Nicotinamide adenine dinucleotide phosphate-dependent 7 alpha-hydroxysteroid dehydrogenase (7 alpha-HSDH) and 7 beta-hydroxysteroid dehydrogenases (7 beta-HSDH) from Clostridium absonum catalyze the epimerization of primary bile acids through 7-keto bile acid intermediates and may be suitable as biocatalysts for the synthesis of bile acids derivatives of pharmacological interest. C. absonum 7 alpha-HSDH has been purified to homogeneity and the N-terminal sequence has been determined by Edman sequencing. After PCR amplifications of a gene fragment with degenerate primers, cloning of the complete gene (786 nt) has been achieved by sequencing of C. absonum genomic DNA. The sequence coding for the 7 beta-HSDH (783 nt) has been obtained by sequencing of the genomic DNA region flanking the 5' termini of 7 alpha-HSDH gene, the two genes being contiguous and presumably part of the same operon. After insertion in suitable expression vectors, both HSDHs have been successfully produced in recombinant form in Escherichia coli, purified by affinity chromatography and submitted to kinetic analysis for determination of Michaelis constants (K (m)) and specificity constants (k (cat)/K (m)) in the presence of various bile acids derivatives. Both enzymes showed a very strong substrate inhibition with all the tested substrates. The lowest K (S) values were observed with chenodeoxycholic acid and 12-ketochenodeoxycholic acid as substrates in the case of 7 alpha-HSDH, whereas ursocholic acid was the most effective inhibitor of 7 beta-HSDH activity.
In search of sustainable chemical processes: cloning, recombinant expression, and functional characterization of the 7 alpha- and 7 beta-hydroxysteroid dehydrogenases from Clostridium absonum
Ferrandi Erica Elisa;Riva Sergio;Monti Daniela
2012
Abstract
Nicotinamide adenine dinucleotide phosphate-dependent 7 alpha-hydroxysteroid dehydrogenase (7 alpha-HSDH) and 7 beta-hydroxysteroid dehydrogenases (7 beta-HSDH) from Clostridium absonum catalyze the epimerization of primary bile acids through 7-keto bile acid intermediates and may be suitable as biocatalysts for the synthesis of bile acids derivatives of pharmacological interest. C. absonum 7 alpha-HSDH has been purified to homogeneity and the N-terminal sequence has been determined by Edman sequencing. After PCR amplifications of a gene fragment with degenerate primers, cloning of the complete gene (786 nt) has been achieved by sequencing of C. absonum genomic DNA. The sequence coding for the 7 beta-HSDH (783 nt) has been obtained by sequencing of the genomic DNA region flanking the 5' termini of 7 alpha-HSDH gene, the two genes being contiguous and presumably part of the same operon. After insertion in suitable expression vectors, both HSDHs have been successfully produced in recombinant form in Escherichia coli, purified by affinity chromatography and submitted to kinetic analysis for determination of Michaelis constants (K (m)) and specificity constants (k (cat)/K (m)) in the presence of various bile acids derivatives. Both enzymes showed a very strong substrate inhibition with all the tested substrates. The lowest K (S) values were observed with chenodeoxycholic acid and 12-ketochenodeoxycholic acid as substrates in the case of 7 alpha-HSDH, whereas ursocholic acid was the most effective inhibitor of 7 beta-HSDH activity.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
