el Hawrani A S, Sessions R B, Moreton K M, Holbrook J J
Molecular Recognition Centre, Department of Biochemistry, Bristol, UK.
J Mol Biol. 1996 Nov 22;264(1):97-110. doi: 10.1006/jmbi.1996.0626.
A gene library was constructed coding for all possible variants of two amino acids (101, 102) in a solvent-exposed surface return loop (alpha E-beta D) of Bacillus stearothermophilus L-lactate dehydrogenase (bsLDH). All but one of 38 enzyme variants examined were thermally stable and had native-like hydrodynamic properties. In this sample, there was no bias detected in either the DNA or amino acid sequences encoded. We argue that the alpha E-beta D surface loop sequence is unimportant for protein folding or stability and can be fully varied to select enzymes with new substrate specificities. The selection of NAD-dependent dehydrogenases with specificity for: malate, phenyllactate, hydroxyisocaproate and 4-phenyl-2-hydroxy-butanoate from two bsLDH libraries is described. This required a highly discriminatory screen for 2-hydroxy acid dehydrogenase activity to select enzymes which, in the absence of the natural allosteric activator fructose-1,6-bisphosphate (FBP), maintained high temperature stability and catalytic activity without substrate inhibition. In general the amino acid residues at positions 101 and 102 which determined substrate specificity were as expected from hydrophobic and ionic complementarity to the substrate. For example, a bsLDH variant with Asn101Va1102 is as efficient with phenylpyruvate as is the wild-type enzyme (Asn101Gln102) with pyruvate. Using molecular modelling, the valine at position 102 can be fitted into the active site without significant structural distortion caused by the aromatic side-chain of the substrate. Similarly, nine out of ten malate dehydrogenases (MDHs) selected had an arginine residue at position 102 to complement the negatively charged carboxyl group in oxaloacetate. One, Arg101Arg102 (Kcat/Kmoxaloacetate = 1.6 x 10(6) M-1 S-1) is 25% more active than the previous best synthetic MDH. There were surprises: present understanding would not have predicted the oxaloacetate transforming activity of Ser101Leu102 or the phenylpyruvate activity of Pro101Lys102. The former is about one-third as efficient as the best malate dehydrogenase selected, whilst the latter had about one-seventh of the best phenylpyruvate dehydrogenase activity.
构建了一个基因文库，其编码嗜热栖热芽孢杆菌L-乳酸脱氢酶（bsLDH）溶剂暴露表面回环（αE-βD）中两个氨基酸（101、102）的所有可能变体。所检测的38种酶变体中，除一种外均具有热稳定性且具有类似天然的流体动力学性质。在该样本中，未检测到编码的DNA或氨基酸序列存在偏差。我们认为，αE-βD表面环序列对蛋白质折叠或稳定性并不重要，可以完全改变以筛选具有新底物特异性的酶。本文描述了从两个bsLDH文库中筛选对苹果酸、苯乳酸、羟基异己酸和4-苯基-2-羟基丁酸具有特异性的NAD依赖性脱氢酶的过程。这需要对2-羟基酸脱氢酶活性进行高度区分性筛选，以选择在没有天然变构激活剂果糖-1,6-二磷酸（FBP）的情况下，仍能保持高温稳定性和催化活性且无底物抑制的酶。一般来说，决定底物特异性的101和102位氨基酸残基符合与底物的疏水和离子互补性预期。例如，Asn101Va1102的bsLDH变体对苯丙酮酸的催化效率与野生型酶（Asn101Gln102）对丙酮酸的催化效率相同。通过分子建模，102位的缬氨酸可以适配到活性位点，而不会因底物的芳香侧链导致明显的结构变形。同样，所筛选的十分之九的苹果酸脱氢酶（MDH）在102位有一个精氨酸残基，以与草酰乙酸中的带负电荷的羧基互补。其中一种，Arg101Arg102（Kcat/Km草酰乙酸 = 1.6×10⁶ M⁻¹ S⁻¹）的活性比之前最好的合成MDH高25%。也有意外发现：目前的认知无法预测Ser101Leu102对草酰乙酸的转化活性或Pro101Lys102对苯丙酮酸的活性。前者的效率约为所筛选出的最佳苹果酸脱氢酶的三分之一，而后者的苯丙酮酸脱氢酶活性约为最佳活性的七分之一。
