Molecular mechanisms of substrate specificities of uridine-cytidine kinase

Abstract

A uridine-cytidine kinase (UCK) catalyzes the phosphorylation of uridine (Urd) and cytidine (Cyd) and plays a significant role in the pyrimidine-nucleotide salvage pathway. Unlike ordinary ones, UCK from Thermus thermophilus HB8 (ttCK) loses catalytic activity on Urd due to lack of a substrate binding ability and possesses an unusual amino acid, i.e. tyrosine 93 (Tyr93) at the binding site, whereas histidine (His) is located in the other UCKs. Mutagenesis experiments revealed that a replacement of Tyr93 by His or glutamine (Gln) recovered catalytic activity on Urd. However, the detailed molecular mechanism of the substrate specificity has remained unclear. In the present study, we performed molecular dynamics simulations on the wild-type ttCK, two mutant ttCKs, and a human UCK bound to Cyd and three protonation forms of Urd to elucidate their substrate specificity. We found three residues, Tyr88, Tyr/His/Gln93 and Arg152 in ttCKs, are important for recognizing the substrates. Arg152 contributes to induce a closed form of the binding site to retain the substrate, and the N3 atom of Urd needed to be deprotonated. Although Tyr88 tightly bound Cyd, it did not sufficiently bind Urd because of lack of the hydrogen bonding. His/Gln93 complemented the interaction of Tyr88 and raised the affinity of ttCK to Urd. The crucial distinction between Tyr and His or Gln was a role in the hydrogen-bonding network. Therefore, the ability to form both hydrogen-bonding donor and accepter is required to bind both Urd and Cyd.

Indispensable residue for uridine binding in the uridine-cytidine kinase family

“…The replacement of Arg152 with Leu reduced the activity towards cytidine (data not shown). These interactions between uridine and ttCK are indispensable for substrate binding, which is consistent with the molecular dynamics simulation [23] .…”

Free energy landscapes of prototropic tautomerism in pyridoxal 5′‐phosphate schiff bases at the active site of an enzyme in aqueous medium

“…To further understand the contributions of different amino acid residues to the stability of ketoenamine and enolimine tautomers at the active site of the SHMT enzyme, we carried out the energy decomposition analysis through MM‐PBSA calculations . It is found that the ARG232 residue, which is hydrogen bonded to the phosphate oxygens of the PLP ring, has most pronounced effect in stabilization of the two tautomers.…”

Purine and pyrimidine salvage pathway in thermophiles: a valuable source of biocatalysts for the industrial production of nucleic acid derivatives

“…When Tyr93 was replaced by histidine (Y93H) or glutamine (Y93Q), TtCK recovered the phosphorolytic activity towards uridine. Conversely, when Tyr93 was replaced by a smaller residue, such as Phe or Leu, uridine could not be accepted by TtCK either (Tanaka et al 2016;Tomoike et al 2011) (Fig. 10).…”

“…Recent studies (Tanaka et al 2016;Tomoike et al 2011) described the presence of an unusual amino acid (Tyr93) in the active site of TtCK, while in other UCKs histidine (His) is commonly found at this position. In view of this finding, the authors suggested that the strict specificity for cytidine can be attributed to this single residue.…”