ACTIVE-SITE-DIRECTED MUTAGENESIS OF 3-BETA 17-BETA-HYDROXYSTEROID DEHYDROGENASE ESTABLISHES DIFFERENTIAL-EFFECTS ON SHORT-CHAIN DEHYDROGENASE/REDUCTASE REACTIONS/

Mutagenetic replacements uf conserved residues within the active site of the short-chain dehydrogenase/reductase (SDR) superfamily were stud ied using prokaryotic 3 beta/17 beta-hydroxysteroid dehydrogenase (3 b eta/17 beta-HSD) from Comamonas testosteroni as a model system. The re sults provide novel data to establish Ser138 as a member of a catalyti cally important ''triad'' of residues also involving Tyr151 and Lys155 . A Ser --> Ala exchange at position 138 results in an almost complete (>99.9%) loss of enzymatic activity, which is not observed with a Ser --> Thr replacement. This indicates that an essential factor for cata lysis is the ability of side chain 138 to form hydrogen bond interacti ons. Mutations in the NAD(H) binding region, in strands beta A, beta D , and adjacent turns, reveal two additional residues, Thr12 and Asn87, which are important for correct binding of the coenzyme aad with a di fferential effect on the reactions catalyzed. Thus, mutation of Thr12 to Ala results in a complete loss of the 3 beta-dehydrogenase activity , whereas the 3-oxoreductase activity remains unchanged. On the other hand, a T12S substitution yields a protein with unaltered catalytic co nstants for both reactions, revealing that a specific hydrogen bond is critical for the dehydrogenase activity. Our interpretation of the av ailable crystal structure of 3 alpha/20 beta-HSD from Streptomyces hyd rogenans suggests a hydrogen her-id in that enzyme between the Thr12 s ide chain and the backbone NW of Asn87 rather than the coenzyme, indic ating that this hydrogen bond to the beta D strand might determine a c rucial difference between the reductive and the oxidative reaction typ es. Similarly, mutation of Asn87 to Ala results in an 80% reduction of K-cat/K-m in the dehydrogenase direction but also unchanged 3-oxoredu ctase propel ties. It appears that the binding of NAD(+) to the protei n is influenced by local structural changes involving strand beta D an d beta A to alpha B.