Stabilization of steroid 11-hydroxylation activity ofCunninghamella elegans protoplasts in organic osmotic stabilizers

Protoplasts ofCunninghamella elegans, showing 11-, and 11-hydroxylating ability of Substance S, preserved high transformation activity when dispersed in glucose-enriched, organic osmotic stabilizers. A joint action of polyoxins and 2-deoxy-d-glucose was necessary to prevent regeneration of the cell wall in long-lasting experiments. Stabilized and active, dispersed protoplasts may be an alternative research model for studying the function of the cell wall and intracellular metabolic pool constituents in steroid hydroxylation.     

Esterification Rates of Main Organic Acids in Wines

The esterification rates (ratio of the concentration of an acid in the neutral ethyl ester form to total concentration of the acid) of main organic acids in wines were determined to study the extent of ethyl ester formation of organic acids. The esterification rates ranged from zero to 24.6%. The averaged values of table wines were from 6 to 16% for acetic and lactic acid, from 0.3 to 3.6% for succinic and malic acid, and from zero to 0.1% for tartaric acid. Sherries had higher esterification rates, about 1.6 to 6 times larger, than table wines. It was found that storage time and temperature influence the formation of ethyl esters, and it was suggested that the aging period required for the ester equilibrium is about one year for acetic and lactic acid, and more than two years for succinic, malic and tartaric acid. The possibility and the procedure to control wine quality during the aging process were discussed.     

Microbial hydroxylation of progesterone with Acremonium strictum

Microbial hydroxylation of progesterone occurred in the culture of Acremonium strictum PTCC 5282 to produce two hydroxylated pregnene-like steroids. The metabolites were purified and characterized using spectroscopic methods and identified as 15alpha-hydroxyprogesterone and 15alpha-hydroxydeoxycorticosterone.     

Biotransformation of diosgenin to nuatigenin-type steroid by a newly isolated strain, <Emphasis Type="Italic">Streptomyces virginiae</Emphasis> IBL-14

An actinomycete strain IBL-14 isolated from soil by utilizing diosgenin as the sole carbon and energy source was identified as Streptomyces virginiae. S. virginiae IBL-14 can transform diosgenin to isonuatigenone. To our knowledge, this is the first reported case of producing rare nuatigenin-type spirosteroid (isonuatigenone) from pyrano-spirosteroid (diosgenin) by microbial transformation. From diosgenin to isonuatigenone, the pathway has been confirmed in this study that diosgenin was first converted to diosgenone, and then diosgenone was transformed to isonuatigenone by the C25 tertiary hydroxylation reaction. It appeared to be favorable to accumulate isonuatigenone when diosgenin was added to the onset of the stationary phase of cell growth, and the yield of isonuatigenone was about 28.4% during 48 h from 1.5 mM diosgenin. The C25 tertiary hydroxylation of diosgenone by S. virginiae IBL-14 is a novel and interesting reaction and will be a practical tool in producing natural nuatigenin-type steroids. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Hydroxylation of cinnamic acids and flavonoids during biosynthesis of anthocyanins in Petunia hybrida Hort.

The effect of hydroxylation genes on the hydroxylation of intermediates of flavonoid biosynthesis in Petunia hybrida is reported. In mutants homozygous recessive, for the gene An9, dihydroflavonols accumulate. The number of hydroxyl groups in the B-ring is determined by the hydroxylation genes Htl and Hfl. A similar effect of Htl and (probably) Hfl occurs in flavanone-accumulating mutants, homozygous recessive for the gene An3. Mutants dominant for Hfl probably accumulate a 5,7,3,4,5-pentahydroxyflavanone. The mutant W43, homozygous recessive for the gene An5, is blocked in an early flavonoid biosynthesis step. It accumulates p-coumaric acid together with caffeic acid. The hydroxylation genes Htl and Hfl, however, are also homozygous recessive, which indicates that the hydroxylation of p-coumaric acid to caffeic acid or derivatives of these compounds is not controlled by Htl. The accumulation of caffeic acid was observed in all mutants investigated so far, regardless of which hydroxylation genes were dominant or recessive. We conclude that hydroxylations involved in anthocyanin biosynthesis occur at the C15 level.Deceased     

Selective aliphatic and aromatic carbon-hydrogen bond activation catalysed by mutants of cytochrome p450cam

The substrate range of the haem monooxygenase cytochrome P450_cam (CYP101) has been broadened by site-directed mutagenesis. The hydroxylation selectivity of five mutants at the 96 position towards a range of substrates has been used to investigate P450_cam -substrate molecular recognition. The substrates contained aromatic and activated and unactivated aliphatic C---H bonds, as well as reactive functional groups. Diphenylmethane, diphenylether, diphenylamine, and 1,1-di-phenylethylene were all hydroxylated regiospecifically at the para position, with no attack at the amine or the olefinic double bond. With benzylcyclohexane the activated benzylic and tertiary C---H bonds were not attacked, and the reactions catalysed by the Y96G and Y96A mutants were highly diastereoselective, with 4-trans-benzylcyclohexanol constituting 90% of the products. 1-Phenyl-1-cyclohexylethylene was oxidised predominantly at the 4-position of the cyclohexane ring without attack at the olefinic double bond, and approximately equal amounts of cis- and trans-4-phenylethenylcyclohexanol were formed. These results show that P450_cam can be engineered to oxidise C---H bonds without attacking more reactive functional groups.     

The Receptor-Mediated Activation of Tyrosine Hydroxylation in the Superior Cervical Ganglion of the Rat

The addition of carbachol to superior cervical ganglia causes a rapid increase in tyrosine hydroxylation in situ. The increase occurs in ganglia from both newborn and adult animals, and in ganglia from animals pretreated with reserpine. The increase is not due to increased transport of the substrate. The increase is dependent upon the presence of calcium, and is additive to the stimulation produced by dibutyryl cyclic AMP. The stimulation seems specific for tyrosine hydroxylation; dopamine beta-hydroxylation is not increased. Preincubation experiments suggest that the carbachol-induced stimulation is due to a change in the availability of, or the affinity of the enzyme for, reduced pterin cofactor. The stimulation is inhibited by atropine and also by low concentrations of phenoxybenzamine or haloperidol, which suggests that it is caused by an action of carbachol on the interneurons in the ganglia.