Active Site Directed Mutagenesis of 3β/17β-Hydroxysteroid Dehydrogenase Establishes Differential Effects on Short-Chain Dehydrogenase/Reductase Reactions

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Abstract
Mutagenetic replacements of conserved residues within the active site of the short-chain dehydrogenase/reductase (SDR) superfamily were studied using prokaryotic 3β/17β-hydroxysteroid dehydrogenase (3β/17β-HSD) from Comamonas testosteroni as a model system. The results provide novel data to establish Ser138 as a member of a catalytically 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 catalysis is the ability of side chain 138 to form hydrogen bond interactions. Mutations in the NAD(H) binding region, in strands βA, βD, and adjacent turns, reveal two additional residues, Thr12 and Asn87, which are important for correct binding of the coenzyme and with a differential effect on the reactions catalyzed. Thus, mutation of Thr12 to Ala results in a complete loss of the 3β-dehydrogenase activity, whereas the 3-oxoreductase activity remains unchanged. On the other hand, a T12S substitution yields a protein with unaltered catalytic constants for both reactions, revealing that a specific hydrogen bond is critical for the dehydrogenase activity. Our interpretation of the available crystal structure of 3α/20β-HSD from Streptomyces hydrogenans suggests a hydrogen bond in that enzyme between the Thr12 side chain and the backbone NH of Asn87 rather than the coenzyme, indicating that this hydrogen bond to the βD strand might determine a crucial difference between the reductive and the oxidative reaction types. Similarly, mutation of Asn87 to Ala results in an 80% reduction of kcat/Km in the dehydrogenase direction but also unchanged 3-oxoreductase properties. It appears that the binding of NAD+ to the protein is influenced by local structural changes involving strand βD and turn βA to αB.

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