Wymer N, Buchanan L V, Henderson D, Mehta N, Botting C H, Pocivavsek L, Fierke C A, Toone E J, Naismith J H
Department of Chemistry, LSRC, Duke University, Durham, NC 27708, USA.
Structure. 2001 Jan 10;9(1):1-9. doi: 10.1016/s0969-2126(00)00555-4.
Aldolases are carbon bond-forming enzymes that have long been identified as useful tools for the organic chemist. However, their utility is limited in part by their narrow substrate utilization. Site-directed mutagenesis of various enzymes to alter their specificity has been performed for many years, typically without the desired effect. More recently directed evolution has been employed to engineer new activities onto existing scaffoldings. This approach allows random mutation of the gene and then selects for fitness to purpose those proteins with the desired activity. To date such approaches have furnished novel activities through multiple mutations of residues involved in recognition; in no instance has a key catalytic residue been altered while activity is retained.
We report a double mutant of E. coli 2-keto-3-deoxy-6-phosphogluconate aldolase with reduced but measurable enzyme activity and a synthetically useful substrate profile. The mutant was identified from directed-evolution experiments. Modification of substrate specificity is achieved by altering the position of the active site lysine from one beta strand to a neighboring strand rather than by modification of the substrate recognition site. The new enzyme is different to all other existing aldolases with respect to the location of its active site to secondary structure. The new enzyme still displays enantiofacial discrimination during aldol addition. We have determined the crystal structure of the wild-type enzyme (by multiple wavelength methods) to 2.17 A and the double mutant enzyme to 2.7 A resolution.
These results suggest that the scope of directed evolution is substantially larger than previously envisioned in that it is possible to perturb the active site residues themselves as well as surrounding loops to alter specificity. The structure of the double mutant shows how catalytic competency is maintained despite spatial reorganization of the active site with respect to substrate.
醛缩酶是形成碳键的酶，长期以来一直被认为是有机化学家的有用工具。然而，它们的效用在一定程度上受到底物利用范围狭窄的限制。多年来，人们对各种酶进行定点诱变以改变其特异性，但通常没有达到预期效果。最近，定向进化已被用于在现有支架上设计新的活性。这种方法允许对基因进行随机突变，然后选择具有所需活性的那些蛋白质以适应特定目的。迄今为止，此类方法通过涉及识别的残基的多个突变提供了新的活性；在任何情况下，关键催化残基都没有被改变而活性得以保留。
我们报道了一种大肠杆菌2-酮-3-脱氧-6-磷酸葡萄糖酸醛缩酶的双突变体，其酶活性降低但仍可测量，并且具有合成上有用的底物谱。该突变体是从定向进化实验中鉴定出来的。底物特异性的改变是通过将活性位点赖氨酸的位置从一条β链改变到相邻链，而不是通过修饰底物识别位点来实现的。就活性位点相对于二级结构的位置而言，这种新酶与所有其他现有的醛缩酶都不同。这种新酶在醛醇加成过程中仍然表现出对映面选择性。我们已经通过多波长方法将野生型酶的晶体结构解析到2.17 Å，将双突变体酶的晶体结构解析到2.7 Å分辨率。
这些结果表明，定向进化的范围比以前设想的要大得多，因为有可能扰动活性位点残基本身以及周围的环来改变特异性。双突变体的结构显示了尽管活性位点相对于底物发生了空间重组，但催化能力是如何得以维持的。
