Abstract
Adenosine kinase (ADK) is the first enzyme in the adenosine remediation pathway that catalyzes adenosine phosphorylation into adenosine monophosphate, thus regulating adenosine homeostasis in cells. To obtain new insights into ADK from Bombyx mori (BmADK), we obtained recombinant BmADK, and analyzed its activity, structure, and function. Gel-filtration showed BmADK was a monomer with molecular weight of approximately 38 kDa. Circular dichroism spectra indicated BmADK had 36.8% α-helix and 29.9% β-strand structures, respectively. The structure of BmADK was stable in pH 5.0–11.0, and not affected under 30 °C. The melting temperature and the enthalpy and entropy changes in the thermal transition of BmADK were 46.51 ± 0.50 °C, 253.43 ± 0.20 KJ/mol, and 0.79 ± 0.01 KJ/(mol·K), respectively. Site-directed mutagenesis demonstrated G68, S201, E229, and D303 were key amino acids for BmADK structure and activity. In particular, S201A mutation significantly increased the α-helix content of BmADK and its activity. BmADK was located in the cytoplasm and highly expressed in the silk gland during the pre-pupal stage. RNA interference revealed the downregulation of BmADK decreased ATG-8, Caspase-9, Ec-R, E74A, and Br-C expression, indicating it was likely involved in 20E signaling, apoptosis, and autophagy to regulate silk gland degeneration and silkworm metamorphosis. Our study greatly expanded the knowledge on the activity, structure, and role of ADK.
Highlights
Adenosine kinase (EC 2.7.1.20, ADK) is the most important enzyme in adenosine metabolism
Our results suggested that S201 is a key amino acid for the enzymatic activity of BmADK
Our results suggested that BmADK is likely involved in 20E signaling pathway, cell apoptosis, and autophagy during the pre-pupal stage of silkworm, regulating silk gland degeneration and silkworm metamorphosis
Summary
Adenosine kinase (EC 2.7.1.20, ADK) is the most important enzyme in adenosine metabolism. It is one of the most common and abundant nucleoside kinases [1,2]. The protein sequence of ADK is conserved among several eukaryotic species [3,4]. ADK is the first enzyme in the adenosine remediation pathway, and an important regulator of extracellular adenosine concentration [5], which has been well elucidated in animal models of epilepsy and ischemia [6,7]. ADK was first isolated from yeast in 1951. ADK properties including substrate specificity [8], inhibitors [9], metal ions [10], ATP, and inorganic phosphate [11] have been extensively investigated
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