Crystal structure of a type III pantothenate kinase: insight into the mechanism of an essential coenzyme A biosynthetic enzyme universally distributed in bacteria

Abstract

Pantothenate kinase (PanK) catalyzes the first step in the five-step universal pathway of coenzyme A (CoA) biosynthesis, a key transformation that generally also regulates the intracellular concentration of CoA through feedback inhibition. A novel PanK protein encoded by the gene coaX was recently identified that is distinct from the previously characterized type I PanK (exemplified by the Escherichia coli coaA-encoded PanK protein) and type II eukaryotic PanKs and is not inhibited by CoA or its thioesters. This type III PanK, or PanK-III, is widely distributed in the bacterial kingdom and accounts for the only known PanK in many pathogenic species, such as Helicobacter pylori, Bordetella pertussis, and Pseudomonas aeruginosa. Here we report the first crystal structure of a type III PanK, the enzyme from Thermotoga maritima (PanK(Tm)), solved at 2.0-A resolution. The structure of PanK(Tm) reveals that type III PanKs belong to the acetate and sugar kinase/heat shock protein 70/actin (ASKHA) protein superfamily and that they retain the highly conserved active site motifs common to all members of this superfamily. Comparative structural analysis of the PanK(Tm) active site configuration and mutagenesis of three highly conserved active site aspartates identify these residues as critical for PanK-III catalysis. Furthermore, the analysis also provides an explanation for the lack of CoA feedback inhibition by the enzyme. Since PanK-III adopts a different structural fold from that of the E. coli PanK -- which is a member of the "P-loop kinase"superfamily -- this finding represents yet another example of convergent evolution of the same biological function from a different protein ancestor.

FIG. 1.

Ribbon diagram of PanK_Tm monomer. The corresponding secondary structure elements in the two duplicate domains (N- and C-terminal domains) are labeled β1 to β5 and β1′ to β5′ for β-strands and α1 to α3 and α1′ for α3′ for α-helices, respectively. The two helices αi1 and αi2 between β3′ and α1′ are considered to be insertions to the core of the fold. Three highly conserved aspartate residues, Asp6, Asp105, and Asp125, are shown in the ball-and-stick representation. The loop regions corresponding to the three conserved motifs PHOSPHATE 1 (between strands β1 and β2), PHOSPHATE 2 (between strands β1′ and β2′), and ADENOSINE (between strands β4′ and α2′) are colored orange.

FIG. 2.

Ribbon diagram of PanK_Tm dimer. The two monomers are colored cyan and magenta, respectively. The active site of each monomer is marked by the ball-and-stick representation of the conserved aspartate residues. Modeled pantothenate (in green; see “Materials and Methods” for details) is also shown to indicate its location near the dimer interface.

FIG. 3.

Fold comparison of PanK-III with representative members of the ASKHA superfamily. The corresponding secondary structure elements in the core of each structure are colored accordingly, with β-strands in magenta and α-helices in cyan. The regions that are considered insertions to the RNase H-like fold core are gray. Bound substrates in each structure are shown in a stick representation. The modeled substrates of PanK_Tm are also shown.

FIG. 4.

Multiple sequence alignment of representative sequences of PanK-III (group I) and actin/hsp70/sugar kinase superfamily with known structures (group II). Sequences are labeled according to the gi number or PDB code and species name. The first and last residue numbers are indicated before and after each sequence, with the lengths of insertions specified in square brackets and the total sequence lengths of proteins following in parentheses. Residue conservation is denoted by the following scheme: uncharged, highlighted in yellow; charged/polar, in gray; small, in red; identical, bold and highlighted in black. The PHOSPHATE 1, PHOSPHATE 2, and ADENOSINE motifs are indicated at the bottom of the alignment. The secondary structure elements (E, β-strand; H, α-helix) for PanK_Tm (gi 15611833) and (PDB 1hux_A) are marked above each sequence block, respectively. Abbreviations of species names are as follows: Hp, Helicobacter pylori; Ps, Pseudomonas syringae, Dv, Desulfovibrio vulgaris; Ch, Cytophaga hutchinsonii; Bc, Bacillus cereus; En, Emericella nidulans; Mm, Mus musculus; Ce, Caenorhabditis elegans; Hs, Homo sapiens; Sa, Staphylococcus aureus; Af, Acidaminococcus fermentans; Tm, Thermotoga maritima; and Ec, Escherichia coli.

FIG. 5.

Model of MgATP and pantothenate binding in PanK_Tm active site. The color scheme is the same as that in Fig. 3 for the first monomer of the dimer, while the region corresponding to the second monomer is in yellow. The substrates ATP and pantothenate are shown as thick bonds, while the side chains of several active site residues are shown in the ball-and-stick representation. The Mg²⁺ ion is shown as a purple ball.