Reshetnyak Andrey V, Armentano Maria Francesca, Ponomarenko Natalia A, Vizzuso Domenica, Durova Oxana M, Ziganshin Rustam, Serebryakova Marina, Govorun Vadim, Gololobov Gennady, Morse Herbert C, Friboulet Alain, Makker Sudesh P, Gabibov Alexander G, Tramontano Alfonso
Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry RAS, 16/10, Miklukho-Maklaya str, Moscow, 117871, Russia.
J Am Chem Soc. 2007 Dec 26;129(51):16175-82. doi: 10.1021/ja076528m. Epub 2007 Nov 29.
Reactivity-based selection strategies have been used to enrich combinatorial libraries for encoded biocatalysts having revised substrate specificity or altered catalytic activity. This approach can also assist in artificial evolution of enzyme catalysis from protein templates without bias for predefined catalytic sites. The prevalence of covalent intermediates in enzymatic mechanisms suggests the universal utility of the covalent complex as the basis for selection. Covalent selection by phosphonate ester exchange was applied to a phage display library of antibody variable fragments (scFv) to sample the scope and mechanism of chemical reactivity in a naive molecular library. Selected scFv segregated into structurally related covalent and noncovalent binders. Clones that reacted covalently utilized tyrosine residues exclusively as the nucleophile. Two motifs were identified by structural analysis, recruiting distinct Tyr residues of the light chain. Most clones employed Tyr32 in CDR-L1, whereas a unique clone (A.17) reacted at Tyr36 in FR-L2. Enhanced phosphonylation kinetics and modest amidase activity of A.17 suggested a primitive catalytic site. Covalent selection may thus provide access to protein molecules that approximate an early apparatus for covalent catalysis.
基于反应性的筛选策略已被用于富集组合文库，以获取具有改变的底物特异性或催化活性的编码生物催化剂。这种方法还可以辅助从蛋白质模板进行酶催化的人工进化，而不会偏向于预定义的催化位点。酶促机制中共价中间体的普遍存在表明共价复合物作为筛选基础具有广泛的实用性。通过膦酸酯交换进行的共价筛选应用于抗体可变片段（单链抗体）的噬菌体展示文库，以探究天然分子文库中化学反应性的范围和机制。筛选出的单链抗体分为结构相关的共价结合剂和非共价结合剂。共价反应的克隆仅将酪氨酸残基用作亲核试剂。通过结构分析鉴定出两个基序，它们招募轻链中不同的酪氨酸残基。大多数克隆在互补决定区轻链1（CDR-L1）中使用酪氨酸32，而一个独特的克隆（A.17）在轻链框架区2（FR-L2）中的酪氨酸36处发生反应。A.17增强的膦酰化动力学和适度的酰胺酶活性表明存在一个原始的催化位点。因此，共价筛选可能提供获取近似早期共价催化装置的蛋白质分子的途径。
