Microbial transformations of steroids-XI. Progesterone transformation by Streptomyces roseochromogenes–purification and characterisation of the 16α-hydroxylase system

Berrie, J. R. H.; Williams, Ruth; Smith, Kelvin E.

doi:10.1016/s0960-0760(99)00132-6

Cited by 40 publications

(29 citation statements)

References 34 publications

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“…It is also noteworthy that in 1957, Streptomyces roseochromogenus was reported to hydroxylate two steroids (9␣-fluorohydrocortisone and 9␣-fluoroprednisolone) at the 16␣ position of the D ring (23), suggesting that S. roseochromogenus has a P450 that catalyzes a reaction similar to that of CYP154C5 of N. francinica. In fact, Berrie et al purified and characterized this P450 enzyme (24). Very recently, Von et al reported on the substrate screening of two CYP154 enzymes, CYP154E1 of T. fusca and CYP154A8 of N. francinica (25).…”

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Regio- and Stereospecific Hydroxylation of Various Steroids at the 16α Position of the D Ring by the Streptomyces griseus Cytochrome P450 CYP154C3

Makino

Katsuyama

Otomatsu³

et al. 2014

Appl Environ Microbiol

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c Cytochrome P450 monooxygenases (P450s), which constitute a superfamily of heme-containing proteins, catalyze the direct oxidation of a variety of compounds in a regio-and stereospecific manner; therefore, they are promising catalysts for use in the oxyfunctionalization of chemicals. In the course of our comprehensive substrate screening for all 27 putative P450s encoded by the Streptomyces griseus genome, we found that Escherichia coli cells producing an S. griseus P450 (CYP154C3), which was fused C terminally with the P450 reductase domain (RED) of a self-sufficient P450 from Rhodococcus sp., could transform various steroids (testosterone, progesterone, ⌬ 4 -androstene-3,17-dione, adrenosterone, 1,4-androstadiene-3,17-dione, dehydroepiandrosterone, 4-pregnane-3,11,20-trione, and deoxycorticosterone) into their 16␣-hydroxy derivatives as determined by nuclear magnetic resonance and high-resolution mass spectrometry analyses. The purified CYP154C3, which was not fused with RED, also catalyzed the regio-and stereospecific hydroxylation of these steroids at the same position with the aid of ferredoxin and ferredoxin reductase from spinach. The apparent equilibrium dissociation constant (K d ) values of the binding between CYP154C3 and these steroids were less than 8 M as determined by the heme spectral change, indicating that CYP154C3 strongly binds to these steroids. Furthermore, kinetic parameters of the CYP154C3-catalyzed hydroxylation of ⌬ 4 -androstene-3,17-dione were determined (K m , 31.9 ؎ 9.1 M; k cat , 181 ؎ 4.5 s ؊1 ). We concluded that CYP154C3 is a steroid D-ring 16␣-specific hydroxylase which has considerable potential for industrial applications. This is the first detailed enzymatic characterization of a P450 enzyme that has a steroid D-ring 16␣-specific hydroxylation activity. C ytochrome P450 monooxygenases (P450s or CYPs) are found in all three domains of life, i.e., archaea, bacteria, and eukarya, and are classified into more than 1,000 families by their amino acid sequence homology (1). They have the ability to hydroxylate inactive carbons of hydrocarbons or aromatic compounds regioand stereospecifically (2, 3). In animals, P450s are mainly involved in xenobiotic metabolism and steroid biosynthesis (4, 5). In plants, many P450s are involved in the biosynthesis of plant hormones and secondary metabolites (6). In contrast, the functions of bacterial P450s are largely unknown, although some bacterial P450s are required for secondary metabolite biosynthesis and assimilation of terpenoids, such as cineol or terpineol (7-10). In spite of their unknown physiological functions, however, bacterial P450s have attracted much attention as a rich resource for new biocatalysts for use in the oxyfunctionalization of chemicals (11, 12). For example, microbial conversion of steroid precursors is widely used for large-scale synthesis of steroid drugs (13-17).CYP154 family members are widely found in actinomycetes, and many putative CYP154 genes have been found in the genomes of Streptomyces species. The structu...

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Regio- and Stereospecific Hydroxylation of Various Steroids at the 16α Position of the D Ring by the Streptomyces griseus Cytochrome P450 CYP154C3

Makino

Katsuyama

Otomatsu³

et al. 2014

Appl Environ Microbiol

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“…We tested (screened) 80 actinomycetes strains, known for their ability to produce various second metabolites and transform various compounds, to convert 1 by microbial conversion and found Amycolatopsis azurea JCM3275 [6] hydroxylate 4-Me to give 2 and Amycolatopsis azurea JCM3275 [5], Streptomyces galiaeus ATCC31649 [7], Saccharothrix aerocolonigenes ATCC39243 [8], Streptomyces capreolus ATCC31963 [9], Streptomyces remosus JCM4073 [10], Streptomyces roseochromogenes NCIMB10984 [11], Actinoplanes utahensis IFO13244 [12] and Nonomuraea reseoviolacea JCM3145 [13] deacetylate to yield 3 ( Table 1). We tested whether the cell extract of 3T3-L1 fibroblasts can convert 1 to 2 or 3, but 1 was not converted.…”

Section: Resultsmentioning

confidence: 99%

FR177391, A New Anti-hyperlipidemic Agent from Serratia

et al. 2005

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FR177391 produced by Serratia liquefaciensNo. 1821 enhances differentiation of mouse 3T3-L1 fibroblasts to adipocytes and reduces the circulating levels of triglyceride in C57BL/KsJ-db/bd mice, an obese noninsulin-dependent diabetes mellitus animal model, although its mechanism of actions remained to be unknown. Its active derivative, 20-hydroxy FR177391, and its inactive derivative, 3-hydroxy FR177391 were produced by microbial conversion of FR177391, and biotin-labeled FR177391 was synthesized from 20-hydroxy FR177391 as an active affinity ligand to identify target molecules of FR177391 by chemical genetic approaches.

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“…However, in most cases, the enzyme responsible for catalysis is not known yet. Only very few steroid hydroxylating bacteria have been described so far, among them Streptomyces roseochromogenes (CYP163A2) [12], Streptomyces griseus (CYP105D1) [13] and Arthrobacter simplex [14]. On the other hand, multistep combinations of chemical and biochemical processes are used nowadays in the industrial production of steroids [15].…”

Section: Introductionmentioning

confidence: 99%

Function and engineering of the 15β-hydroxylase CYP106A2

Virus

Lisurek

Simgen

et al. 2006

Biochemical Society Transactions

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CYP106A2 from Bacillus megaterium ATCC 13368 is a bacterial cytochrome P450 that is capable of transforming steroid hormones. It can be easily expressed in Escherichia coli with a high yield. Its activity in vitro can be achieved by using the adrenal redox proteins adrenodoxin and adrenodoxin reductase. So far, it was not possible to crystallize CYP106A2 because of degradation during the crystallization process. Nevertheless, CYP106A2 is an interesting enzyme for biotechnological use. It hydroxylates pharmaceutically important steroids such as progesterone and 11-deoxycortisol. However, it will be necessary for efficient application of CYP106A2 in biotechnology to improve the hydroxylation activity and manipulate the regiospecificity. The present paper gives an overview of recent developments in protein engineering of CYP106A2.

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Microbial transformations of steroids-XI. Progesterone transformation by Streptomyces roseochromogenes–purification and characterisation of the 16α-hydroxylase system

Cited by 40 publications

References 34 publications

Regio- and Stereospecific Hydroxylation of Various Steroids at the 16α Position of the D Ring by the Streptomyces griseus Cytochrome P450 CYP154C3

Regio- and Stereospecific Hydroxylation of Various Steroids at the 16α Position of the D Ring by the Streptomyces griseus Cytochrome P450 CYP154C3

FR177391, A New Anti-hyperlipidemic Agent from Serratia

Function and engineering of the 15β-hydroxylase CYP106A2

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