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首页> 外文期刊>Biochemistry >Altering Substrate Specificity of Phosphatidylcholine-Preferring Phospholipase C of Bacillus cereus by Random Mutagenesis of the Headgroup Binding Site.
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Altering Substrate Specificity of Phosphatidylcholine-Preferring Phospholipase C of Bacillus cereus by Random Mutagenesis of the Headgroup Binding Site.

机译：通过头基团结合位点的随机诱变来改变蜡样芽胞杆菌首选磷脂酰胆碱的磷脂酶C的底物特异性。

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摘要

PLC(Bc)() is a 28.5 kDa monomeric enzyme that catalyzes the hydrolysis of the phosphodiester bond of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine to provide a diacylglycerol and the corresponding phosphorylated headgroup. Because single replacements of Glu4, Tyr56, and Phe66 in the headgroup binding pocket led to changes in substrate specificity [Martin et al. (2000) Biochemistry 39, 3410-3415], a combinatorial library of approximately 6000 maltose binding protein-PLC(Bc)() fusion protein mutants containing random permutations of these three residues was generated to identify PLC(Bc)() mutants with altered specificity profiles and high catalytic activities. Members of this library were screened for hydrolytic activity toward the water soluble substrates C6PC, C6PE, and C6PS using a novel protocol that was conducted in a 96-well format and featured the in situ cleavage of the fusion protein to release the mutant PLC(Bc)()s. Ten mutant enzymes that exhibited significant preferences toward C6PE or C6PS were selected and analyzed by steady-state kinetics to determine their specificity constants, k(cat)/K(M). The C6PS selective clones E4G, E4Q/Y56T/F66Y, and E4K/Y56V exhibited higher specificity constants toward C6PS than wt, whereas Y56T, F66Y, and Y56T/F66Y were C6PE selective and had comparable or higher specificity constants than wt for C6PE. The corresponding wt residues were singly reinserted back into the E4Q/Y56T/F66Y and E4K/Y56V mutants via site-directed mutagenesis, and the E4Q/F66Y mutant thus obtained exhibited a 10-fold higher specificity constant toward C6PS than wt, a value significantly higher than other PLC(Bc)() mutants. On the basis of available data, an aromatic residue at position 66 appears important for significant catalytic activity toward all three substrates, especially C6PC and C6PE. The charge of residue 4 also appears to be a determinant of enzyme specificity as a negatively charged residue at this position endows the enzyme with C6PC and C6PE preference, whereas a polar neutral or positively charged residue results in C6PS selectivity. Replacing Tyr56 with Val, Ala, Thr, or Ser greatly reduces activity toward C6PC. Thus, the substrate specificity of PLC(Bc)() can be modulated by varying three of the amino acid residues that constitute the headgroup binding pocket, and it is now apparent that this enzyme is not evolutionarily optimized to hydrolyze phospholipids with ethanolamine or serine headgroups.
机译：PLC（Bc）（）是一种28.5 kDa的单体酶，可催化磷脂酰胆碱，磷脂酰乙醇胺和磷脂酰丝氨酸的磷酸二酯键水解，从而提供二酰基甘油和相应的磷酸化头基。因为在头基结合袋中单次替换Glu4，Tyr56和Phe66会导致底物特异性的改变[Martin等。 （2000）Biochemistry 39，3410-3415]，产生了包含这三个残基的随机排列的大约6000个麦芽糖结合蛋白-PLC（Bc）（）融合蛋白突变体的组合文库，以鉴定具有改变的PLC（Bc）（）突变体特异性和高催化活性。使用以96孔格式进行的新颖方案筛选该文库中成员对水溶性底物C6PC，C6PE和C6PS的水解活性，并以融合蛋白原位裂解为特征来释放突变型PLC（Bc ）（）。选择十种对C6PE或C6PS表现出显着偏好的突变酶，并通过稳态动力学进行分析，以确定其特异性常数k（cat）/ K（M）。 C6PS选择性克隆E4G，E4Q / Y56T / F66Y和E4K / Y56V对C6PS表现出比wt高的特异性常数，而Y56T，F66Y和Y56T / F66Y具有C6PE选择性，并且具有比wt更高或更高的C6PE特异性常数。通过定点诱变将相应的wt残基单独重新插入E4Q / Y56T / F66Y和E4K / Y56V突变体中，如此获得的E4Q / F66Y突变体对C6PS的特异性常数比wt高10倍，这一值非常明显高于其他PLC（Bc）（）突变体。根据可获得的数据，对于对于所有三种底物，特别是C6PC和C6PE而言，显着的催化活性，66位的芳族残基显得很重要。残基4的电荷也似乎是酶特异性的决定因素，因为在此位置带负电荷的残基使酶具有C6PC和C6PE偏好，而极性中性或带正电荷的残基导致C6PS选择性。用Val，Ala，Thr或Ser取代Tyr56大大降低了对C6PC的活性。因此，PLC（Bc）（）的底物特异性可以通过改变构成头基结合袋的三个氨基酸残基来调节，现在很明显，该酶没有经过进化优化以水解乙醇胺或丝氨酸头基的磷脂。 。

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