Studying the Role of a Single Mutation of a Family 11 Glycoside Hydrolase Using High-Resolution X-ray Crystallography

XynII is a family 11 glycoside hydrolase that uses the retaining mechanism for catalysis. In the active site, E177 works as the acid/base and E86 works as the nucleophile. Mutating an uncharged residue (N44) to an acidic residue (D) near E177 decreases the enzyme’s optimal pH by ~ 1.0 unit. D44 was previously suggested to be a second proton carrier for catalysis. To test this hypothesis, we abolished the activity of E177 by mutating it to be Q, and mutated N44 to be D or E. These double mutants have dramatically decreased activities. Our high-resolution crystallographic structures and the microscopic.pKa calculations show that D44 has similar position and pKa value during catalysis, indicating that D44 changes electrostatics around E177, which makes it prone to rotate as the acid/base in acidic conditions, thus decreases the pH optimum. Our results could be helpful to design enzymes with different pH optimum.

木聚糖酶II（XynII）是一种第11家族糖苷水解酶，它采用保留机制进行催化。在活性位点，E177作为酸/碱，E86作为亲核试剂。将靠近E177的一个不带电残基（N44）突变为酸性残基（D）会使酶的最适pH降低约1.0个单位。先前有人认为D44是催化过程中的第二个质子载体。为了验证这一假设，我们通过将E177突变为Q使其失活，并将N44突变为D或E。这些双突变体的活性显著降低。我们的高分辨率晶体结构和微观pKa计算表明，D44在催化过程中具有相似的位置和pKa值，这表明D44改变了E177周围的静电环境，使其在酸性条件下更容易作为酸/碱发生旋转，从而降低了最适pH。我们的研究结果可能有助于设计具有不同最适pH的酶。