One-Pot Multienzymatic Synthesis of 12-Ketoursodeoxycholic Acid: Subtle Cofactor Specificities Rule the Reaction Equilibria of Five Biocatalysts Working in a Row

Received: November 21, 2008; Published online: March 17, 2009Dedicated to Prof. Karl “Kalle” Hult on the occasion of his 65th birthday.Abstract: The hydroxysteroid dehydrogenases(HSDHs)-catalyzed one-pot enzymatic synthesis of12-ketoursodeoxycholic acid (3a,7b-dihydroxy-12-oxo-5b-cholanoic acid), a key intermediate for thesynthesis of ursodeoxycholic acid, from cholic acidhas been investigated. This goal has been achievedby alternating oxidative and reductive steps in a one-pot system employing HSDHs with different cofactorspecificity, namely NADH-dependent HSDHs in theoxidative step and an NADPH-dependent 7b-HSDHin the reductive one. Coupled in situ regenerationsystems have been exploited not only to allow theuse of catalytic amounts of the cofactors, but also toprovide the necessary driving force to opposite reac-tions (i.e., oxidation and reduction) acting on differ-ent sites of the substrate molecule. Biocatalysts suita-ble for the set-up of this process have been selectedand their kinetic behaviour in respect of the reac-tions of interest has been evaluated. Finally, the pro-cess has been studied employing the enzymes both infree and compartmentalized form.Keywords: bile acids; hydroxysteroid dehydrogenas-es; one-pot reaction; oxido-reductions; regioselectiv-ity

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