Catalytic enhancements in cytochrome P450 2C19 by cytochrome b5.

Human cytochrome P450 2C19 catalyzes P450 enzyme reactions of various substrates, including steroids and clinical drugs. Recombinant P450 2C19 enzyme with histidine tag was successfully expressed in Escherichia coli and purified using affinity column chromatography. Ultra-performance liquid chromatography-tandem mass (UPLC-MS/MS) spectrometry showed that the purified P450 2C19 enzyme catalyzed 5-hydroxylation reaction of omeprazole. The purified enzyme displayed typical type I binding spectra to progesterone with a Kd value of 4.5 ± 0.2 µM, indicating a tight substrate binding. P450 2C19 catalyzed the hydroxylation of progesterone to produce 21-hydroxy (OH) as a major and 17-OH product as a minor product. Steady-state kinetic analysis of progesterone 21-hydroxylation indicated that the addition of cytochrome b5 stimulated a five-times catalytic turnover number of P450 2C19 with a kcat value of 1.07 ± 0.08 min-1. The molecular docking model of progesterone in the active site of P450 2C19 displayed that the 21-carbon of progesterone was located close to the heme with a distance of 4.7 Å, suggesting 21-hydroxylation of progesterone is the optimal reaction of P450 2C19 enzyme for a productive orientation of the substrate. Our findings will help investigate the extent to which cytochrome b5 affects the metabolism of P450 2C19 to drugs and steroids. Supplementary Information: The online version contains supplementary material available at 10.1007/s43188-023-00219-8.

Functional Characterization of Allelic Variations of Human Cytochrome P450 2C8 (V181I, I244V, I331T, and L361F).

The human cytochrome P450 2C8 is responsible for the metabolism of various clinical drugs as well as endogenous fatty acids. Allelic variations can significantly influence the metabolic outcomes. In this study, we characterize the functional effects of four nonsynonymous single nucleotide polymorphisms *15, *16, *17, and *18 alleles recently identified in cytochrome P450 2C8. The recombinant allelic variant enzymes V181I, I244V, I331T, and L361F were successfully expressed in Escherichia coli and purified. The steady-state kinetic analysis of paclitaxel 6-hydroxylation revealed a significant reduction in the catalytic activities of the V181I, I244V, and L361F variants. The calculated catalytic efficiency (kcat/Km) of these variants was 5-26% of that of the wild-type enzyme. The reduced activities were due to both decreased kcat values and increased Km values of the variants. The epoxidation of arachidonic acid by the variants was analyzed. The L361F variant only exhibited 4-6% of the wild-type catalytic efficiency in ω-9- and ω-6-epoxidation reactions to produce 11,12-epoxyeicosatrienoic acid (EET) and 14,15-EET, respectively. These reductions were mainly due to a decrease in the kcat value of the L361F variant. The binding titration analysis of paclitaxel and arachidonic acid showed that all variants had similar affinities to those of the wild-type (10-14 µM for paclitaxel and 20-49 µM for arachidonic acid). The constructed paclitaxel docking model of the variant enzyme suggests that the L361F substitution leads to the incorrect orientation of paclitaxel in the active site, with the 6'C of paclitaxel displaced from the productive catalytic location. This study suggests that individuals carrying the newly identified P450 2C8 allelic variations are likely to have an altered metabolism of clinical medicines and production of fatty acid-derived signal molecules.

Hydroxylation and lyase reactions of steroids catalyzed by mouse cytochrome P450 17A1 (Cyp17a1).

Cytochrome P450 17A1 (CYP17A1) catalyzes 17α-hydroxylation and 17,20-lyase reactions with steroid hormones. Mice contain an orthologous Cyp17a1 enzyme in the genome, and its amino acid sequence has high similarity with human CYP17A1. We purified recombinant mouse Cyp17a1 and characterized its oxidation reactions with progesterone and pregnenolone. The open reading frame of the mouse Cyp17a1 gene was inserted and successfully expressed in Escherichia coli and then purified using Ni2+-nitrilotriacetic acid (NTA) affinity column chromatography. Purified mouse Cyp17a1 displayed typical Type I binding titration spectral changes upon the addition of progesterone, 17α-OH progesterone, pregnenolone, and 17α-OH pregnenolone, with similar binding affinities to those of human CYP17A1. Catalytic activities for 17α-hydroxylation and 17,20-lyase reactions were studied using ultra-performance liquid chromatography (UPLC)-mass spectrometry analysis. Mouse Cyp17a1 showed cytochrome b5 stimulation in catalysis. In comparison to human enzyme, much higher specificity constants (kcat/Km) were observed with mouse Cyp17a1. In the reactions of Δ4-steroids (progesterone and 17α-OH progesterone), the specificity constants were 2100 times higher than the human enzyme. The addition of cytochrome b5 produced significant stimulation of 17,20-lyase activities of mouse Cyp17a1. Two Arg mutants of mouse Cyp17a1 (R347H and R358Q) displayed a larger decrease in 17,20-lyase reaction (from 17α-OH pregnenolone to dehydroepiandrosterone, DHEA) than 17α-hydroxylation, indicating that -as in human CYP17A1-these basic residues in mouse Cyp17a1 are important in interacting with the cytochrome b5 protein in the lyase reactions.

Xanthogranuloma formation after endoscopic sinus surgery: A case report.

INTRODUCTION: Although xanthogranuloma is known to be related to trauma or mucosa, possibly developing around a periorbital or oral lesion, xanthogranuloma related to sinusitis urgery has not been reported. We present a case of xanthogranuloma formation after endoscopic sinus surgery (ESS). PRESENTAION OF CASE: A 54-year-old man with pain and swelling in the right periorbital area presented to our clinic. He had had a blowout fracture treated by ESS 2 years prior. Physical examination and computed tomography revealed an ∼1-cm × 0.7-cm cystic mass on the right lower eyelid. Subciliary exploration found a fat-like mass that we completely excised. A histological examination revealed xanthogranuloma. No recurrence was observed for 1 year. DISCUSSION: If the wall between the sinuses and the orbit and the mucosa of the maxillary sinus are injured during ESS, infectious material and hematoma could develop into chronic granulomatous inflammation. In addition, a large antrostomy and/or a damaged nasolacrimal duct are risk factors for xanthogranuloma. Antibiotics can treat the disease and prevent infection. Progressive growth of the lesion and its infiltration into surrounding tissues may result in surgical resection. CONCLUSION: Because many masses are idiopathic, the development of xanthogranuloma after simple ESS or a nondisplaced blowout fracture is possible. Although xanthogranuloma progression usually is benign and without specific complications, it may be sight- or life-threatening. Antibiotics and surgical resection are the treatments of choice and the latter can be a diagnostic tool. Physicians should be aware of the possibility of granuloma formation in patients who have undergone ESS.

Retromandibular reduction of medially dislocated condylar process fractures.

BACKGROUND: Condylar process fractures account for one-third of all mandibular fractures, and the distal fragment is prone to dislocate to the medial side due to the pulling of the lateral pterygoid muscle. Retromandibular approaches are commonly used, but the intraoperative view becomes limited in medially dislocated fractures. This study summarized a series of cases of retromandibular reduction for medially dislocated condylar process fractures and described our supplementary procedure to realign the dislocated condylar process. METHODS: Nine patients with medially dislocated condylar process fractures underwent surgical correction from January 2012 to December 2016. In 6 of them, it was possible to realign the fractures with a conventional retromandibular approach, but for 3 cases of severe dislocation to the middle cranial fossa, a supplementary transoral procedure was carried out. The angle difference between the ramus and condyle, ramus height, and maximal mouth opening (MMO) were evaluated. RESULTS: All 9 cases were restored to the proper anatomical alignment without any major complications, and postoperative images revealed successful union. The angle difference was 8.94°±4.11° preoperatively, and 0.99±0.49° at the 6-month follow-up. The pretreatment ramus height difference was 6.12±6.09 mm, and the postoperative difference was 0.18±0.10 mm. These changes after surgery were statistically significant. The MMO before surgery was 11.44±3.0 mm, and the postoperative MMO was 37.2±2.9 mm, reflecting a significant increase after reduction. CONCLUSIONS: Retromandibular reduction is a useful method in medially dislocated condylar process fractures, and additional transoral assistance should be considered to realign condylar processes that severely dislocate to the middle cranial fossa.