Tomschy A, Tessier M, Wyss M, Brugger R, Broger C, Schnoebelen L, van Loon A P, Pasamontes L
F. Hoffmann-La Roche Ltd, Basel, Switzerland.
Protein Sci. 2000 Jul;9(7):1304-11. doi: 10.1110/ps.9.7.1304.
Previously, we determined the DNA and amino acid sequences as well as biochemical and biophysical properties of a series of fungal phytases. The amino acid sequences displayed 49-68% identity between species, and the catalytic properties differed widely in terms of specific activity, substrate specificity, and pH optima. With the ultimate goal to combine the most favorable properties of all phytases in a single protein, we attempted, in the present investigation, to increase the specific activity of Aspergillus fumigatus phytase. The crystal structure of Aspergillus niger NRRL 3135 phytase known at 2.5 A resolution served to specify all active site residues. A multiple amino acid sequence alignment was then used to identify nonconserved active site residues that might correlate with a given favorable property of interest. Using this approach, Gln27 of A. fumigatus phytase (amino acid numbering according to A. niger phytase) was identified as likely to be involved in substrate binding and/or release and, possibly, to be responsible for the considerably lower specific activity (26.5 vs. 196 U x mg protein at pH 5.0) of A. fumigatus phytase when compared to Aspergillus terreus phytase, which has a Leu at the equivalent position. Site-directed mutagenesis of Gln27 of A. fumigatus phytase to Leu in fact increased the specific activity to 92.1 U x (mg protein)(-1), and this and other mutations at position 27 yielded an interesting array of pH activity profiles and substrate specificities. Analysis of computer models of enzyme-substrate complexes suggested that Gln27 of wild-type A. fumigatus phytase forms a hydrogen bond with the 6-phosphate group of myo-inositol hexakisphosphate, which is weakened or lost with the amino acid substitutions tested. If this hydrogen bond were indeed responsible for the differences in specific activity, this would suggest product release as the rate-limiting step of the A. fumigatus wild-type phytase reaction.
此前，我们测定了一系列真菌植酸酶的DNA和氨基酸序列以及生化和生物物理特性。不同物种间的氨基酸序列一致性为49%-68%，且在比活性、底物特异性和最适pH值方面，催化特性差异很大。为了将所有植酸酶最有利的特性整合到一种蛋白质中，我们在本研究中试图提高烟曲霉植酸酶的比活性。黑曲霉NRRL 3135植酸酶的晶体结构在2.5埃分辨率下已知，用于确定所有活性位点残基。然后通过多氨基酸序列比对来识别可能与特定有利特性相关的非保守活性位点残基。使用这种方法，烟曲霉植酸酶的Gln27（根据黑曲霉植酸酶的氨基酸编号）被确定可能参与底物结合和/或释放，并且可能是烟曲霉植酸酶与土曲霉植酸酶相比比活性显著较低（在pH 5.0时为26.5 U x mg蛋白质 对196 U x mg蛋白质）的原因，土曲霉植酸酶在等效位置为Leu。将烟曲霉植酸酶的Gln27定点突变为Leu实际上将比活性提高到了92.1 U x (mg蛋白质)(-1)，并且在第27位的这个突变和其他突变产生了一系列有趣的pH活性曲线和底物特异性。对酶-底物复合物计算机模型的分析表明，野生型烟曲霉植酸酶的Gln27与肌醇六磷酸的6-磷酸基团形成氢键，在所测试的氨基酸取代中该氢键减弱或消失。如果这个氢键确实是比活性差异的原因，这将表明产物释放是烟曲霉野生型植酸酶反应的限速步骤。
