The pimeloyl-CoA synthetase BioW defines a new fold for adenylate-forming enzymes.

Reactions that activate carboxylates through acyl-adenylate intermediates are found throughout biology and include acyl- and aryl-CoA synthetases and tRNA synthetases. Here we describe the characterization of Aquifex aeolicus BioW, which represents a new protein fold within the superfamily of adenylating enzymes. Substrate-bound structures identified the enzyme active site and elucidated the mechanistic strategy for conjugating CoA to the seven-carbon α,ω-dicarboxylate pimelate, a biotin precursor. Proper position of reactive groups for the two half-reactions is achieved solely through movements of active site residues, as confirmed by site-directed mutational analysis. The ability of BioW to hydrolyze adenylates of noncognate substrates is reminiscent of pre-transfer proofreading observed in some tRNA synthetases, and we show that this activity can be abolished by mutation of a single residue. These studies illustrate how BioW can carry out three different biologically prevalent chemical reactions (adenylation, thioesterification, and proofreading) in the context of a new protein fold.

Proofreading of noncognate acyl adenylates by an acyl-coenzyme a ligase.

Aminoacyl-tRNA synthetases remove (proofread) incorrect substrates and thereby prevent errors in protein synthesis. We report enzyme-catalyzed pretransfer editing by pimeloyl-coenzyme A (CoA) ligase (BioW), a biotin synthetic enzyme that converts pimelate, a seven-carbon dicarboxylic acid, to its CoA ester. The noncognate BioW substrate glutaric acid results in hydrolysis of ATP to AMP with formation of only trace amounts of glutaryl-CoA, thereby mimicking pretransfer editing of incorrect aminoacyl-adenylates by aminoacyl-tRNA synthetases.