Moretti Rocco, Thorson Jon S
Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706, USA.
J Biol Chem. 2007 Jun 8;282(23):16942-7. doi: 10.1074/jbc.M701951200. Epub 2007 Apr 12.
Nucleotidyltransferases are central to nearly all glycosylation-dependent processes and have been used extensively for the chemoenzymatic synthesis of sugar nucleotides. The determination of the NTP specificity of the model thymidylyltransferase RmlA revealed RmlA to utilize all eight naturally occurring NTPs with varying levels of catalytic efficiency, even in the presence of nonnative sugar-1-phosphates. Guided by structural models, active site engineering of RmlA led to alterations of the inherent pyrimidine/purine bias by up to three orders of magnitude. This study sets the stage for engineering single universal nucleotidyltransferases and also provides new catalysts for the synthesis of novel nucleotide diphosphosugars.
核苷酸转移酶几乎参与了所有依赖糖基化的过程，并已被广泛用于糖核苷酸的化学酶法合成。对模型胸苷酸转移酶RmlA的NTP特异性的测定表明，即使存在非天然糖-1-磷酸，RmlA也能以不同的催化效率利用所有八种天然存在的NTP。在结构模型的指导下，对RmlA的活性位点进行工程改造，使固有的嘧啶/嘌呤偏好性改变了多达三个数量级。这项研究为工程化单一通用核苷酸转移酶奠定了基础，也为新型核苷二磷酸糖的合成提供了新的催化剂。
