The Flexibility of the Non-Conservative Region at the C Terminus of d-Hydantoinase from Pseudomonas putida YZ-26 is Extremely Limited

Abstract

We previously reported that a deletion mutant (P478) with a residue Arg deleted at the C terminus of D-hydantoinase (P479) from Pseudomonas putida YZ-26 was dissociated into the monomer from its dimeric state. Based on the above result, a series of mutants of the enzyme with the C-terminal residues either deleted or substituted were prepared. The size-exclusion chromatography and bioactivity assay show that a C-terminal-substituted enzyme (R479D) and several truncated mutants (P478, P477, P476, and P475) are dissociated into the monomeric state as well, but their activities are largely retained. In contrast, two other mutants (R474 and R479A) are expressed in the form of random aggregates without any activity. Our experiments demonstrate that only the last four amino acids (-PVQR) at the C terminus of the enzyme can be deleted without seriously affecting its activity, although the enzyme is dissociated from a dimer into a monomer. These mutants also reveal some unique properties such as the enzymatic activity in vivo or in vitro, the effect of divalent metal ions, and the thermostability etc. in comparison to wild-type enzyme (P479). In addition, the three-dimensional structural modeling shows that the intact structure of the enzyme is essential, and the flexibility of the non-conservative region at the C terminus of the enzyme is quite limited.

Crystal structure of dihydropyrimidinase from Pseudomonas aeruginosa PAO1: Insights into the molecular basis of formation of a dimer

“…1), only the hydrogen bonds of D163eN186, E206eR479, E206eV477, and Y275eR479 as well as the salt bridges of E206eR479, E219eR479, and R223eE219 are highly conserved. Previous works indicated that the C-terminal Arg479 in bacterial hydantoinase plays an important role in dimerization [22,23]. In the present study, the structure provides the evidence that the subunit association of P. aeruginosa dihydropyrimidinase is mediated by the C-terminal Arg479 through hydrogen bonds and salt bridges.…”

“…The native molecular mass of P. aeruginosa dihydropyrimidinase is approximately two times higher than the molecular mass of a monomer (53 kDa). Thus, we conclude that P. aeruginosa dihydropyrimidinase in solution is a stable dimer, similar to bacterial hydantoinase from P. putida YZ-26 [22,23].…”

“…Previous studies indicated that hydantoinase from Pseudomonas putida YZ-26 functions as a dimer [22,23]. To confirm their results and determine how this enzyme can form a dimer, instead of tetramer, the structure is needed to obtain more information in molecular level.…”

Crystal Structures of Vertebrate Dihydropyrimidinase and Complexes from Tetraodon nigroviridis with Lysine Carbamylation

“…Previous reports have shown that modifications to the C termini of DHP/HYD enzymes could decrease the enzyme activity (9, 48,[53][54][55]. In the D-HYD from B. stearothermophilus and Bacillus thermocatenulatus, the truncated enzymes with a deletion of 40 amino acids on the C terminus have lost 64% of their enzymatic activities (48).…”

“…In the D-HYD from B. stearothermophilus and Bacillus thermocatenulatus, the truncated enzymes with a deletion of 40 amino acids on the C terminus have lost 64% of their enzymatic activities (48). In Pseudomonas putida YZ-26, a series of deletions were performed in the D-HYD and all of the truncated enzymes exhibited either low or non-detectable activities (53,54). However, CD spectroscopy showed that these deletions of the C-terminal fragment did not alter the secondary structures of the overall folding (48,54).…”