Modification of Ribonuclease T1 Specificity by Random Mutagenesis of the Substrate Binding Segment^,

Abstract

Attempts to modify the guanine specificity of ribonuclease T1 (RNase T1) by rationally designed amino acid substitutions failed so far. Therefore, we applied a semirational approach by randomizing the guanine binding site. A combinatorial library of approximately 1.6 million RNase T1 variants containing permutations of 6 amino acid positions within the recognition loop was screened on RNase indicator plates. The specificity profiles of 180 individual clones showing RNase activity revealed that variant K41S/N43W/N44H/Y45A/E46D (RNaseT1-8/3) exhibits an altered preference toward purine nucleotides. The ApC/GpC preference in the cleavage reaction of this variant was increased 4000-fold compared to wild-type. Synthesis experiments of dinucleoside monophosphates from cytidine and the corresponding 2'3'-cyclic diesters using the reverse reaction of the transesterification step showed a 7-fold higher ApC synthesis rate of RNase 8/3 than wild-type, whereas the GpC synthesis rates for both enzymes were comparable. This study shows that site-directed random mutagenesis is a powerful additional tool in protein design in order to achieve new enzymatic specificities.

Probing Functional Perfection in Substructures of Ribonuclease T1: Double Combinatorial Random Mutagenesis Involving Asn43, Asn44, and Glu46 in the Guanine Binding Loop

“…A previous attempt to alter the specificity of RNase T 1 employed random mutagenesis involving six residues at the PRS from position 41 to 46 (14). The activity of 180 variants in periplasmic extracts were studied with GpC and ApC as substrates.…”

“…Substitutions at Asn43 and Asn44 yielded enzymes with slightly lower or markedly lower catalytic activities, respectively (8); also, substitution of Glu46 with Asp, Gln, and Ala substantially decreased activity as did nonaromatic substitutions at Tyr42. Last, a random mutagenesis study of residues 41 through 46 did not reveal any transformants with activity greater than wild type (14). All of these findings suggest that guanine loop may have achieved optimization in RNase T 1 even though the enzyme has not achieved catalytic perfection since the maximal value of k cat / K M for small oligonucleotide substrates [about (2-5) × 10 6 M -1 s -1 (3,15)] is considerably less than the theoretical diffusion control limit (10 9 -10 10 M -1 s -1 ), which has essentially been achieved by RNase Ba (Barnase; a bacterial member of the RNase T 1 superfamily) (16).…”

Identification of Substrate Binding Sites in Enzymes by Computational Solvent Mapping

“…the NH group of N43 interacts with guanine N7, and the NH of Y45 hydrogen bonds to O6 of guanidine moiety. 33 The Y45 side-chain is also important, and this residue, often referred to as the lid of the guaninebinding site, interacts with the highest number of probes. 32 Side-chain effects are also important for F100, which frequently interacts both with the probes in the mapping ( Figure 3b) and with the ligands in the ribonuclease complexes, and is assumed to enhance the electrostatic interactions between substrate and active site via a change in the dielectric constant.…”

Molecular Basis of the Recognition Process: Hydrogen-Bonding Patterns in the Guanine Primary Recognition Site of Ribonuclease T1

“…3,5,6,12,14 All of these findings suggest that the PRS may have achieved its optimal structure for guanine recognition. The free energy change through mutation of Tyr45 (Tyr45Ala) of the guanine-RNase T1 complex suggests that the stacking interaction between the phenolic side chain of Tyr45 and the aromatic ring of guanine is less important compared with the multiple H-bonding between the PRS and the guanine moiety.…”