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

Abstract

Combinatorial random mutageneses involving either Asn43 with Asn44 (set 1) or Glu46 with an adjacent insertion (set 2) were undertaken to explore the functional perfection of the guanine recognition loop of ribonuclease T(1) (RNase T(1)). Four hundred unique recombinants were screened in each set for their ability to enhance enzyme catalysis of RNA cleavage. After a thorough selection procedure, only six variants were found that were either as active or more active than wild type which included substitutions of Asn43 by Gly, His, Leu, or Thr, an unplanned Tyr45Ser substitution and Glu46Pro with an adjacent Glu47 insertion. Asn43His-RNase T(1) has the same loop sequence as that for RNases Pb(1) and Fl(2). None of the most active mutants were single substitutions at Asn44 or double substitutions at Asn43 and Asn44. A total of 13 variants were purified, and these were subjected to kinetic analysis using RNA, GpC, and ApC as substrates. Modestly enhanced activities with GpC and RNA involved both k(cat) and K(M) effects. Mutants having low activity with GpC had proportionately even lower relative activity with RNA. Asn43Gly-RNase T(1) and all five of the purified mutants in set 2 exhibited similar values of k(cat)/K(M) for ApC which were the highest observed and about 10-fold that for wild type. The specificity ratio [(k(cat)/K(M))(GpC)/(k(cat)/K(M))(ApC)] varied over 30 000-fold including a 10-fold increase [Asn43His variant; mainly due to a low (k(cat)/K(M))(ApC)] and a 3000-fold decrease (Glu46Ser/(insert)Gly47 variant; mainly due to a low (k(cat)/K(M))(GpC)) as compared with wild type. It is interesting that k(cat) (GpC) for the Tyr45Ser variant was almost 4-fold greater than for wild type and that Pro46/(insert)Glu47 RNase T(1) is 70-fold more active than the permuted variant (insert)Pro47-RNase T(1) which has a conserved Glu46. In any event, the observation that only 6 out of 800 variants surveyed had wild-type activity supports the view that functional perfection of the guanine recognition loop of RNase T(1) has been achieved.

Retinal cone topography of artiodactyl mammals: Influence of body height and habitat

“…The H‐bonding interactions between proteins and nucleic acids have been found to be a vital factor in the process of repairing oxidative damage to the purines 7–9 and mediating the glycosidic‐bond cleavage 6. Multiple H‐bonging has been proposed to be the driving force in the molecular recognition process in protein‐nucleic acid complexes 10–14. Besides, multiple HBs between an amino acid side chain and bases are expected to alter in different ways the electronic properties of nucleic acid bases 15, which act an important role in understanding DNA and RNA damages 16–19.…”

Conserved asparagine residue 54 of α-sarcin plays a role in protein stability and enzyme activity

“…In this structure, loop 2 extends from Thr 53 to Tyr 93 joining strands b3 and b4 (Figure 1). For example, in RNase T1 the equivalent residue would be Asn 44, which has been demonstrated to be directly involved in recognition of the substrate guanine moiety (Kumar and Walz, 2001). It shows a lack of repetitive secondary structure, and is one of the most mobile parts of the protein.…”

Density functional theory study on the ionization potentials and electron affinities of thymine–formamide complexes

“…Furthermore, plenty of these protein–nucleic acid interactions are directly dominated by hydrogen bonds (HBs). For example, the HBs have been proposed to be the driving force in the molecular recognition process in protein–nucleic acid complexes 21–25. More interesting, multiple HBs between an amino acid side chain and bases are expected to alter in different ways the electronic properties of nucleic acid bases 26, which we have mentioned above act as an important role in understanding DNA and RNA damages.…”