Reaction Intermediate Structures of 1-Aminocyclopropane-1-carboxylate Deaminase

Abstract

The pyridoxal 5 -phosphate-dependent enzymes have been evolved to catalyze diverse substrates and to cause the reaction to vary. 1-Aminocyclopropane-1-carboxylate deaminase catalyzes the cyclopropane ring-opening reaction followed by deamination specifically. Since it was discovered in 1978, the enzyme has been widely investigated from the mechanistic and physiological viewpoints because the substrate is a precursor of the plant hormone ethylene and the enzymatic reaction includes a cyclopropane ring-opening. We have previously reported the crystal structure of the native enzyme. Here we report the crystal structures of the two reaction intermediates created by the mutagenesis complexed with the substrate. The substrate was validated in the active site of two forms: 1) covalent-bonded external aldimine with the coenzyme in the K51T form and 2) the non-covalent interaction around the coenzyme in the Y295F form. The orientations of the substrate in both structures were quite different form each other. In concert with other site-specific mutation experiments, this experiment revealed the ingenious and unique strategies that are used to achieve the specific activity. The substrate incorporated into the active site is reactivated by a two-phenol charge relay system to lead to the formation of a Schiff base with the coenzyme. The catalytic Lys 51 residue may play a novel role to abstract the methylene proton from the substrate in cooperation with other factors, the carboxylate group of the substrate and the electron-adjusting apparatuses of the coenzyme.The ␣-amino acid with a cyclopropane ring, 1-aminocyclopropane-1-carboxylate (ACC), 1 is known to be a key intermediate in the biosynthesis of the plant hormone ethylene (1), which is responsible for the initiation of fruit-ripening and for regulating many other plant developmental processes. In higher plants, the pathway of ethylene biosynthesis ( Fig. 1) is initiated with the conversion of the methionine to S-adenosylmethionine (2) by methionine adenosyltransferase followed by the conversion of S-adenosylmethionine to ACC by ACC synthase, pyridoxal 5Ј-phosphate (PLP)-dependent enzyme. ACC is oxidized by mononuclear iron dependent-ACC oxydase (3) to ethylene in the final step.In a soil bacterium, Pseudomonas sp. strain ACP, ACC was also converted to ␣-ketobutyrate and ammonia by a bacterial enzyme when ACC was the sole nitrogen source for the growth of this bacterium (4, 5). This degradation is catalyzed by another PLP-dependent enzyme, ACC deaminase (ACCD, EC 4.1.99.4), which has a special ability to break the cyclopropane ring preceding deamination (Fig. 2). The possibility of symbiosis between the plants and the soil bacterium has also been mentioned (6, 7). The introduction of ACCD in higher plants by gene technology reduced the production level of ethylene and delayed the ripening progression of fruits (8, 9). This enzyme has been isolated from a few strains of Pseudomonas species (5, 8, 10, 11), yeast Hansenula saturnus (12), and fungus Penicillium citrinum (13)...

Structural Analysis of 1‐Aminocyclopropane‐1‐Carboxylate Deaminase: Observation of an Aminyl Intermediate and Identification of Tyr 294 as the Active‐Site Nucleophile

“…In fact, the authors favored Route A as the mechanism for the yeast enzyme and assigned Lys 51, whose side chain orientation could only be deduced by modeling, as the base responsible for abstraction of the b proton. [15] This interpretation clearly differs from ours and warrants additional comment. A ringopening mechanism initiated by deprotonation is inconsistent with the results obtained from studies of 2-methylene-ACC (9).…”

“…[18] Ose et al have reported the crystal structure of the yeast ACC deaminase Y295F mutant complexed with ACC, which revealed that the bound ACC adopts a very different, nonproductive conformation in the mutant complex. [15] This result suggests that, in addition to its proposed catalytic role, the Tyr 294 residue of the Pseudomonas enzyme (or Tyr 295 in the yeast enzyme) may help to correctly position the bound substrate in the active site.…”

“…The catalytically more relevant structure 5 was recently observed by Ose et al in a complex of the yeast ACC deaminase K51T mutant and ACC (1) [15] in which a two-phenol relay system (Y269 and Y295 in the yeast enzyme) exists that involves a similar juxtaposition of residues to that shown in Figure 1 B. Comparison of the active site structure of the Pseudomonas enzyme (see Figure 1 B) with structure 5 of the yeast enzyme [15] reveals little difference in the protein backbone and a shift of about 1 on the part of PLP-ACC adduct. The Tyr 295 residue of the yeast enzyme is also found in proximity to the cyclopropane ring and could easily perform the proposed nucleophilic role.…”

The interconversion of ACC deaminase and d-cysteine desulfhydrase by directed mutagenesis

“…ACP and has since been characterized in a few other species, including the yeast Hansenula saturnus. A large portion of known biochemical properties of this enzyme can be attributed to the work done by Honma and co-workers (Walsh et al 1981;Honma 1985;Honma et al 1993;Minami et al 1998;Jia et al 1999;Ose et al 2003), supplemented by a few biochemical studies by other groups (Liu et al 1984;Jacobson et al 1994;Li et al 2001;Zhao et al 2003;Hontzeas et al 2004). ACC deaminase binds one pyridoxal phosphate (PLP) molecule at each subunit via a conserved lysine residue.…”

Methods to Study 1-Aminocyclopropane-1-carboxylate (ACC) Deaminase in Plant Growth-Promoting Bacteria

“…Honna and co-workers have intensively studied the biochemical properties of ACC deaminase (Honma and Shimomura 1978 ;Walsh et al 1981 ;Honma 1985 ;Honma et al 1993a , b ;Minami et al 1998 ;Jia et al 1999 ;Ose et al 2003 ). In addition a few other studies on this enzyme and its biochemical properties have been conducted by other groups (Liu et al 1984 ;Jacobson et al 1994 ;Li et al 1996 ;Zhao et al 2003 ;Hontzeas et al 2004 ).…”