A new enzyme, 1,2-dihydrovomilenine reductase (E.C. 1.3.1), has been detected in Rauvolfia cell suspension cultures. The enzyme specifically converts 2beta( R)-1,2-dihydrovomilenine through an NADPH-dependent reaction into 17-O-acetylnorajmaline, a close biosynthetic precursor of the antiarrhythmic alkaloid ajmaline from Rauvolfia. A five-step purification procedure using SOURCE 30Q chromatography, hydroxyapatite chromatography, 2',5'-ADP Sepharose 4B affinity chromatography and ion exchange chromatography on DEAE Sepharose and Mono Q delivered an approximately 200-fold enriched enzyme in a yield of approximately 6%. SDS-PAGE showed an M r for the enzyme of approximately 48 kDa. Optimum pH and optimum temperature of the reductase were at pH 6.0 and 37 degrees C. The enzyme shows a limited distribution in cell cultures expressing ajmaline biosynthesis, and is obviously highly specific for the ajmaline pathway.
Fifteen noncardioform bacteria strains, capable of transforming steroid compounds were investigated with regard to their range of inducible steroid-1-dehydrogenase (St1DH)1 activities. The St1DHs of these bacteria were compared due to their immuno reactivity in Western blot experiments with a rabbit antiserum raised against the purified St1DH of Rhodococcus rhodochrous 7030. Four strains exhibited a strong immuno reactivity, irrespective of differences in the electrophoretic mobility of the enzymes. Five strains revealed significantly diminished reactivities, and in five strains with a very low St1DH content, no reactivity was found. One strain, designated as Nocardiaspec. 7151, exhibited a high, inducible St1DH activity, but no immunoreaction was found. The absence of immuno reactivity is discussed in connection with the considerably diminished electrophoretic mobility of this enzyme.
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