Reaction mechanism and structural model of ADP-forming Acetyl-CoA synthetase from the hyperthermophilic archaeon Pyrococcus furiosus: evidence for a second active site histidine residue

Abstract

In Archaea, acetate formation and ATP synthesis from acetyl-CoA is catalyzed by an unusual ADP-forming acetyl-CoA synthetase (ACD) (acetyl-CoA + ADP + P(i) acetate + ATP + HS-CoA) catalyzing the formation of acetate from acetyl-CoA and concomitant ATP synthesis by the mechanism of substrate level phosphorylation. ACD belongs to the protein superfamily of nucleoside diphosphate-forming acyl-CoA synthetases, which also include succinyl-CoA synthetases (SCSs). ACD differs from SCS in domain organization of subunits and in the presence of a second highly conserved histidine residue in the beta-subunit, which is absent in SCS. The influence of these differences on structure and reaction mechanism of ACD was studied with heterotetrameric ACD (alpha(2)beta(2)) from the hyperthermophilic archaeon Pyrococcus furiosus in comparison with heterotetrameric SCS. A structural model of P. furiosus ACD was constructed suggesting a novel spatial arrangement of the subunits different from SCS, however, maintaining a similar catalytic site. Furthermore, kinetic and molecular properties and enzyme phosphorylation as well as the ability to catalyze arsenolysis of acetyl-CoA were studied in wild type ACD and several mutant enzymes. The data indicate that the formation of enzyme-bound acetyl phosphate and enzyme phosphorylation at His-257alpha, respectively, proceed in analogy to SCS. In contrast to SCS, in ACD the phosphoryl group is transferred from the His-257alpha to ADP via transient phosphorylation of a second conserved histidine residue in the beta-subunit, His-71beta. It is proposed that ACD reaction follows a novel four-step mechanism including transient phosphorylation of two active site histidine residues:

FIGURE 1.

Comparative illustration of subunit composition and domain organization in E. coli SCS (a) and ACD from P. furiosus (b). The domains are specified and numbered as described in the introduction. The conservation of the histidine residues in α- and β-subunit are highlighted with the bar. c, detailed sequence alignment of the sequence region of the conserved histidine residues in α- and β-subunit of ACD and SCS. The histidine in the α-subunit conserved in SCS and ACD is highlighted by a red background, the conserved second histidine in the β-subunit of ACD is shown on a green background.

FIGURE 2.

Ribbon representation of the P. furiosus ACD model (top) in comparison to the crystal structure of the E. coli SCS (bottom). The domain organization of ACD and SCS is illustrated as in Fig. 1, and the same colors are used. The connected CoA binding and CoA ligase domains corresponding to the α-subunit in SCS are colored red, and the ATP-grasp fold comprising the domains 3 and 4, which represent the β-subunit in ACD, is shown in green. The second CoA ligase domain (domain 5) C-terminally attached to the α-subunit in ACD, whereas comprising the C terminus of the β-subunit in SCS, is shown in cyan. One of the two active sites in each of both proteins is highlighted by a circle. The figure was drawn using the RIBBON program (26).

FIGURE 3.

Close-up view of the active site in the model of the P. furiosus ACD. Colors are the same as used in Figs. 1 and 2. The two active site histidine residues are shown as ball-and-stick models as well as the Glu-218α and the Asp-212β. Also, coenzyme A and the phosphate ion bound to the α-subunit as well as ADP bound to the β-subunit are represented by ball-and-stick models. The magnesium ion is shown as a shaded sphere. The figure was drawn using the RIBBON program (26).

FIGURE 4.

Time course for the phosphorylation of wild type (wt) ACD and the wild type α-subunit alone as well as of the His²⁵⁷αD and His⁷¹βA mutant proteins as well as the Glu²¹⁸α, and the Asp²¹²β mutants. The proteins were incubated in the presence of succinyl-CoA and ³²P_i (left panel) and of [γ-³²P]ATP (right panel). At the times indicated samples were taken and applied to SDS-PAGE. Gels were dried on Whatman paper and analyzed by phosphorimaging (see “Experimental Procedures”).

FIGURE 5.

Superimposition of the loop containing the conserved His⁷¹β and Lys⁷²β and the neighboring β sheets of the P. furiosus ACD model (green) on the crystal structure of SCS (PDB code 1CQI) (yellow). The side chains of His-71β and K⁷²β of ACD and the Arg-54β of SCS are highlighted as ball-and-stick models.