1-Aminocyclopropane-1-carboxylic acid oxidase: insight into cofactor binding from experimental and theoretical studies.
1-Aminocyclopropane-1-carboxylic acid oxidase (ACCO) is a nonheme Fe(II)-containing enzyme that is related to the 2-oxoglutarate-dependent dioxygenase family. The binding of substrates/cofactors to tomato ACCO was investigated through kinetics, tryptophan fluorescence quenching, and modeling studies. α-Aminophosphonate analogs of the substrate (1-aminocyclopropane-1-carboxylic acid, ACC), 1-aminocyclopropane-1-phosphonic acid (ACP) and (1-amino-1-methyl)ethylphosphonic acid (AMEP), were found to be competitive inhibitors versus both ACC and bicarbonate (HCO(3)(-)) ions. The measured dissociation constants for Fe(II) and ACC clearly indicate that bicarbonate ions improve both Fe(II) and ACC binding, strongly suggesting a stabilization role for this cofactor. A structural model of tomato ACCO was constructed and used for docking experiments, providing a model of possible interactions of ACC, HCO(3)(-), and ascorbate at the active site. In this model, the ACC and bicarbonate binding sites are located close together in the active pocket. HCO(3)(-) is found at hydrogen-bond distance from ACC and interacts (hydrogen bonds or electrostatic interactions) with residues K158, R244, Y162, S246, and R300 of the enzyme. The position of ascorbate is also predicted away from ACC. Individually docked at the active site, the inhibitors ACP and AMEP were found coordinating the metal ion in place of ACC with the phosphonate groups interacting with K158 and R300, thus interlocking with both ACC and bicarbonate binding sites. In conclusion, HCO(3)(-) and ACC together occupy positions similar to the position of 2-oxoglutarate in related enzymes, and through a hydrogen bond HCO(3)(-) likely plays a major role in the stabilization of the substrate in the active pocket.
Aix-Marseille Université and CNRS, Institut des Sciences Moléculaires de Marseille, UMR 7313, Marseille, France.
Lydie
L
Brisson
Nadia
N
El Bakkali-Taheri
Michel
M
Giorgi
Antoine
A
Fadel
József
J
Kaizer
Marius
M
Réglier
Thierry
T
Tron
El Hassan
el H
Ajandouz
A Jalila
AJ
Simaan
D000594:Amino Acid Oxidoreductases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D000598:Amino Acids, Cyclic / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D001205:Ascorbic Acid / Q000737:chemistry / Q000494:pharmacology; D001665:Binding Sites / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D006860:Hydrogen Bonding; D007700:Kinetics; D018551:Solanum lycopersicum / Q000201:enzymology; D008958:Models, Molecular; D015394:Molecular Structure; D010757:Phosphorous Acids / Q000737:chemistry / Q000494:pharmacology; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D017693:Sodium Bicarbonate / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship
D000598:Amino Acids, Cyclic; D010757:Phosphorous Acids; D011994:Recombinant Proteins; C023863:1-aminocyclopropane-1-carboxylic acid; D017693:Sodium Bicarbonate; D000594:Amino Acid Oxidoreductases; C059460:1-aminocyclopropane-1-carboxylic acid oxidase; D001205:Ascorbic Acid