Dual activities of ACC synthase: Novel clues regarding the molecular evolution of ACS genes

Abstract

Ethylene plays profound roles in plant development. The rate-limiting enzyme of ethylene biosynthesis is 1-aminocyclopropane-1-carboxylate (ACC) synthase (ACS), which is generally believed to be a single-activity enzyme evolving from aspartate aminotransferases. Here, we demonstrate that, in addition to catalyzing the conversion of S-adenosyl-methionine to the ethylene precursor ACC, genuine ACSs widely have C_β-S lyase activity. Two N-terminal motifs, including a glutamine residue, are essential for conferring ACS activity to ACS-like proteins. Motif and activity analyses of ACS-like proteins from plants at different evolutionary stages suggest that the ACC-dependent pathway is uniquely developed in seed plants. A putative catalytic mechanism for the dual activities of ACSs is proposed on the basis of the crystal structure and biochemical data. These findings not only expand our current understanding of ACS functions but also provide novel insights into the evolutionary origin of ACS genes.

Fig. 1.. AtACS7 has ACS and C_β-S lyase dual enzymatic activities both in vitro and in planta.

(A) Chemical reactions catalyzed by ACSs (top) or C_β-S lyases (bottom). (B) Chromatograms of NH4⁺ produced from the C_β-S bond cleavage of l-cystine, which is catalyzed by either PpACL1 or AtACS7 in vitro. (C) In vitro pyruvate production assays of PpACL1- or AtACS7-containing C_β-S lyase reaction systems. (D) In vitro ACS activity assays of PpACL1 and AtACS7. (E to G) The prominent accumulation of AtACS7 protein resulted in a strong triple-response phenotype, an elevated ethylene production, and a significant increase in the content of pyruvate in the etiolated seedlings of two independent AtACS7-overexpressing lines (AtACS7-ox-3-4 and AtACS7-ox-7-5). (H) The C_β-S lyase activity of purified AtACS7 was PLP dependent. (I) Exogenous AVG totally suppressed the C_β-S lyase activity of purified AtACS7. (J and K) Determination of the optimal temperature and pH for C_β-S lyase activity of purified AtACS7. (L) Estimation of kinetic parameters of C_β-S lyase activity of purified AtACS7 under the optimal temperature and pH conditions. The C_β-S lyase activities were measured by the generation of pyruvate using l-cystine as substrate. Data represent means ± SE (n ≥ 3, biological replicates). The number of biological replicates for each experiment is indicated in Materials and Methods. Asterisks indicate statistically significant differences based on Student’s t test (α = 0.01). For negative control (NC), blank buffer was used instead of purified proteins.

Fig. 2.. Structure of AtACS7.

(A) Illustration of AtACS7 dimer. (B) The active site of AtACS7. (C) Superposition of the AtACS7 dimer and MdACS1 dimer. (D) Comparison of the active sites of AtACS7 and MdACS1. In all the panels, the two AtACS7 subunits are colored yellow (chain C) and cyan (chain D), and the PPG molecules of AtACS7 are colored violet. The contents from MdACS1 are colored white for (C) and (D). See also table S1.

Fig. 3.. The C_β-S lyase activity of ACS proteins may be a common phenomenon.

(A) Apple MdACS1 has both ACS and C_β-S lyase activities, although they are both lower than those of AtACS7. (B to E) ACS proteins in other plant species, such as GmACS7-like in soybean (Phytozome: Glyma.05G223000), OsACS5 (GenBank: X97066.1) and OsACS1 (GenBank: M96673.1) in rice, and SlACS4 in tomato (GenBank: M88487.1), have ACS and C_β-S lyase dual activities. (F to H) ACS proteins in the model plant Arabidopsis, such as the type I AtACS6 (TAIR Locus: AT4G11280), type II AtACS8 [TAIR (The Arabidopsis Information Resource) Locus: AT4G37770], and AtACS11 (TAIR Locus: AT4G08040), have ACS and C_β-S lyase activities. Purified ACS proteins were used in all in vitro assays. Data are means ± SE (n ≥ 3, biological replicates). The number of biological replicates for each ACS protein is indicated in Materials and Methods. AtACS7 (TAIR Locus: AT4G26200) was used as a positive control, and protein extracts from E. coli harboring the empty vector were used as an NC.

Fig. 4.. The Q98 residue plays a substantial role in conferring ACS activity.

(A) In vitro C_β-S lyase and ACS activity assays of purified AtACS7 mutants. The activities of wild-type AtACS7 were regarded as 100%. Data represent means ± SE (n ≥ 3, biological replicates). The number of biological replicates for each protein is indicated in Materials and Methods. (B) Superimposition of the overall structure of AtACS7-R6 and wild-type AtACS7. The two AtACS7-R6 subunits are colored yellow and cyan, respectively, and the PPG molecules are colored violet. Wild-type AtACS7 is colored white, except for the 91-to-104 region, which is colored red. (C) Comparison of the active sites of AtACS7-R6 (colored) and wild-type AtACS7 (white). (D) Determination of in planta C_β-S lyase and ACS activities of the AtACS7^Q98A mutant by measuring the contents of pyruvate or ACC in the Agrobacterium-infiltrated tobacco leaves as described in Materials and Methods. Free eYFP was injected as an NC, while wild-type AtACS7 was used as a positive control. Data represent means ± SE (n = 6, biological replicates). Black asterisks indicate statistically significant differences in pyruvate contents, while a red asterisk indicates statistically significant difference in ACC contents compared with the eYFP control based on Student’s t test (α = 0.05). (E) Phylogenetic analysis of functional ACS proteins, ACS-like proteins, aminotransferases, and C_β-S lyases from a wide variety of organisms. The phylogenetic tree was constructed using the neighbor-joining method in MEGA X software. Numbers at each interior branch indicate the bootstrap values of 1000 replicates. The bar indicates a genetic distance of 0.2 cM. Detailed organisms and locus numbers or PDB IDs of all protein sequences are listed in table S2. All functional ACS proteins form a separate clade (red) containing the glutamine residue corresponding to Q98 of AtACS7. (F) Docking for SAM at the AtACS7 active site.

Fig. 5.. Catalytic mechanism of ACSs.

Reactions catalyzed by the dual activities of ACS diverge after the formation of external substrate aldimine. For the C_β-S lyase activity, the K285 of AtACS7 extracts the Cα proton of l-cystine and transfers it to the Sγ atom, thereby breaking the bond between Cβ and Sγ of l-cystine. A quinonoid intermediate is required in this process. Whereas for the ACS activity, a residue other than K285, probably Y160, is required to break the Cγ-Sδ bond of SAM. Following deprotonation of Cα by K285, a new covalent bond is formed between the Cα and Cγ to form ACC. In this process, the quinonoid intermediate is not essential.

Fig. 6.. The acquisition of two N-terminal motifs is a key event for ACS activity.

(A) MEME analysis was performed using Arabidopsis ACS proteins and the functionally confirmed ACS sequences from soybean, rice, and tomato in addition to P. patens C_β-S lyase PpACL1 and Arabidopsis aminotransferases AtACS10 and AtACS12 as controls. The results revealed nine motifs (ACS motifs 1 to 9) that are collectively required in a specific order for ACS activity. The motif present only in the two Arabidopsis aminotransferases AtACS10 and AtACS12 was named AAT. (B) Proposed structural model of the genuine ACS proteins contains nine conserved ACS motifs and the glutamine residue corresponding to Q98 of AtACS7 (indicated with a star and an arrow) in the second ACS motif. (C) Schematic diagram of N-terminal substitution between AtACS7 and PpACL1. The N7-PpACL1 recombinant protein is composed of the N-terminal sequence of AtACS7 and the C-terminal sequence of PpACL1. The purified recombinant protein N7-PpACL1 had no C_β-S lyase activity in vitro (D) but gained in vitro ACS activity (E). The empty vector pET28a and C_β-S lyase PpACL1 were used as controls. Data represent means ± SE (n = 3, biological replicates). See also table S3.

Fig. 7.. Validation of the proposed ACS model.

(A) Validation of the proposed ACS model using ACS-like proteins from plant species ranging from chlorophytes to angiosperms. (B) Validation of the proposed ACS model using functionally confirmed ACS proteins in the literatures. The plus symbols (+) represent the presence of identical ACS motifs or Q98 key residue and/or exhibiting ACS or C_β-S lyase activity, while the minus symbols (−) represent the absence of identical motifs or Q98 residue and/or no ACS or C_β-S lyase activity detected, respectively. Data shown for ACS activity determination were based on at least four biological replicates, while those for C_β-S lyase activity measurements were from at least three biological replicates. Raw data are presented in figs. S5 to S8. * indicates the special issue of AtACS1, which meets the model requirements but lacks a key asparagine residue as discussed in the text.