Oliynyk Z, Briseño-Roa L, Janowitz T, Sondergeld P, Fersht A R
Centre for Protein Engineering, MRC, Hills Road, Cambridge CB2 2QH, UK.
Protein Eng Des Sel. 2004 Apr;17(4):383-90. doi: 10.1093/protein/gzh046. Epub 2004 May 27.
KDO8PS (3-deoxy-d-manno-octulosonate-8-phosphate synthase) and DAH7PS (3-deoxy-d-arabino-heptulosonic acid-7-phosphate synthase) enzymes catalyse analogous condensation reactions between phosphoenolpyruvate and arabinose 5-phosphate or erythrose 4-phosphate, respectively. All known DAH7PS and some of KDO8PS enzymes (Aquifex aeolicus KDO8PS) require a metal ion for activity whereas another class of KDO8PS (including Escherichia coli KDO8PS) does not. Based on sequence alignment of all known KDO8PS and DAH7PS enzymes, we identified a single amino acid residue that might define the metal dependence of KDO8PS activity. One of the four metal-binding residues, a cysteine, is conserved only among metal-binding KDO8PS and DAH7PS enzymes and is replaced by an asparagine residue in other KDO8PS enzymes. We introduced a metal binding site into E.coli KDO8PS by a single N26C and a double M25P N26C mutation, which led to an increased k(cat) of the enzymes in the presence of activating Mn(2+) ions. The M25P N26C mutant of E.coli KDO8PS had a value of k(cat)/K(M) in the presence of Mn(2+) ions four times higher than A.aeolicus KDO8PS. KDO8PS and DAH7PS may have evolved from a common ancestor protein that required a divalent metal ion for activity. A non-metal-binding KDO8PSs may have evolved from an ancestor protein that was able to bind Mn(2+) but no longer required Mn(2+) to function and eventually lost one of metal-binding residues.
KDO8PS（3-脱氧-D-甘露糖辛酮酸-8-磷酸合酶）和DAH7PS（3-脱氧-D-阿拉伯庚酮糖酸-7-磷酸合酶）分别催化磷酸烯醇丙酮酸与5-磷酸阿拉伯糖或4-磷酸赤藓糖之间类似的缩合反应。所有已知的DAH7PS和部分KDO8PS酶（嗜泉古菌KDO8PS）的活性需要金属离子，而另一类KDO8PS（包括大肠杆菌KDO8PS）则不需要。基于所有已知KDO8PS和DAH7PS酶的序列比对，我们鉴定出一个可能决定KDO8PS活性对金属依赖性的单个氨基酸残基。四个金属结合残基之一的半胱氨酸，仅在金属结合型KDO8PS和DAH7PS酶中保守，在其他KDO8PS酶中被天冬酰胺残基取代。我们通过单个N26C和双突变M25P N26C在大肠杆菌KDO8PS中引入了一个金属结合位点，这导致在存在激活的Mn(2+)离子时酶的k(cat)增加。大肠杆菌KDO8PS的M25P N26C突变体在存在Mn(2+)离子时的k(cat)/K(M)值比嗜泉古菌KDO8PS高四倍。KDO8PS和DAH7PS可能从一个活性需要二价金属离子的共同祖先蛋白进化而来。非金属结合型KDO8PS可能从一个能够结合Mn(2+)但不再需要Mn(2+)发挥功能并最终失去一个金属结合残基的祖先蛋白进化而来。
