Crystal structure and characterization of nucleoside diphosphate kinase from Vibrio cholerae

Highlights

• Structure elucidation of Vibrio Cholera Nucleoside Diphosphate Kinase (VNDK).

• VNDK shows highest affinity for GTP amongst NTPs.

• VNDK possesses kinase and DNase activities.

• VNDK is maximally stable between pH 8 and 9, and exhibits elevated thermolability.

Abstract

Nucleoside diphosphate kinases (NDK) are ubiquitous enzymes that catalyse the transfer of the γ phosphate from nucleoside triphosphates (NTPs) to nucleoside diphosphate (NDPs), to maintain appropriate NTP levels in cells. NDKs are associated with signal transduction, cell development, proliferation, differentiation, tumor metastasis, apoptosis and motility. The critical role of NDK in bacterial virulence renders it a potential drug target. The present manuscript reports crystal structure and functional characterization of Vibrio cholerae NDK (VNDK). The 16 kDa VNDK was crystallized in a solution containing 30% PEG 4000, 100 mM Tris-HCl pH 8.5 and 200 mM sodium acetate in orthorhombic space group P2₁2₁2₁ with unit cell parameters a = 48.37, b = 71.21, c = 89.14 Å, α = β = γ = 90° with 2 molecules in asymmetric unit. The crystal structure was solved by molecular replacement and refined to crystallographic R_factor and R_free values of 22.8% and 25.8% respectively. VNDK exists as both dimer and tetramer in solution as confirmed by size exclusion chromatography, glutaraldehyde crosslinking and small angle X-ray scattering while the crystal structure appears to be a dimer. The biophysical characterization states that VNDK has kinase and DNase activity with maximum stability at pH 8–9 and temperature up to 40 °C. VNDK shows elevated thermolability as compared to other NDK and shows preferential binding with GTP rationalized using computational studies.

Introduction

Nucleoside diphosphate kinase (NDK) is an important enzyme that helps maintain the concentration of nucleoside triphosphate (NTP) pool inside the cell by the transfer of γ-phosphate from NTPs to NDPs [1]. The NTP production by pathogens is vital for pathogenesis as NTPs are precursors of RNA, DNA and polysaccharides which in turn are critical for energy metabolism and protein synthesis, making this enzyme a suitable drug candidate being actively pursued for therapeutic strategies [[2], [3], [4]]. NDKs work via a ‘‘ping-pong” mechanism, in which the native protein removes the gamma-phosphate of NTP by nucleophilic attack, forming a phospho-enzyme, which then binds to NDP, facilitating phosphate transfer [5]. Although the main source of high-energy phosphate is ATP, these enzymes have broad specificity range and can utilize both purine and pyrimidine nucleotides in their ribose and deoxyribose forms. NDK thus are a source of most of the DNA and RNA precursors except ATP. NDKs are highly conserved enzymes with the only prominent distinction among organisms is the quaternary association which can be dimeric, tetrameric or hexameric [6,7]. Differences in quaternary associations are probably linked to the diverse functions of the enzyme in that particular organism. Structural studies showed at least two kinds of tetramers (Type I and Type II) in NDKs from gram-negative bacteria. Type I and Type II tetramers differ in the relative orientation of the interacting dimers, either through their convex surface (Type I) or the concave side of their central sheet (Type II). Type I tetramers, as in Myxococcus NDK (MxNDK) [8] interact through their C-terminal region while Type II tetramers, as in E. coli NDK (EcNDK), interact via a loop comprising residue positions 90–114, also known as the kpn loop [9].

Here in this manuscript, we report the structure of Vibrio cholerae NDK (VNDK), its biochemical and biophysical characterization in comparison with Leishmania amazonensis (LaNDK) and other NDKs. The recombinant protein possesses nucleoside diphosphate kinase, phosphotransferase and DNase activity and preferentially binds GTPs.