Hypoxanthine-Guanine Phosphoribosyltransferase/adenylate Kinase From Zobellia galactanivorans: A Bifunctional Catalyst for the Synthesis of Nucleoside-5'-Mono-, Di- and Triphosphates

Abstract

In our search for novel biocatalysts for the synthesis of nucleic acid derivatives, we found a good candidate in a putative dual-domain hypoxanthine-guanine phosphoribosyltransferase (HGPRT)/adenylate kinase (AMPK) from Zobellia galactanivorans (ZgHGPRT/AMPK). In this respect, we report for the first time the recombinant expression, production, and characterization of a bifunctional HGPRT/AMPK. Biochemical characterization of the recombinant protein indicates that the enzyme is a homodimer, with high activity in the pH range 6-7 and in a temperature interval from 30 to 80°C. Thermal denaturation experiments revealed that ZgHGPRT/AMPK exhibits an apparent unfolding temperature (Tm) of 45°C and a retained activity of around 80% when incubated at 40°C for 240 min. This bifunctional enzyme shows a dependence on divalent cations, with a remarkable preference for Mg²⁺ and Co²⁺ as cofactors. More interestingly, substrate specificity studies revealed ZgHGPRT/AMPK as a bifunctional enzyme, which acts as phosphoribosyltransferase or adenylate kinase depending upon the nature of the substrate. Finally, to assess the potential of ZgHGPRT/AMPK as biocatalyst for the synthesis of nucleoside-5'-mono, di- and triphosphates, the kinetic analysis of both activities (phosphoribosyltransferase and adenylate kinase) and the effect of water-miscible solvents on enzyme activity were studied.

Keywords: dual domain protein; enzymatic synthesis; nucleoside-5cpsdummy′-monophosphate kinase; nucleotides; phosphoribosyltransferase.

FIGURE 1

(A) Enzymatic synthesis of 6-oxopurine purine nucleosides monophosphate catalyzed by 6-oxopurine PRTs. (B) Enzymatic synthesis adenine nucleotides catalyzed by AMPKs.

FIGURE 2

Cartoon representation of the ZgHGPRT/AMPK monomer model comprising HGPRT (yellow) and AMPK (green) domains. The figure was prepared with PyMOL (Delano, 2002).

FIGURE 3

Cartoon representation of the overall ZgHGPRT/AMPK model, comprising both subunits (green and blue). The figure was prepared with PyMOL (Delano, 2002).

FIGURE 4

(A) Multiple sequence alignment of amino acid sequences of 6-oxopurine PRTs from Zobellia galactanivorans (ZgHGPRT/AMPK), Leptospira interrogans (LiHGXPRT, PDB id 4QRI), Trypanosoma cruzi (TcHPRT, PDB id 1P19), Bacillus anthracis (BaHPRT, PDB id 6D9Q), Salmonella typhimurium (StHPRT, PDB id 1J7J), Escherichia coli (EcHPRT, PDB id 5KNR), (B) Overall representation of the active site architecture of ZgHGPRT domain based on the structural alignment of ZgHGPRT/AMPK homology model (green) with Trypanosoma cruzi HRPT (gray) complexed with PRPP and 7-hydroxypyrazolo[4,3-D]pyrimidine (PDB id 1TC2) (sticks), and Mg²⁺ (black sphere). PRPP binding site, PPi and flexible loops (blue) and purine pocket (violet) are encircled with dotted lines. The figure was prepared with PyMOL (Delano, 2002).

FIGURE 5

(A) Multiple sequence alignment of amino acid sequences of AMPK from Zobellia galactanivorans (ZgHGPRT/AMPK), Aquifex aeolicus (AaAMPK, PDB id 2RGX), Mycobacterium tuberculosis (MtAMPK, PDB id 1P4S), Geobacillus stearothermophilus (GsAMPK, PDB id 1ZIN), Marinibacillus marinus (MmAMPK, PDB id 3FB4), Sporosarcina globispora (SgAMPK, PDB id 5X6J). (B) Overall representation of the active site architecture of ZgAMPK domain based on the structural alignment of ZgHGPRT/AMPK homology model with AMPK from Homo sapiens (gray) complexed with P1,P4-Bis(5′-adenosyl) tetraphosphate (PDB id 2C95) (sticks). P-Loop site (red), AMP binding site (blue) and LID domain (yellow) are encircled with dotted lines. The core domain (green), including P-loop site and AMPK fingerprint, is also shown. The figure was prepared with PyMOL (Delano, 2002).

FIGURE 6

SDS-PAGE analysis of soluble ZgHGPRT/AMPK. Lane 1. Precision Plus Protein^TM prestained standard from Bio-Rad used as a molecular weight marker. Lane 2. Supernatant obtained after centrifugation of the lysed cells. Lane 3. Pellet obtained after centrifugation of the lysed cells. Lane 4. ZgHGPRT/AMPK (15 μg protein) after chromatography purification.

FIGURE 7

Biochemical characterization of ZgHGPRT/AMPK. (A) Effect of temperature on ZgHGPRT/AMPK activity (•). (B) Effect of pH on ZgHGPRT/AMPK activity, (•) 50 mM sodium citrate (pH 4–6), (∘) 50 mM MES (pH 5.5–7), (■) 50 mM sodium phosphate (pH 6–8.5), (△) 50 mM Tris–HCl (pH 7–9), (▲) 50 mM sodium borate (pH 8–11). All determinations were carried out in triplicate and the maximum standard deviation value was 2%.

FIGURE 8

(A) Thermal inactivation of ZgHGPRT/AMPK at (■) 40°C, (∘) 50°C and (•) 60°C. (B) Melting temperature of ZgHGPRT/AMPK. All determinations were carried out in triplicate and the maximum standard deviation value was 1.8%.

FIGURE 9

Influence of divalent salts on ZgHGPRT/AMPK activity. (•) MgSO₄, (∘) CoSO₄, (△) MgSO₄, (▲) ZnSO₄ and (■) CaCl₂. All determinations were carried out in triplicate and the maximum standard deviation value was 1.7%.

FIGURE 10

Effect of organic co-solvents (20% v/v) on ZgHGPRT/AMPK activity. (A) Aprotic polar solvents. (B) Alcohols and polyols. All determinations were carried out in triplicate and the maximum standard deviation value was 2%.

FIGURE 11

Structural alignment of ZgHGPRT/AMPK model (green) and TtHGXPRT (yellow) complexed with IMP (atom-type coloring sticks). Hydrogen bonds formed between the protein residues and IMP are shown as yellow dotted lines. Active site residues in the model are represented by sticks with the atom-type coloring (green). The figure was prepared with PyMOL (Delano, 2002).