Crystal Structure of a Hyperthermophilic Archaeal Acylphosphatase from Pyrococcus horikoshiiStructural Insights into Enzymatic Catalysis, Thermostability, and Dimerization^,

Abstract

Acylphosphatases catalyze the hydrolysis of the carboxyl-phosphate bond in acyl phosphates. Although acylphosphatase-like sequences are found in all three domains of life, no structure of acylphosphatase has been reported for bacteria and archaea so far. Here, we report the characterization of enzymatic activities and crystal structure of an archaeal acylphosphatase. A putative acylphosphatase gene (PhAcP) was cloned from the genomic DNA of Pyrococcus horikoshii and was expressed in Escherichia coli. Enzymatic parameters of the recombinant PhAcP were measured using benzoyl phosphate as the substrate. Our data suggest that, while PhAcP is less efficient than other mammalian homologues at 25 degrees C, the thermophilic enzyme is fully active at the optimal growth temperature (98 degrees C) of P. horikoshii. PhAcP is extremely stable; its apparent melting temperature was 111.5 degrees C and free energy of unfolding at 25 degrees C was 54 kJ mol(-)(1). The 1.5 A crystal structure of PhAcP adopts an alpha/beta sandwich fold that is common to other acylphosphatases. PhAcP forms a dimer in the crystal structure via antiparallel association of strand 4. Structural comparison to mesophilic acylphosphatases reveals significant differences in the conformation of the L5 loop connecting strands 4 and 5. The extreme thermostability of PhAcP can be attributed to an extensive ion-pair network consisting of 13 charge residues on the beta sheet of the protein. The reduced catalytic efficiency of PhAcP at 25 degrees C may be due to a less flexible active-site residue, Arg20, which forms a salt bridge to the C-terminal carboxyl group. New insights into catalysis were gained by docking acetyl phosphate to the active site of PhAcP.

Stabilization of a protein conferred by an increase in folded state entropy

“…3 and 4 and our own contact analysis). The latter contacts are formed predominantly with residues located in the first loop of the protein (L1), which runs along L4 and is involved in the binding of the phosphate group of the substrate (4,(11)(12)(13)(14)(15).Characterizing the properties of hmAcP mutants carrying deletions or insertions in L4 we found that the thermodynamic stability of mutants in which the loop was shortened is increased to an extent significantly larger than that predicted by polymer models for loop closure entropy. The increased stability is predominantly due to a decrease in the unfolding rate and is attained despite the fact that shortening of the loop is accompanied by considerable losses in enthalpy.…”

Solution structure and conformational heterogeneity of acylphosphatase from Bacillus subtilis

“…Structures of several AcPs from different organisms have been solved, most of which are in the ligand-bound state [6][7][8][9][10][11][12][13]. The first AcP (muscle type AcP) structure was determined by Nuclear magnetic resonance (NMR), and the structure in the ligand-free state revealed the presence of mobility in loop regions, although the overall RMS difference (RMSD) for the structure ensemble is rather high ($1.5 Å) [6,12,13].…”

Structural Basis for the Reaction Mechanism of S-Carbamoylation of HypE by HypF in the Maturation of [NiFe]-Hydrogenases

“…In addition, the carboxamide of Asn-44 interacts with the phosphate ion through a water molecule. Previous mutagenic and structural studies on mammalian ACP have revealed that Arg-26 and Asn-44 are essential to the catalytic reaction (32,33), and the binding manner of the phosphate ion to the enzyme revealed in this study resembled that of the proposed catalytic transition state (21). So far, several structures of ACPs in complex that mimic the substrate-bound states are available.…”

“…Based on the observed arrangement, deprotonation of the water molecule (termed W1 in Fig. 2B) by a phosphate oxygen should be enhanced to produce the nucleophile hydroxide for dephosphorylation of carbamoylphosphate, as in the proposed catalytic mechanism (21,33). The absence of catalytic activity for the ACP domain of HypF without the Zn finger-like domain (18) can be explained by the observed difference in the structure of the acylphosphate-binding site.…”

“…In addition, the structure of the ACP domain in complex with the phosphate ion indicated that the binding manner is inconsistent with the proposed transition state for the hydrolysis reaction (21). Recently, a partial structure of HypF including the other three domains revealed that the enzyme harbors two nucleotide-binding sites (one each at the YrdC-like and Kae1-like domains) facing each other in a parallel fashion and separated by about 14 Å (17).…”