RNase T1 Variant RV Cleaves Single-Stranded RNA after Purines Due to Specific Recognition by the Asn46 Side Chain Amide

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• 作者：Rico Czaja ; Marc Struhalla ; Katja Hö ; schler ; Wolfram Saenger ; Norbert Str&auml ; ter ; and Ulrich Hahn
• 刊名：Biochemistry
• 出版年：2004
• 出版时间：March 16, 2004
• 年：2004
• 卷：43
• 期：10
• 页码：2854 - 2862
• 全文大小：637K
• 年卷期：v.43,no.10(March 16, 2004)
• ISSN：1520-4995

文摘

Attempts to alter the guanine specificity of ribonuclease T1 (RNase T1) by rational or randommutagenesis have failed so far. The RNase T1 variant RV (Lys41Glu, Tyr42Phe, Asn43Arg, Tyr45Trp,and Glu46Asn) designed by combination of a random and a rational mutagenesis approach, however,exhibits a stronger preference toward adenosine residues than wild-type RNase T1. Steady state kineticsof the cleavage reaction of the two dinucleoside phosphate substrates adenylyl-3',5'-cytidine and guanylyl-3',5'-cytidine revealed that the ApC/GpC ratio of the specificity coefficient (k_cat/K_m) was increased ~7250-fold compared to that of the wild-type. The crystal structure of the nucleotide-free RV variant has beenrefined in space group P6₁ to a crystallographic R-factor of 19.9% at 1.7 Å resolution. The primaryrecognition site of the RV variant adopts a similar conformation as already known from crystal structuresof RNase T1 not complexed to any nucleotide. Noteworthy is a high flexibility of Trp45 and Asn46within the three individual molecules in the asymmetric unit. In addition to the kinetic studies, these dataindicate the participation of Asn46 in the specific recognition of the base and therefore a specific bindingof adenosine.