Tigloyl-CoA:Pseudotropine (cas 135-97-7) acyl transferase--an enzyme of tropane alkaloid biosynthesis

Add time:10/01/2019    Source:infona.pl

Tigloyl-CoA:pseudotropine acyl transferase esterifies the 3β-hydroxyl group of pseudotropine (3β-hydroxytropane) with the tiglic acid moiety of tigloyl-Coenzyme A. This enzyme was purified to near electrophoretic homogeneity--about 330-fold--from transformed root cultures of Datura stramonium. The protocol involved the sequential use of ammonium sulphate precipitation, hydrophobic interaction, anion-exchange, chromatofocusing and gel filtration chromatography. The enzyme has a M r of 65 000, as determined by gel filtration chromatography on Sepharose 6 and SDS-PAGE electrophoresis, indicating it to be active as a monomer. Maximal activity occurs at pH 9.0 but the enzyme is still about 30% active at pH 7.0. The purified protein shows simple Michaelis-Menten kinetic behaviour, with K m values of 0.36 and 1.31 mM for pseudotropine and tigloyl-CoA, respectively. The enzyme is specific for the acyl group receptor. Of a range of potential acceptors tested, only pseudotropine and 4-hydroxy-1-methylpiperidine (14%) were used: tropine (3α-hydroxytropane) and norpseudotropine were not acylated. In contrast, the enzyme possesses the ability to transfer the acyl moiety to pseudotropine from a wide range of aliphatic acyl-CoA thioesters. Tigloyl-CoA and acetyl-CoA act reciprocally as competitive inhibitors, suggesting that they compete for the same active site on the enzyme. With acetyl-CoA the K m is 0.33 mM, indicating that the enzyme has a higher affinity for this acyl donor than for tigloyl-CoA. Neither CoA nor any of the alkamine acceptors tested were able to inhibit the acylation of pseudotropine.

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