Ostermeier M, Nixon A E, Shim J H, Benkovic S J
Department of Chemistry, The Pennsylvania State University, University Park, PA 16802-6300, USA.
Proc Natl Acad Sci U S A. 1999 Mar 30;96(7):3562-7. doi: 10.1073/pnas.96.7.3562.
We have developed a combinatorial approach, using incremental truncation libraries of overlapping N- and C-terminal gene fragments, that examines all possible bisection points within a given region of an enzyme that will allow the conversion of a monomeric enzyme into its functional heterodimer. This general method for enzyme bisection will have broad applications in the engineering of new catalytic functions through domain swapping and chemical synthesis of modified peptide fragments and in the study of enzyme evolution and protein folding. We have tested this methodology on Escherichia coli glycinamide ribonucleotide formyltransferase (PurN) and, by genetic selection, identified PurN heterodimers capable of glycinamide ribonucleotide transformylation. Two were chosen for physical characterization and were found to be comparable to the wild-type PurN monomer in terms of stability to denaturation, activity, and binding of substrate and cofactor. Sequence analysis of 18 randomly chosen, active PurN heterodimers revealed that the breakpoints primarily clustered in loops near the surface of the enzyme, that the breaks could result in the deletion of highly conserved residues and, most surprisingly, that the active site could be bisected.
我们开发了一种组合方法，利用重叠的N端和C端基因片段的递增截短文库，该方法可检测酶给定区域内所有可能的二分点，这些二分点能使单体酶转化为其功能性异二聚体。这种酶二分的通用方法在通过结构域交换和修饰肽片段的化学合成来设计新的催化功能以及酶进化和蛋白质折叠研究方面将有广泛应用。我们已在大肠杆菌甘氨酰胺核糖核苷酸甲酰基转移酶（PurN）上测试了该方法，并通过基因筛选鉴定出了能够进行甘氨酰胺核糖核苷酸甲酰化反应的PurN异二聚体。选择了两个进行物理特性分析，发现它们在对变性的稳定性、活性以及底物和辅因子的结合方面与野生型PurN单体相当。对18个随机选择的活性PurN异二聚体的序列分析表明，断点主要聚集在酶表面附近的环中，这些断点可能导致高度保守残基的缺失，最令人惊讶的是，活性位点可能被一分为二。
