A single Gly114Arg mutation stabilizes the hexameric subunit assembly and changes the substrate specificity of halo‐archaeal nucleoside diphosphate kinase

Abstract

Nucleoside diphosphate kinase from extremely halophilic archaeon (HsNDK) requires above 2 M NaCl concentration for in vitro refolding. Here an attempt was made to isolate mutations that allow HsNDK to refold in low salt media. Such a screening resulted in isolation of an HsNDK mutant, Gly114Arg, which efficiently refolded in the presence of 1 M NaCl. This mutant, unlike the wild type enzyme, was expressed in Escherichia coli as an active form. The residue 114 is in close proximity to Glu155 of the neighboring subunit in the three dimensional hexameric structure of the HsNDK. It is thus possible that the attractive electrostatic interactions occur between Arg114 and Glu155 in the mutant HsNDK, stabilizing the hexameric subunit assembly.

Effects of mutations at Gly114 on the stability and refolding of haloarchaeal nucleoside diphosphate kinase in low salt solution

“…On the contrary, G114R mutant recently isolated efficiently refolded in the presence of 1 M NaCl, in which the wild-type HsNDK showed marginal refolding; i.e., G114R mutation made the enzyme less dependent on salt concentration for refolding. The residue 114 of the mutant is in close proximity to Glu155 of the neighboring subunit in the three-dimensional hexameric structure of HsNDK [6,7]. We thus speculated that the attractive electrostatic interactions occur between Arg114 and Glu155 in the mutant HsNDK hexamer, and stabilize the subunit assembly of this mutant protein in low salt.…”

“…We have previously reported that mutation of G114R results in about 10 • C higher thermal stability than the wild-type HsNDK [7]. Thermal stability was therefore examined in 0.2 M NaCl by measuring the resistance to heat treatment at 20-60 • C. Fig.…”

“…Forward primer 5 -GGCGACTACGGCAACG-ACCTCAGCCACAACCTCATCCACGGCAGC-3 (changed codon was underlined) and reverse primer 5 -GCTGCCGTGGATGAGGTTGTG GCTGAGGTCGTTGCCGTAGTCGCC-3 were used for G114S mutation. Plasmid pG114R DNA [7] was used as a template, and PCR product, treated by DpnI for 1 h, was transformed to E. coli JM109 to construct pG114S. Likewise, for G114K mutation, forward primer 5 -GGCGACTACGGCAACGACCTCAAACACAACCTCATCCACGGCAGC-3 and reverse primer 5 -GCTGCCGTGGATGAGGTTGTGTTTGAGGT-CGTTGCCGTAGTCGCC-3 were used to construct pG114K.…”

Dissecting the Unique Nucleotide Specificity of Mimivirus Nucleoside Diphosphate Kinase

“…There are a number of natural NDK sequence variants, with an extreme case being the P. aerophilum NDK (Fig. 1), where this residue is replaced by a 10-amino-acid insert, which also affects the substrate specificity (17). Interestingly, a randomly generated G3R mutant of the archaeal H. salinarum NDK (67% GϩC) also resulted in an increase in the enzyme affinity for pyrimidine nucleotides (35), thus confirming the critical role of this amino acid for substrate specificity.…”

Halophilic Properties and their Manipulation and Application

“…4.1). Furthermore, it resulted in about 10 • C higher thermal stability in a low-salt solution than that of the parent molecule (Ishibashi et al, 2007). We thus speculated that the attractive electrostatic interactions occurred between Arg114 and Glu155 in G114R mutant and were reduced to the extent that the subunit assembly of this mutant protein can occur in low salt.…”