A broad specificity nucleoside kinase from Thermoplasma acidophilum

Abstract

The crystal structure of Ta0880, determined at 1.91 Å resolution, from Thermoplasma acidophilum revealed a dimer with each monomer composed of an α/β/α sandwich domain and a smaller lid domain. The overall fold belongs to the PfkB family of carbohydrate kinases (a family member of the Ribokinase clan) which include ribokinases, 1-phosphofructokinases, 6-phosphofructo-2-kinase, inosine/guanosine kinases, fructokinases, adenosine kinases, and many more. Based on its general fold, Ta0880 had been annotated as a ribokinase-like protein. Using a coupled pyruvate kinase/lactate dehydrogenase assay, the activity of Ta0880 was assessed against a variety of ribokinase/pfkB-like family substrates; activity was not observed for ribose, fructose-1-phosphate, or fructose-6-phosphate. Based on structural similarity with nucleoside kinases (NK) from Methanocaldococcus jannaschii (MjNK, PDB 2C49, and 2C4E) and Burkholderia thailandensis (BtNK, PDB 3B1O), nucleoside kinase activity was investigated. Ta0880 (TaNK) was confirmed to have nucleoside kinase activity with an apparent KM for guanosine of 0.21 μM and catalytic efficiency of 345,000 M(-1) s(-1) . These three NKs have significantly different substrate, phosphate donor, and cation specificities and comparisons of specificity and structure identified residues likely responsible for the nucleoside substrate selectivity. Phylogenetic analysis identified three clusters within the PfkB family and indicates that TaNK is a member of a new sub-family with broad nucleoside specificities. Proteins 2013. © 2012 Wiley Periodicals, Inc.

Figure 1

Sequence alignment of TaNK, MjNK, BtNK, and MtAK, and EcRK. Secondary elements are indicated by arrows (β-strands), gray curvy lines (α-helices), light gray curvy lines (3₁₀ helices), and straight lines (loops); the swapped β-stand is indicated with a black outline. Residues involved in the TaNK dimer interface are colored in light gray font. Residues involved in substrate binding are highlighted pale yellow (ribose interactions) or yellow (base interactions); phosphate donor binding, green; Mg²⁺ coordination, purple; and unconserved residues within these categories are boxed with a thin line. Regions boxed with a thick line correspond to the three characteristic motifs: the glycine-glycine dipeptide sequence (G37, G38 in TaNK), the NXXE motif (N173-E176 in TaNK), and the anion-hole motif (G225-D228 in TaNK). The asterisks indicate residues involved in the binding site that are contributed by the other subunit. Conserved residues in ribokinases are underlined.

Figure 2

Structure of TaNK. (A) Ribbon diagram of the TaNK dimer (subunits colored gray and orange). The TaNK monomer has 14 β-strands (β1- β14), nine α-helices (α1-α9), and two 3₁₀- helices (H_3/101 and H_3/102). One of the β-strands (β3) is swapped between the monomers. (B) The NKs are homodimers (MjNK; rendered with a semi-transparent surface and the backbone as a cartoon and the subunits are colored dark and light gray) that bind a phosphate-donor (e.g. ATP), a divalent cation (e.g. Mg²⁺), and a nucleoside substrate (e.g. adenosine).

Figure 3

TaNK nucleoside kinase activity. (A) TaNK Activity was demonstrated with inosine, guanosine, cytidine and adenosine as phosphate acceptor substrates. (B) TaNK activity was tested with ATP, GTP, ITP, TTP, and UTP as phosphate donors. (C) TaNK activity was modulated by the presence of various metal ions: Mn ²⁺, Mg²⁺, Ni²⁺, and Co²⁺. (D) Michaelis- Menten plots of TaNK activity with varying ATP (◆) and GTP (■) concentrations. Data points are averages of three replicates and the error bars represent the standard deviation.

Figure 4

TaNK nucleoside kinase kinetic parameters: (A) Michaelis-Menten and (B) Hanes-Woolf plots with phosphate acceptor substrates adenosine (●), inosine (◆), guanosine (□), and cytidine (Δ). Data points are averages of three replicates and the error bars represent the standard deviation.

Figure 5

The pH dependency of TaNK. TaNK shows optimal inosine kinase activity at pH 6.6 and significant activity from pH 6.4 to 7.8. Activity dramatically decreases at pH values less than 6.4 and above 7.8. Data points are averages of three replicates and the error bars represent the standard deviation.

Figure 6

Cation coordination and phosphate donor interactions. (A) Cation binding site (stereo image). (B) Phosphate donor binding site (stereo image). The backbone of TaNK is rendered as a cartoon and the side chains involved are shown and labeled. These figures were generated by structurally aligning TaNK using DaliLite with the adenosine-Mg²⁺-AMP-PNP complexed MjNK structure (PDB ID 2C49). The residues are labeled according to the TaNK sequence (Fig. 1).

Figure 7

Nucleoside specificity of TaNK and MjNK. Residues (side chain atoms only) involved in interacting with the nitrogenous base of the nucleoside are rendered as sticks and labeled in the left panel. The nucleosides, also rendered as sticks, are labeled at the top of each column. Boxes indicate activity was observed for the nucleoside substrate.

Figure 8

Nucleoside specificity of BtNK and MtAK. Residues (side chain atoms only) involved in interacting with the nitrogenous base of the nucleoside are rendered as sticks and labeled in the left panel. The nucleosides, also rendered as sticks, are labeled at the top of each column. Boxes indicate activity was observed for the nucleoside substrate.

Figure 9

Cladogram of nucleoside and sugar kinases. Phylogenetic analysis of ribokinases (RK), adenosine kinases (AK), fructokinases (FRK), inosine-guanosine kinases (INGK), broad range kinase (BR) and nucleoside kinases (NK). TaNK is highlighted with a box and the associated broad nucleoside PfkB subfamily in bold font. Information about each sequence can be found in Table S1. Branch support values are given for each branch and only values greater than 50% are considered.