1-Aminocyclopropane-1-carboxylic acid oxidase: insight into cofactor binding from experimental and theoretical studies

Abstract

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.

Identification and active site analysis of the 1-aminocyclopropane-1-carboxylic acid oxidase catalysing the synthesis of ethylene in Agaricus bisporus

“…6A). These results are similar to those reported for tomato ACO 523 [46]. Lehrer [54] and Horrocks [55] proposed that the mechanism of the tryptophan fluorescence quenching upon iron binding was strong.…”

“…Additions of 45 μM Fe 2+ led to an~60% decrease of fluorescence inten-532 sity, which is comparable to the results in tomato ACO and TfdA (~80% 533 reduction) [46,47]. The data showed that Ab-ACO protein should have a 534 motif for Fe 2+ binding.…”

“…Intrinsic fluorescence of proteins is a powerful tool for 367 monitoring the microenvironment of Trp residues in proteins [44]. This368tool has been successfully used to study the binding of metals to several 369 proteins[45], including tomato ACO[46] and an ACO-related enzyme,370 2-OG-dependent dioxygenase TfdA [47]. Fig.…”

Catalytic Mechanisms of Fe(II)- and 2-Oxoglutarate-dependent Oxygenases

“…These enzymes possess a vicinal oxygen chelate fold that is unrelated to the DSBH fold, do not require 2OG, and carry out oxidative decarboxylation of the 2-oxo acid moiety of the substrate to generate the ferryl intermediate that is used in subsequent chemistry (81). Furthermore, three Fe(II)-dependent oxygenases possess the DSBH fold, but act independent of 2OG; isopenicillin N synthase converts the tripeptide ␦-(L-␣-aminoadipoyl)-L-cysteinyl-D-valine to isopenicillin N (20, 82), 1-aminocyclopropane-1-carboxylate oxygenase catalyzes the synthesis of ethylene (83), and the epoxide-forming enzyme HppE catalyzes the formation of fosfomycin from (2S)-hydroxypropylphosphonate (84,85). New members of the of Fe(II)/2OG-dependent oxygenase superfamily are certain to be discovered, and it is likely they too will utilize a ferryl intermediate to catalyze their novel oxidative transformations.…”

Ligand-dependent oxidation of copper bound α-amino-isobutyric acid as 1-aminocyclopropane-1-carboxylic acid oxidase mimics

“…For example, 1-aminocyclopropane-1-carboxylate (ACC) oxidase (ACCO) is a key enzyme that catalyzes the final step in the biosynthesis of the plant hormone ethylene [1][2][3][4][5][6][7][8][9][10]. ACCO has been classified as a member of a family of non-heme iron proteins, and suggested that the substrate oxidation proceeds through two successive monoelectronic oxidation steps including the formation of peroxoiron(III) and oxoiron(IV) intermediates [11][12][13][14][15][16][17]. However, there are only few reported functional models of ACCO.…”