ABSTRACT
9α-hydroxy-4-androstene-3,17-dione (9-OH-AD) is an important intermediate in the steroidal drugs production. 3-ketosteroid-9α-hydroxylase (KSH), a two protein system of KshA and KshB, is a key-enzyme in the microbial steroid ring B-opening pathway. KSH catalyzes the transformation of 4-androstene-3,17-dione (AD) into 9-OH-AD specifically. In the present study, the putative KshA and KshB genes were cloned from Mycobacterium smegmatis mc(2)155 and Gordonia neofelifaecis NRRL B-59395 respectively, and were inserted into the expression vector pNIT, the co-expression plasmids of kshA-kshB were obtained and electroporated into Mycobacterium sp. NRRL B-3805 cells. The recombinants were used to transform steroids, the main product was characterized as 9α-hydroxy-4-androstene-3,17-dione (9-OH-AD), showing that kshA and kshB were expressed successfully. Different from the original strain Mycobacterium sp. NRRL B-3805 that accumulates 4-androstene-3,17-dione, the recombinants accumulates 9α-hydroxy-4-androstene-3,17-dione as the main product. This results indicates that the putative genes kshA, kshB encode active KshA and KshB, respectively. The process of biotransformation was investigated and the results show that phytosterol is the most suitable substrate for biotransformation, kshA and kshB from M. smegmatis mc(2)155 seemed to exhibit high activity, because the resultant recombinant of them catalyzed the biotransformation of phytosterol to 9-OH-AD in a percent conversion of 90%, which was much higher than that of G. neofelifaecis NRRL B-59395. This study on the manipulation of the ksh genes in Mycobacterium sp. NRRL B-3805 provides a new pathway for producing steroid medicines.
Subject(s)
Androstenedione , Bacterial Proteins , Genetics , Metabolism , Biotransformation , Ketosteroids , Mixed Function Oxygenases , Genetics , Metabolism , Mycobacterium , Metabolism , Mycobacterium smegmatis , PlasmidsABSTRACT
Comparamos as respostas dos corticosteróides urinários:17-hidroxicorticosteróides (17-OHCS), 17-cetosteróides (17-KS) e cortisol livre (FC) à supressäo com dexametasona (baixa dose - 2 mg e alta dose - 8mg) em 71 pacientes com síndrome de Cushing (45 com doença de Cushing, 12 com carcinoma adrenal, oito com adenoma adrenocortical e seis com hiperplasia nodular da córtex adrenal). Os pacientes com doença de Cushing e hiperplasia nodular da córtex adrenal näo apresentam supressäo suficiente com 2 mg de dexametasona,porém o fazem com 8 mg. Os tumores adrenais näo demonstram supressäo adequada. A resposta hiperativa dos 17-OHCS, 17-KS e FC ao hormônio adrenocorticotrópico (ACTH) exógeno foi comparada na doença de Cushing. Concluimos que a determinaçäo urinária dos 17-OHCS, 17-KS e FC säo parâmetros diagnósticos úteis, embora a dosagem do FC mostrou ser um índice mais sensível da hiperfunçäo adrenocortical
Subject(s)
Humans , Male , Female , Child , Adolescent , Adult , Middle Aged , Cushing Syndrome/diagnosis , 17-Hydroxycorticosteroids/urine , Adrenocorticotropic Hormone , Dexamethasone , Diagnosis, Differential , Hydrocortisone/urine , Ketosteroids/urine , SuppressionABSTRACT
The effect of acute non-fatal haemorrhage on adrenocortical function was assessed in 19 Burmese subjects. The results indicated that acute non-fatal haemorrhage as produced by blood donation stimulated the adrenal cortex to increase its secretion.