RNase T1 variant RV cleaves single-stranded RNA after purines due to specific recognition by the Asn46 side chain amide.
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.
Czaja
Rico
R
Department of Chemistry, Division of Biochemistry and Molecular Biology, Hamburg University, Martin-Luther-King-Platz 6, 20146 Hamburg, Germany.
Struhalla
Marc
M
Höschler
Katja
K
Saenger
Wolfram
W
Sträter
Norbert
N
Hahn
Ulrich
U
D000225:Adenine / Q000737:chemistry*; D000227:Adenine Nucleotides / Q000737:chemistry / Q000235:genetics; D000577:Amides / Q000737:chemistry*; D019943:Amino Acid Substitution / Q000235:genetics; D001216:Asparagine / Q000235:genetics*; D001236:Aspergillus oryzae / Q000201:enzymology / Q000235:genetics; D020134:Catalytic Domain / Q000235:genetics; D002412:Cations; D003460:Crystallization; D018360:Crystallography, X-Ray; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics; D014644:Genetic Variation; D006868:Hydrolysis; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding / Q000235:genetics; D012331:RNA, Fungal / Q000737:chemistry* / Q000235:genetics; D006163:Ribonuclease T1 / Q000737:chemistry* / Q000235:genetics; D013379:Substrate Specificity / Q000235:genetics; D014867:Water / Q000737:chemistry
D000227:Adenine Nucleotides; D000577:Amides; D002412:Cations; D005656:Fungal Proteins; D012331:RNA, Fungal; D014867:Water; D001216:Asparagine; D006163:Ribonuclease T1; D000225:Adenine