scope of 139-3 enzyme, thereby expanding its potential applications as a biocatalyst on steroids hydroxylation in pharmaceutical industry.

Regio- and stereo-selective hydroxylations of ingenane diterpenoids by Mortierella ramanniana and Gibberella fujikuroi / 中国天然药物

The regio- and stereo-selective hydroxylations of two ingenane diterpenoids, 20-deoxyingenol (1) and 13-oxyingenol dodecanoat (2), by the filamentous fungi Mortierella ramanniana and Gibberella fujikuroi were investigated in the present study. Four undescribed metabolites (3-6) of substrate 1 and two undescribed metabolites (7 and 8) of substrate 2 were isolated. All the metabolites were identified as hydroxylated ingenane derivatives by extensive NMR and HR-ESI-MS data analyses. All the biotransformed compounds and the substrates were evaluated for their cytotoxicities against three human cancer cell lines, including human colon cancer Caco-2, breast cancer MCF-7, and adriamycin (ADM)-resistant MCF-7/ADM cell lines. All ingenane alcohols (1, and 3-6) displayed no significant cytotoxic activities. The substrate 13-oxyingenol dodecanoat (2) showed moderate cytotoxicity with IC values being 35.59 ± 5.37 μmol·L (Caco-2), 24.04 ± 4.70 μmol·L (MCF-7), and 22.24 ± 5.19 μmol·L (MCF-7/ADM). However, metabolites 7 and 8 displayed no significant cytotoxicity. These results indicated that the hydroxylation at the C-13 aliphatic acid ester of substrate 2 can significantly reduce the cytotoxic activity.

Validation of a liquid chromatographic method for the pharmaceutical quality control of products containing elacridar$ / 药物分析学报

Many anticancer drugs have an impaired bioavailability and poor brain penetration because they are sub-strates to drug efflux pumps such as P-glycoprotein and Breast Cancer Resistance Protein. Elacridar is a strong inhibitor of these two drug efflux pumps and therefore has great potential to improve oral absorption and brain penetration of many anticancer drugs. Currently, a clinical formulation of elacridar is unavailable and therefore the pharmaceutical development of a drug product is highly warranted. This also necessitates the availability of an analytical method for its quality control. A reverse-phase high-performance liquid chro-matographic method with ultraviolet detection was developed for the pharmaceutical quality control of products containing elacridar as the active pharmaceutical ingredient. The analytical method was validated for linearity, accuracy, precision, selectivity, carry-over, stability of stock and reference solutions, stability of the final extract, stability-indicating capability and impurity testing. We found that elacridar is unstable in aqueous solutions that are exposed to light because a hydroxylation product of elacridar is formed. Therefore, sample solutions with elacridar must be protected from light.

Long-term clinical outcome and the identification of homozygous CYP27B1 gene mutations in a patient with vitamin D hydroxylation-deficient rickets type 1A

Vitamin D hydroxylation-deficient rickets type 1A (VDDR1A) is an autosomal recessively-inherited disorder caused by mutations in CYP27B1 encoding the 1α-hydroxylase enzyme. We report on a female patient with VDDR1A who presented with hypocalcemic seizure at the age of 13 months. The typical clinical and biochemical features of VDDR1A were found, such as hypocalcemia, increased alkaline phosphatase, secondary hyperparathyroidism and normal 25-hydroxyvitamin D3 (25(OH)D₃). Radiographic images of the wrist showed metaphyseal widening with cupping and fraying of the ulna and distal radius, suggesting rickets. A mutation analysis of the CYP27B1 gene identified a homozygous mutation of c.589+1G>A in the splice donor site in intron 3, which was known to be pathogenic. Since that time, the patient has been under calcitriol and calcium treatment, with normal growth and development. During the follow-up period, she did not develop genu valgum, scoliosis, or nephrocalcinosis.

Synthesis of 6-benzyl-1-[ (benzyloxy) methyl ]-3-hydroxy-5-(hydroxymethyl) pyrimi-dine-2,4(1H,3H)-dione / 北京大学学报(医学版)

Objective:To find the best synthesis method of 6-benzyl-1-[ ( benzyloxy ) methyl ]-3-hydro-xy-5-(hydroxymethyl)pyrimidine-2,4(1H,3H)-dione e for observing the change of its biological activity after N-3 hydroxylation .Methods:After trying some N-hydroxylation methods , the target compound was successfully synthesized via one-pot oxidizing process by sodium hydride ( NaH) and 3-chloroperbenzoic acid( m-CPBA);the anti-HIV reverse transcriptase ( RT) activity and integrase ( IN) activity of the tar-get compound was assayed via enzyme-linked immunesorbent assay ( ELISA) and phosphorylation of DNA package method .Results:The target compound could be obtained through the improved m-CPBA oxida-tive method by only one step , and the yield of the reaction could reach 60%-70%.And the structure of this compound was identified by 1 H NMR, 13 C NMR and MS;The activity result showed it added the an-ti-HIV IN activity after N-3 hydroxylation as well as retained the anti-HIV RT activity.Conclusion:The improved m-CPBA oxidative method is a convenient and efficient way to prepare the compound 6-benzyl-1-[(benzyloxy)methyl]-3-hydroxy-5-(hydroxymethyl)pyrimidine-2,4(1H,3H)-dione e which has both anti-HIV RT and IN activity .

16α-Hydroxycholic Acid: In Vivo Microbial Transformation Product of Cholic Acid.

Aims: This study describes the transformation of cholic acid to hydroxylated cholic acid metabolites that could not be easily synthesized. Study Design: The transformation was catalyzed by thermophilic Geobacillus stearothermophilus comb. nov., isolated from oil contaminated soil in Kuwait. Cholic acid, as the sole source of carbon, was added to G. stearothermophilus cells in phosphate buffer pH 7 and shaken at 65ºC for 5 days. Methodology: The cholic acid transformation products were extracted with ethyl acetate, purified on preparative TLC plates and their molecular structures were established from their spectral data. Results: The bacterium could selectively oxidize hydroxyl-groups at C3 and C7 while leaving the C12-hydroxyl-group unoxidized, in cholic acid. Five commonly found metabolites of cholic acid and a novel transformation product, 16α-hydroxycholic acid, were identified. Conclusion: Our results indicate that G. stearothermophilus can hydroxylate/oxidize a steroid nucleus at various ring positions, and has a unique ability for hydroxylation at C16α in cholic acid.

CYP1A2 activity as a risk factor for bladder cancer

CYP1A2, CYP2D6 and N-acetyltransferase activities were estimated in 100 patients with bladder cancer and 84 control subjects from measurements of theophylline, metoprolol and isoniazid and their metabolites in urine, respectively. The frequency of occurrence of slow acetylators of isoniazid and poor metabolizers of metoprolol were 16.7% and 1.2% in the control group and 16.3% and 2.0% in the cancer patient group. These differences were not significant. The recovery ratio of 1-methyluric acid(1-MU) from theophylline was significantly higher in patients with bladder cancer than in control subjects(0.340 +/- 0.016 versus 0.260 +/- 0.020, p< 0.05). The 1-MU recovery ratio was a significant, independent risk factor among the metabolic capacities tested as shown by logistic regression analysis, controlling for N-acetylation of isoniazid, hydroxylation of metoprolol, age, sex, and smoking. We concluded that the capacity for 3-demethylation of theophylline, as a reflection of CYP1A2 activity, is significantly associated with increased risk of nonoccupational urinary bladder cancer.

Study on the Fermentation Condition of Steroid 15?-Hydroxylation by Penicillium raistrickii / 微生物学通报

The fermentation conditions which affect C-15? hydroxylation o f 18-methyl-estr- 4-ene-3,17-dione were investigated. As the key step in the Hydroxylation, the dissolution of substrate was focused on. Tween80, MeOH, DMSO,?-CD and 2-HP?CD were studied to improve the dissolvability of 18-met hyl-estr-4-ene-3,17-dione.The other factors such as pH, substrate concent ration and aeration strategies which affected conversion rate were also resea rched. As a result, the conversion rate can be up to 60% in shake flask and ach i(eve 50% in fermentor,which would overcome the disadvantage of 15?-hydroxyl -18-methyl-estr-4-ene-3,17-dione chemosynthesis and provide a good techn ics to industry.

Radical intermediates in peroxide-dependent reactions catalyzed by cytochrome P-450.

Thus, cumyl hydroperoxide yields acetophenone and methane, and 13-hydroperoxyoctadeca- 9,11-dienoic acid yields pentane and an as yet unidentified additional product. Since hydroperoxide reduction does not produce the corresponding alcohol, it is concluded that homolytic cleavage of the oxygen-oxygen bond occurs with rearrangement of the resulting alkoxy radical. Studies are in progress to determine how broad a role the new hydroperoxide cleavage reaction plays in the biological peroxidation of lipids.