Secondary and quaternary structural transition of the halophilic archaeon nucleoside diphosphate kinase under high- and low-salt conditions

Abstract

Most halophilic enzymes from extremely halophilic archaea are denatured immediately after transfer from high-salt to low-salt medium. However, nucleoside diphosphate kinase (HsNDK) from the extremely halophilic archaeon Halobacterium salinarum seems to be exceptional, since the enzyme exhibited catalytic activity even under the low-salt condition. Here we show the mechanism how HsNDK is active under both high- and low-salt conditions that the HsNDK hexamer in high-salt medium dissociates into a dimer in the low-salt medium without denaturation. The observed change of the subunit structure was accompanied by a large decrease of alpha-helical content and lowered thermal sensitivity, yet keeping the conformations. This novel hexamer to dimer conversion under high- and low-salt conditions, respectively, seems to be the mechanism by which HsNDK is avoided from the irreversible denaturation.

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

“…The wild type protein completely lost the activity at 40°C, indicating that the mutant is more stable in 0.2 M NaCl as well. A puzzling data is the CD melting of the wild type; i.e., it begins to melt at $40°C [25], similarly to the mutant. We have shown before that HsNDK is a dimer in 0.2 M NaCl by sedimentation equilibrium analysis [25].…”

“…A puzzling data is the CD melting of the wild type; i.e., it begins to melt at $40°C [25], similarly to the mutant. We have shown before that HsNDK is a dimer in 0.2 M NaCl by sedimentation equilibrium analysis [25]. However, we showed here that the HsNDK is a hexamer at 25°C and dimer at 35°C.…”

“…4A). However, in 0.2 M NaCl, the melting profile of the mutant is similar to the wild type HsNDK [25], which showed the onset temperature at 40°C and end temperature at 53°C. Thus, under the low-salt conditions, the wild and mutant HsNDKs showed the almost same denaturation profile.…”

“…dependency We have previously reported that HsNDK forms dimer at 0.2 M NaCl and hexamer at 3.8 M NaCl by sedimentation equilibrium [25]. In this study, we used a high performance size exclusion chromatography-multi-angle laser light scattering (SEC-MALLS) in low salt buffer containing 0.2 M NaCl.…”

Effects of Salt and Ligand Concentrations on the Thermal Unfolding and Refolding of Halophilic Starch-Binding Domain from Kocuria varians α-Amylase

“…It is thus evident that His-SBD12 derived from secretory -amylase of moderately halophilic bacteria is stable and active in the wide range of salinity conditions, 0 to high salt concentrations. Nucleoside diphosphate kinase from extremely halophilic archaea, Halobacterium salinarum (HsNDK), showed extremely halophilic profile: it was active at wide range of salt concentrations with maximum activity at 2 M NaCl [16] and the HsNDK was stabilized by ~30 o C by the addition of 2 M salt [17]. In addition, the thermally unfolded HsNDK required NaCl over 2 M concentration and 2~3 days for complete refolding [18].…”