Shrimp oncoprotein nm23 is a functional nucleoside diphosphate kinase

Abstract

Biosynthesis of nucleoside triphosphates is critical for bioenergetics and nucleic acid replication, and this is achieved by nucleoside diphosphate kinase (NDK). As an emerging biological model and the global importance of shrimp culture, we have addressed the study of the Pacific whiteleg shrimp (Litopenaeus vannamei) NDK. We demonstrated its activity and affinity towards deoxynucleoside diphosphates. Also, the quaternary structure obtained by gel filtration chromatography showed that shrimp NDK is a trimer. Affinity was in the micro-molar range for dADP, dGDP, dTDP and except for dCDP, which presented no detectable interaction by isothermal titration calorimetry, as described previously for Plasmodium falciparum NDK. This information is particularly important, as this enzyme could be used to test nucleotide analogs that can block white spot syndrome virus (WSSV) viral replication and to study its bioenergetics role during hypoxia and fasting.

Structure of nucleoside diphosphate kinase from pacific shrimp (Litopenaeus vannamei) in binary complexes with purine and pyrimidine nucleoside diphosphates

“…Recombinant nucleoside diphosphate kinase from L. vannamei (GenBank DQ907945) was obtained from a synthetic gene optimized for expression in E. coli and its purification has been described elsewhere (Quintero-Reyes et al, 2012). A screening of crystallization conditions of LvNDK was performed using Crystal Screen from Hampton Research (Aliso Viejo, California, USA) by the microbatch method in Greiner plates.…”

“…The dissociation constant (K d ) was determined using ITC: the K d for dADP was 14.6 mM (QuinteroReyes et al, 2012) and the K d for ADP was 17 mM (unpublished data). The binding of dCDP is atypical, because although LvNDK phosphorylated the nucleotide no heat of binding was observed by ITC (Quintero-Reyes et al, 2012).…”

“…We have studied shrimp NDK in solution: it is a homotrimer of $57 kDa and binds and phosphorylates nucleoside diphosphates (Quintero-Reyes et al, 2012). The aim of this work is to experimentally characterize the structural interaction of an invertebrate NDK with either acceptor (dADP and dCDP) or donor (ADP) phosphate substrates in their binary complexes.…”

“…Expression of the NDK from Litopenaeus vannamei (LvNDK) is known to be regulated under viral infection. Also, as determined by isothermal titration calorimetry, LvNDK binds both purine and pyrimidine deoxynucleoside diphosphates with high binding affinity for dGDP and dADP and with no heat of binding interaction for dCDP [Quintero-Reyes et al (2012), J. Bioenerg. Biomembr.…”

Cloning, Expression and Comparative Study of White Spot Syndrome Virus (WSSV) Thymidine Kinase Gene during Infection in Penaeus monodon

“…The WSSV genome (Van Hulten et al, 2001) et al, 2012 andTopalis et al, 2005], These enzymes are helps to maintain proliferation of viral replication. Nucleoside kinase which catalyze the nucleotide phosphorylation which is necessary for the formation of new DNA segment.…”