1q9e
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(New page: 200px<br /><applet load="1q9e" size="450" color="white" frame="true" align="right" spinBox="true" caption="1q9e, resolution 1.70Å" /> '''RNase T1 variant wit...)
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Revision as of 22:23, 20 November 2007
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RNase T1 variant with adenine specificity
Overview
Attempts to alter the guanine specificity of ribonuclease T1 (RNase T1) by, rational or random mutagenesis 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 kinetics of 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 approximately 7250-fold compared, to that of the wild-type. The crystal structure of the nucleotide-free RV, variant has been refined in space group P6(1) to a crystallographic, R-factor of 19.9% at 1.7 A resolution. The primary recognition site of the, RV variant adopts a similar conformation as already known from crystal, structures of RNase T1 not complexed to any nucleotide. Noteworthy is a, high flexibility of Trp45 and Asn46 within the three individual molecules, in the asymmetric unit. In addition to the kinetic studies, these data, indicate the participation of Asn46 in the specific recognition of the, base and therefore a specific binding of adenosine.
About this Structure
1Q9E is a Single protein structure of sequence from Aspergillus oryzae with TRS as ligand. Active as Ribonuclease T(1), with EC number 3.1.27.3 Full crystallographic information is available from OCA.
Reference
RNase T1 variant RV cleaves single-stranded RNA after purines due to specific recognition by the Asn46 side chain amide., Czaja R, Struhalla M, Hoschler K, Saenger W, Strater N, Hahn U, Biochemistry. 2004 Mar 16;43(10):2854-62. PMID:15005620
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