Oppermann U C, Filling C, Berndt K D, Persson B, Benach J, Ladenstein R, Jörnvall H
Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
Biochemistry. 1997 Jan 7;36(1):34-40. doi: 10.1021/bi961803v.
Mutagenetic replacements of conserved residues within the active site of the short-chain dehydrogenase/reductase (SDR) superfamily were studied using prokaryotic 3 beta/17 beta-hydroxysteroid dehydrogenase (3 beta/17 beta-HSD) from Comamonas testosteroni as a model system. The results provide novel data to establish Ser 138 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 beta A, beta D, 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 3 beta-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 3 alpha/20 beta-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 beta D 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 beta D and turn beta A to alpha B.
以睾丸酮丛毛单胞菌的原核3β/17β-羟基类固醇脱氢酶（3β/17β-HSD）作为模型系统，研究了短链脱氢酶/还原酶（SDR）超家族活性位点内保守残基的诱变替代。研究结果提供了新的数据，确定Ser 138是一个催化重要的“三联体”残基的成员，该三联体还涉及Tyr151和Lys155。138位的Ser→Ala交换导致酶活性几乎完全丧失（>99.9%），而Ser→Thr替代则未观察到这种情况。这表明催化的一个关键因素是138位侧链形成氢键相互作用的能力。NAD(H)结合区域（βA、βD链及相邻转角）的突变揭示了另外两个残基Thr12和Asn87，它们对辅酶的正确结合很重要，且对催化反应有不同影响。因此，Thr12突变为Ala会导致3β-脱氢酶活性完全丧失，而3-氧还原酶活性保持不变。另一方面，T12S替代产生的蛋白质对两种反应的催化常数均未改变，这表明特定的氢键对脱氢酶活性至关重要。我们对氢化链霉菌3α/20β-HSD现有晶体结构的解读表明，该酶中Thr12侧链与Asn87的主链NH之间存在氢键，而非与辅酶之间，这表明与βD链的这种氢键可能决定了还原型和氧化型反应类型之间的关键差异。同样，Asn87突变为Ala会导致脱氢酶方向的kcat/Km降低80%，但3-氧还原酶特性不变。看来NAD+与蛋白质的结合受到涉及βD链和βA到αB转角的局部结构变化的影响。
