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 alpha-ketobutyric acid and ammonia as the products. This ring cleavage reaction is unusual because the substrate, ACC, contains no abstractable alpha-proton and the carboxyl group is retained in the product. How the reaction is initiated to generate an alpha-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 alpha-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 A) 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 alpha-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 beta-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.
Karthikeyan
Subramanian
S
Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9038, USA.
Zhou
Qingxian
Q
Zhao
Zongbao
Z
Kao
Chai-Lin
CL
Tao
Zhihua
Z
Robinson
Howard
H
Liu
Hung-wen
HW
Zhang
Hong
H
D001665:Binding Sites / Q000235:genetics; D002087:Butyrates / Q000737:chemistry; D019755:Carbon-Carbon Lyases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D002384:Catalysis; D003460:Crystallization; D018360:Crystallography, X-Ray; D003521:Cyclopropanes / Q000737:chemistry*; D003959:Diamines / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000737:chemistry; D005998:Glycine / Q000031:analogs & derivatives* / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D010843:Pichia / Q000201:enzymology; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics; D011732:Pyridoxal Phosphate / Q000737:chemistry*; D040681:Structural Homology, Protein; D013379:Substrate Specificity / Q000235:genetics; D014443:Tyrosine / Q000235:genetics; D015091:beta-Alanine / Q000031:analogs & derivatives* / Q000737:chemistry
D002087:Butyrates; D003521:Cyclopropanes; D003959:Diamines; D004791:Enzyme Inhibitors; D015091:beta-Alanine; C006977:3-chloroalanine; D014443:Tyrosine; C006507:vinylglycine; D011732:Pyridoxal Phosphate; C005087:alpha-ketobutyric acid; C030797:cyclopropane; C033634:1-aminocyclopropane-1-carboxylate deaminase; D019755:Carbon-Carbon Lyases; D005998:Glycine