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 ²,‡ Subramanian Karthikeyan, § Qingxian Zhou, § Zongbao Zhao, | Chai-Lin Kao, | Zhihua Tao, | Howard Robinson, ⊥ Hung-wen Liu,* ,| and Hong Zhang* ,§ Department of Biochemistry, UniVersity of Texas Southwestern Medical Center, Dallas, Texas 75390-9038, DiVision of Medicinal Chemistry, College of Pharmacy, and Department of Chemistry and Biochemistry, UniVersity of Texas, Austin, Texas 78712, and National Synchrotron Light Source, BrookhaVen National Laboratory, Upton, New York 11973 ReceiVed June 1, 2004; ReVised Manuscript ReceiVed August 12, 2004 ABSTRACT: 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 R-ketobutyric acid and ammonia as the products. This ring cleavage reaction is unusual because the substrate, ACC, contains no abstractable R-proton and the carboxyl group is retained in the product. How the reaction is initiated to generate an R-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 R-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 R-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 -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. 1-Aminocyclopropane-1-carboxylate (ACC, 1 1, Scheme 1) is the precursor for the biosynthesis of ethylene, a plant hormone that regulates fruit ripening and other aspects of plant growth and development (1, 2). In higher plants, ACC is formed from S-adenosylmethionine (SAM) via a ring- closing γ-displacement reaction catalyzed by ACC synthase (3, 4). ACC is then oxidized by ACC oxidase to form ethylene (2, 5). In several bacteria and yeast species, ACC can also be converted to R-ketobutyrate (R-KB, 2) and ammonia by ACC deaminase (6, 7). It has been shown that introduction of bacterial ACC deaminase into higher plants by gene technology reduces the production of ethylene, delays the ripening progression of fruits, and extends the shelf life of fruits and vegetables (8, 9). ACC deaminase is a pyridoxal 5′-phosphate (PLP) de- pendent enzyme catalyzing an unusual cyclopropane ring- opening reaction. For most PLP-dependent enzymes, the reaction starts with the conversion of an internal aldimine between PLP and an active site lysine residue (such as 3) to an external aldimine between PLP and the amino group of the substrate (such as 5). The next step is the removal of the R-proton of the substrate, which triggers subsequent trans- ² This work was supported in part by grants from the National Institutes of Health (GM63689 to H.Z. and GM40541 to H.-w.L.). ‡ The atomic coordinates and the structure factors for Pseudomonas ACC deaminase complexes have been deposited in the Protein Data Bank (PDB; http://www.rcsb.org/pdb/). The accession codes are 1tyz (native enzyme), 1tz2 (complex with ACC), 1tzm (complex with -Cl- D-Ala), 1tzj (complex with D-vinylglycine), and 1tzk (complex with R-ketobutyric acid). * To whom correspondence should be addressed. Tel: 214-648-9299 (H.Z.) and 512-232-7811 (H.-w.L.). Fax: 214-648-9099 (H.Z.) and 512- 471-2746 (H.-w.L.). E-mail: zhang@chop.swmed.edu (H.Z.) and h.w.liu@mail.utexas.edu (H.-w.L.). § University of Texas Southwestern Medical Center. | University of Texas. ⊥ Brookhaven National Laboratory. 1 Abbreviations: ACC, 1-aminocyclopropane-1-carboxylic acid; ACP, 1-aminocyclopropane phosphonate; -Cl-D-Ala, -chloro-D- alanine ((2S)-3-chloroalanine); DMAPP, dimethylallyl diphosphate; HOMO, highest occupied molecular orbital; IPP, isopentenyl diphos- phate; R-KB, R-ketobutyric acid; MACC, 1-amino-2-methylenecyclo- propane-1-carboxylic acid; NADH, -nicotinamide adenine dinucle- otide, reduced form; PCR, polymerase chain reaction; PEG, poly(ethylene glycol); PLP, pyridoxal 5′-phosphate; SAM, S-adenosylmethionine; SDS, sodium dodecyl sulfate; TMS, tetramethylsilane; D-VG, D- vinylglycine ((2R)-vinylglycine); rmsd, root-mean-square deviation. 13328 Biochemistry 2004, 43, 13328-13339 10.1021/bi048878g CCC: $27.50 © 2004 American Chemical Society Published on Web 09/29/2004