Structural analysis of Pseudomonas 1-aminocyclopropane-1-carboxylate deaminase complexes: Insight into the mechanism of a unique pyridoxal-5′- phosphate dependent cyclopropane ring-opening reaction

1-Aminocyclopropane-1-carboxylate (ACC) deaminase is a pyridoxal 5′-phosphate (PLP) dependent enzyme catalyzing the opening of the cyclopropane ring of ACC to give α-ketobutyric acid and ammonia as the products. This ring cleavage reaction is unusual because the substrate, ACC, contains no abstractable α-proton and the carboxyl group is retained in the product. How the reaction is initiated to generate an α-carbanionic intermediate, which is the common entry for most PLP-dependent reactions, is not obvious. To gain insight into this unusual ring-opening reaction, we have solved the crystal structures of ACC deaminase from Pseudomonas sp. ACP in complex with substrate ACC, an inhibitor, 1-aminocyclopropane-1-phosphonate (ACP), the product α-ketobutyrate, and two D-amino acids. Several notable observations of these structural studies include the following: (1) a typically elusive gem-diamine intermediate is trapped in the enzyme complex with ACC or ACP; (2) Tyr294 is in close proximity (3.0 Å) to the pro-S methylene carbon of ACC in the gem-diamine complexes, implicating a direct role of this residue in the ring-opening reaction; (3) Tyr294 may also be responsible for the abstraction of the α-proton from D-amino acids, a prelude to the subsequent deamination reaction; (4) the steric hindrance precludes accessibility of active site functional groups to the L-amino acid substrates and may account for the stereospecificity of this enzyme toward D-amino acids. These structural data provide evidence favoring a mechanism in which the ring cleavage is induced by a nucleophilic attack at the pro-S β-methylene carbon of ACC, with Tyr294 as the nucleophile. However, these observations are also consistent with an alternative mechanistic possibility in which the ring opening is acid-catalyzed and may be facilitated by charge relay through PLP, where Tyr294 functions as a general acid. The results of mutagenesis studies corroborated the assigned critical role for Tyr294 in the catalysis.