Structure of the acetophenone carboxylase core complex: prototype of a new class of ATP-dependent carboxylases/hydrolases

Abstract

Degradation of the aromatic ketone acetophenone is initiated by its carboxylation to benzoylacetate catalyzed by acetophenone carboxylase (Apc) in a reaction dependent on the hydrolysis of two ATP to ADP and Pi. Apc is a large protein complex which dissociates during purification into a heterooctameric Apc(αα′βγ)2 core complex of 482 kDa and Apcε of 34 kDa. In this report, we present the X-ray structure of the Apc(αα′βγ)2 core complex from Aromatoleum aromaticum at ca. 3 Å resolution which reveals a unique modular architecture and serves as model of a new enzyme family. Apcβ contains a novel domain fold composed of two β-sheets in a barrel-like arrangement running into a bundle of eight short polyproline (type II)-like helical segments. Apcα and Apcα′ possess ATP binding modules of the ASKHA superfamily integrated into their multidomain structures and presumably operate as ATP-dependent kinases for acetophenone and bicarbonate, respectively. Mechanistic aspects of the novel carboxylation reaction requiring massive structural rearrangements are discussed and criteria for specifically annotating the family members Apc, acetone carboxylase and hydantoinase are defined.

A rare polyglycine type II‐like helix motif in naturally occurring proteins

“…The structure of Apc of Aromatoleum aromaticum αα′βγ revealed an unusual structural motif in the β‐subunit (ApcD) (Figure ) which is reminiscent to the basic pattern of a hexagonal PG II ‐helix array of synthetic polyglycines . It is composed of eight short and approximately linear chains of amino acids (Figure A) exhibiting typical PG II ‐helical dihedral angles, as verified by DSSP, PROSS and SEGNO (Figure ).…”

“…It is composed of eight short and approximately linear chains of amino acids (Figure A) exhibiting typical PG II ‐helical dihedral angles, as verified by DSSP, PROSS and SEGNO (Figure ). A central PG II ‐like helix (residues 581–585) is flanked by six further PG II ‐like helices (389–393, 421–427, 469–473, 476–480, 507–510, and 512–517) arranged as a distorted hexagon (note that some helices were attributed with slightly deviating start and end positions compared to Weidenweber et al 2017, due to a more thorough analysis of the hydrogen bonding networks and sequence conservation). Three of the surrounding PG II ‐like helices are oriented in parallel and three in antiparallel directions to the central one in an alternating manner (Figure B).…”

“…(A) Prediction of local conformations in the domain of Apcβ carrying the eight PG II helixes, as calculated by three different methods using the http://www.dsimb.inserm.fr/dsimb_tools/polyproline server. The individual PG II ‐helices are indicated by yellow shading (coordinates are from pdb 5L9W), α‐helices (H, h), 3 10 ‐helices (G) and π‐helices in red, strands (E, e) and isolated β‐bridge (B, b) in green, PP II ‐ and PG II ‐helices (P, p) in blue as well as loop/coils/turns (T) and bends (S) in gray. (B) Ramachandran‐Plot of the chain segments 389–393, 421–427, 430–435, 469–473, 476–480, 507–510, 512–517 and 581–585…”

“…Sequence comparisons among all members of the hydantoinase/carboxylase family comprising acetophenone carboxylases, acetone carboxylases and hydantoinases, indicate a high degree of conservation of the glycines in the predicted eight‐helix bundles of the orthologous subunits, but less conservation of the other residues of the helices (Supporting Information Figure S2). Therefore, the glycine‐rich segments serve as signature motif for the enzymes of the hydantoinase/carboxylase family …”

“…After crystallization and data collection at the Swiss‐Light source (Villigen) the X‐ray structure of the Apc core complex was determined at a resolution of ca. 3 Å by the SIRAS method . Refinement converged to R/R free factors of 20.2/24.0% and the root‐mean‐square deviations for bond length and bond angles were 0.007 Å and 1.01°, respectively.…”