The active site and substrate-binding mode of 1-aminocyclopropane-1-carboxylate oxidase determined by site-directed mutagenesis and comparative modelling studies

Abstract

The active site and substrate-binding mode of MD-ACO1 (Malus domestica Borkh. 1-aminocyclopropane-1-carboxylate oxidase) have been determined using site-directed mutagenesis and comparative modelling methods. The MD-ACO1 protein folds into a compact jelly-roll motif comprised of eight a-helices, 12 b-strands and several long loops. The active site is well defined as a wide cleft near the C-terminus. The co-substrate ascorbate is located in cofactor Fe2+-binding pocket, the so-called '2-His-1-carboxylate facial triad'. In addition, our results reveal that Arg244 and Ser246 are involved in generating the reaction product during enzyme catalysis. The structure agrees well with the biochemical and site-directed mutagenesis results. The three-dimensional structure together with the steady-state kinetics of both the wild-type and mutant MD-ACO1 proteins reveal how the substrate specificity of MD-ACO1 is involved in the catalytic mechanism, providing insights into understanding the fruit ripening process at atomic resolution.

Ethylene in induced conifer defense: cDNA cloning, protein expression, and cellular and subcellular localization of 1-aminocyclopropane-1-carboxylate oxidase in resin duct and phenolic parenchyma cells

“…1b; numbering of amino acid positions according to spruce ACO) and is believed to interact by strong charge-charge interactions (Seo et al 2004;Zhang et al 2004). The RXS motif is another common region of the family and binds 2-oxoglutarate (Roach et al 1995), but has been modeled by Seo et al (2004) to bind ascorbate in ACO (Arg-258-X-Ser-260; Fig. 1b).…”

“…For further structural comparison of conifer and angiosperm ACOs, we modeled the spruce and Douglas Wr ACO proteins with ACO from petunia, for which a crystal structure is available (Zhang et al 2004), and ACO from apple, which has previously been modeled on the crystal structure of a related isopenicillin-N-synthase (Seo et al 2004) and structurally characterized (Rocklin et al 1999;Zhou et al 2002;Tierney et al 2005) (Fig. 1b).…”

“…Nomenclature and GenBank protein accession numbers for all sequences shown in this alignment are provided in Table 2. b Schematic model of the spruce and Douglas Wr ACO active site modeled based on data for petunia and apple ACO (Rocklin et al 1999;Zhang et al 2004;Seo et al 2004). The conifer ACO model was created with MDL ISIS/Draw 2.5 software (http://www.mdl.com/index.jsp) indicated that the side chains corresponding to Tyr-304/ 300 are pointed toward the metal and may be involved in mediating electron transfer.…”

Ethylene Interacts with Abscisic Acid to Regulate Endosperm Rupture during Germination: A Comparative Approach UsingLepidium sativumandArabidopsis thaliana

“…The rooting was based on midpoint. Molecular phylogenetic analysis of the 13 Arabidopsis ACO and the three Lepidium ACO sequences was achieved by comparison of the amino acid sequence of the iron binding conserved domain, which is specific for the 2OG-Fe(II) oxygenase family (see Supplemental Figure 3 and Supplemental Data Set 9 online; Seo et al, 2004).…”

Regioselective alkylation of 1,3,4,5-tetrahydrobenzo[d]azepin-2-one and biological evaluation of the resulting alkylated products as potentially selective $$\hbox {5-HT}_{\mathrm{2C}}$$ 5-HT 2 C agonists

“…The obtained model was energy minimized by macro model module from Schrodinger and is evaluated for its quality. To evaluate the stereochemical quality of the minimized structure, PROCHECK [51] software was utilized in the study which showed the superiority of the optimized model over the initial model with an appreciable distribution of ψ and ϕ angels in Ramachandran plot. With the Sitemap module of Schrodinger, the active site of the receptor was identified [33].…”