Archaeal acetoacetyl-CoA thiolase/HMG-CoA synthase complex channels the intermediate via a fused CoA-binding site

Abstract

Many reactions within a cell are thermodynamically unfavorable. To efficiently run some of those endergonic reactions, nature evolved intermediate-channeling enzyme complexes, in which the products of the first endergonic reactions are immediately consumed by the second exergonic reactions. Based on this concept, we studied how archaea overcome the unfavorable first reaction of isoprenoid biosynthesis-the condensation of two molecules of acetyl-CoA to acetoacetyl-CoA catalyzed by acetoacetyl-CoA thiolases (thiolases). We natively isolated an enzyme complex comprising the thiolase and 3-hydroxy-3-methylglutaryl (HMG)-CoA synthase (HMGCS) from a fast-growing methanogenic archaeon, HMGCS catalyzes the second reaction in the mevalonate pathway-the exergonic condensation of acetoacetyl-CoA and acetyl-CoA to HMG-CoA. The 380-kDa crystal structure revealed that both enzymes are held together by a third protein (DUF35) with so-far-unknown function. The active-site clefts of thiolase and HMGCS form a fused CoA-binding site, which allows for efficient coupling of the endergonic thiolase reaction with the exergonic HMGCS reaction. The tripartite complex is found in almost all archaeal genomes and in some bacterial ones. In addition, the DUF35 proteins are also important for polyhydroxyalkanoate (PHA) biosynthesis, most probably by functioning as a scaffold protein that connects thiolase with 3-ketoacyl-CoA reductase. This natural and highly conserved enzyme complex offers great potential to improve isoprenoid and PHA biosynthesis in biotechnologically relevant organisms.

Structure and Catalytic Mechanism of a Bacterial Friedel–Crafts Acylase

“…Recently, the structure of an archaeal acetoacetyl‐CoA thiolase/HMG‐CoA synthase (HMGCS) complex from Methanothermococcus thermolithotrophicus was reported . The arrangement of the individual protein subunits within this complex is very similar to that in Pp ATase: the HMGCS subunit corresponds to PhlA in Pp ATase (sequence identity 38.5 %), whereas the thiolase subunit corresponds to PhlC (sequence identity 28.9 %).…”

“…The presence of the active Cys residue in the thiolase subunit is preserved in both complexes (Cys88 for PhlC and Cys85 in the thiolase subunit). Soaking of the thiolase/HMGCS complex with acetyl‐CoA (PDB ID: 6ESQ) revealed the binding of CoA at the subunit interface comprised by residues from all three proteins . This region is significantly different from its counterpart in the structure of Pp ATase (Figure S9), showing altered relative positions of individual secondary structure elements, the presence of additional residues in the PhlB region of Pp ATase, and two additional short β‐strands in HMGCS, as well as a lack of CoA‐binding residues in Pp ATase.…”

Evolution of a xenobiotic degradation pathway: formation and capture of the labile phthaloyl‐CoA intermediate during anaerobic phthalate degradation

“…Channeling of intermediates between different active sites in enzyme complexes has been described for several enzymatic reactions where labile or toxic intermediates are prevented from being released to the cytoplasm (Weeks et al ., ). A channeling of CoA ester intermediates has recently been reported for an acetoacetyl‐CoA thiolase/HMG‐CoA synthase complex (Vögeli et al ., ). In this case substrate channeling via complex formation has evolved to facilitate a thermodynamically unfavorable enzymatic reaction.…”

Unveiling the Binding Modes of the Crystallophore, a Terbium‐based Nucleating and Phasing Molecular Agent for Protein Crystallography

“…Such interaction mapping analyses demonstrate the versatile binding behavior of the Crystallophore and pave the way to ab etter understanding of its unique properties.Crystallophore:t his term represents an ew class of lanthanide complexes that act as powerful auxiliary for protein crystallography,t ackling simultaneously the two main bottlenecks of the field:o btaining good qualityc rystals and solving the phase problem. [1] Indeed the Crystallophore combines (i)astrong nucleatinge ffect, providing new crystallization conditions and leadingt oh igh-quality diffracting crystals; (ii)luminescence properties under UV-irradiation facilitating the derived protein crystalsi dentification,a nd (iii)straightforwardp hasing effect due to the exceptional anomalous properties of f-elements. Recently,t he first Crystallophore, noted Tb-Xo4 (Scheme 1) has been reported.I tconsists of acationic terbium complex featuring am acrocyclict riazacyclononane bis-picolinate ligand (L) exhibiting an excellent solubility and stabilityi nt he crystallization media.The search for additives to promote nucleationa nd production of single crystals is al ong-runnings tory in macromolecular crystallography.S everal solid additives have been tested to induce the nucleation including minerals dust, [2] naturalmaterials such as horse and humanh air powder, [3] or carbon nanotubes.…”