JXB Advance Access originally published online on June 27, 2005
Journal of Experimental Botany 2005 56(418):2203-2210; doi:10.1093/jxb/eri220
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Published by Oxford University Press [2005] on behalf of the Society for Experimental Biology.
RESEARCH PAPER |
Tyr152 plays a central role in the catalysis of 1-aminocyclopropane-1-carboxylate synthase
1Department of Biology, The Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong SAR, China
2Key Laboratory of Gene Engineering of the Ministry of Education, Zhongshan University, Guangzhou 510275, China
* To whom correspondence should be addressed. Fax: +852 2358 1559. E-mail: boningli{at}ust.hk
Received 20 January 2005; Accepted 5 May 2005
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Abstract |
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1-Aminocyclopropane-1-carboxylate (ACC) synthase is a key enzyme in the regulation of ethylene biosynthesis in higher plants. To investigate the catalytic significances of two conserved tyrosine residues, Tyr151 and Tyr152, of a tomato ACC synthase isozyme (LeACS2), five ACC synthase mutants (Y151F, Y151G, Y152F, Y152G, and Y151F/Y152F) were constructed and over-expressed in Escherichia coli. Subsequent kinetic analysis indicated that these point mutations in mutants Y152F, Y152G, and Y151F/Y152F, either reduced the catalytic efficiency more than 98% or fully inactivated ACC synthase, while Y151F and Y151G mutants reduced the enzymatic activities by 27% and 83%, respectively. It is therefore concluded that Tyr152, especially its hydroxyl group, plays an essential role in the catalysis of ACC synthase. Thus, a revised catalytic model is hereby proposed for functional ACC synthase.
Key words: ACC formation, ACC synthase, catalysis, kinetic analysis, site-directed mutagenesis, tyrosine
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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The simplest phytohormone, ethylene, has profound and diverse effects on the plant growth and development (Yang and Hoffman, 1984
; Johnson and Ecker, 1998). One of the key enzymes that regulate ethylene biosynthesis is 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (Kende, 1993). This enzyme converts the ubiquitous precursor S-adenosyl-L-methionine (SAM) to ACC, which is then converted to ethylene by ACC oxidase (Yang and Hoffman, 1984). ACC synthase was found to be encoded by a multigene family and the expression of these enzymes was regulated differentially by various developmental, environmental, and hormonal signals (Barry et al., 2000; Ge et al., 2000; Llop-Tous et al., 2000). However, the primary amino acid sequences of ACC synthase isomers retain the conservation from 50–96% (Rottmann et al., 1991).
As a member of pyridoxal 5'-phosphate (PLP)-dependent enzymes, ACC synthase catalyses ACC formation through 
, 
-elimination (Adams and Yang, 1979; Ramalingam et al., 1985). Based upon the crystal structure of apple ACC synthase and the active-site identified by radio-labelling, a catalytic mechanism for the production of ACC from SAM was proposed which involves the following steps (Yip et al., 1990; Capitani et al., 1999): the 
-amino group of the active site lysine of ACC synthase forms a Schiff base with the cofactor PLP, and then the amino group of the substrate SAM replaces the active site lysine to form the Schiff base with PLP. The C proton of SAM is subsequently attracted by the -amino group of active site lysine to form a carbanion, which in turn attacks the C of SAM to generate the imine of ACC. The resultant ACC is eventually released by the transimination of the active site lysine of ACC synthase. In this catalytic model, the protonation on the C of SAM may be stabilized efficiently by the quinonoid intermediate of pyridine as it is in most of other PLP-dependent enzymes (McCarthy et al., 2001). However, the -amino group of active site lysine of ACC synthase or the electron pair of the C-N double bond in the quinonoid intermediate is less likely to have a significant impact on the -elimination that is located three bonds away from the C of SAM. Therefore, a revised catalytic model was adopted according to the results from a recent X-ray crystallography study of a tomato ACC synthase, LeACS2 (Huai et al., 2001). In this model, the oxygen of the hydroxyl group of Tyr152 is located 3.7 Å away from C of SAM, and thus its lone pair of electrons may make the C slightly electron rich so as to weaken the C-S bond and facilitate the , -elimination. Although Tyr152 has been reported to be necessary for ACC synthase activity, as shown by a loss of >95% activity in the Y152N mutant (Tarun et al., 1998), the participation of Tyr152 in the catalysis rather than the folding of ACC synthase remained to be elucidated.
Furthermore, Tyr151 and Tyr152 are highly conserved among all functional ACC synthases reported so far. Since the catalytic roles of critical residues such as Tyr92, Lys278, and Arg286 of ACC synthase (LeACS2), have been successfully elucidated by site-directed or random mutagenesis investigations (White et al., 1994; Tarun et al., 1998; Zhou et al., 1999; McCarthy et al., 2001), the site-directed mutagenesis approach was followed in order to study the roles of Tyr151 and Tyr152 in ACC synthase catalysis and the biochemical data on both the wild-type and mutants of tomato ACC synthase (LeACS2) is presented. These results provide novel insights into the general mechanism underlying the catalysis of ACC synthase.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Modelling of SAM to the active site
The substrate SAM was modelled on the basis of the structures of LeACS2 in complex with AVG (Huai et al., 2001
) and apple ACS in complex with an amino-oxy analogue (Capitani et al., 2003). SAM was manually fitted into the active site of LeACS2 by program O (Jones et al., 1991) and the conformation of SAM is refined by program CNS (Brunger et al., 1998).
Construction of the recombinant plasmids pET-ACS-GST and pET-mACS-GST
A cDNA fragment encoding LeACS2 was synthesized using RT-PCR with a pair of primers: ACSN (forward), 5'-CGCATATGGGATTTGAGATTGCAAAG-3' (the underlined is a NdeI site), ACSB (reverse), 5'-AAGGATCCACGAACTAATGGTGAGGGAGGA-3' (the underlined is a BamHI site). The LeACS2 cDNA was digested with NdeI/BamHI and inserted into the pET30a vector (Novagen) to produce pET-ACS-GST.
To carry out the site-directed mutagenesis in the LeACS2 enzyme, the RT-PCR product of the LeACS2 gene was cloned into the pMD18-T vector (Takara) and point mutations were introduced into the LeACS2 protein using the MutanBEST Kit (Takara) according to the manufacturer's protocol. The PCR primers used in the mutagenesis are: Y151F(F): 5'-GTACCTTCACCATTCTACCCAGCATTT-3', Y151G(F): 5'-GTACCTTCACCAGGCTACCCAGCATTT-3', Y152F(F): 5'-GTACCTTCACCATACTTCCCAGCATTT-3', Y152G(F): 5'-GTA CCTTCACCATACGGCCCAGCATTT-3', Y151FY152F(F): 5'-GTACCTTCACCATTCTTCCCAGATTT-3'; Mutant(R): 5'-TAAAAATGCATCGCCAGGATCAGC-3' (the underlined are the mutated nucleotides). EcoRI/BamHI fragment of the wild-type LeACS2 cDNA on pET-ACS-GST was replaced individually with EcoRI/BamHI fragments of pMD18-T-mACS containing different point mutations.
Over-expression and purification of the wild-type and mutant enzymes
E. coli BL21 starTM (DE3) pLysS cells (Invitrogen) carrying the recombinant plasmids pET-ACS-GST and pET-mACS-GST were grown in 2x YT liquid medium containing 20 mg ml–1 kanamycin and 0.1 mM IPTG at 30 °C for 5 h. The cells were harvested by centrifugation and then disrupted by sonication on ice. The supernatant of the cell lysate was subjected to GST affinity column purification. The resultant wild-type and mutant LeACS2-GST fusion proteins were digested by Factor Xa and the consequent LeACS2 enzyme was further purified by MonoQ chromatography.
Determination of protein concentration and western blot analysis
Protein concentration was determined using the method of Bradford (Bradford, 1976). Bovine serum albumin was used as a protein standard. For western blot analysis, GST hybrid proteins were resolved on SDS-PAGE and followed by immunoblotting using anti-GST antibodies (Cell Signalling Technology) as primary antibodies. The signals of the target proteins on the western blots were visualized by DAB Chromogen (Sigma).
Enzyme kinetic analysis
The enzyme samples were incubated individually with various concentrations of SAM in an assay buffer containing 50 mM EPPS, pH 8.5, 10 µM PLP, and 2 mM DTT at 30 °C for 30 min. The reaction was terminated with 10 µl of 100 mM HgCl2. ACC was determined using the method of Lizada and Yang (Lizada and Yang, 1979). Five repetitions were performed at each data point. The kinetic parameters were calculated using the software Enzyme Kinetics Pro (ChemSW, Fairfield, CA) according to the Michaelis-Menten equation.
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Model for ACC synthase–SAM complex and alignment analysis of Tyr151 and Tyr152 of LeACS2
How ACC synthase binds to SAM has been an unresolved issue even though several crystal structures of ACC synthase have been deciphered (Capitani et al., 1999
, 2002, 2003; Huai et al., 2001). Based on the crystal structure of tomato ACS (LeACS2) in complex with cofactor PLP and inhibitor AVG (Huai et al., 2001) and that of apple ACS in complex with a SAM analogue (Capitani et al., 2003), the substrate SAM was modelled into the active site of LeACS2 (Fig. 1A). The adenine ring of SAM stacks against the Tyr92 residue from the neighbouring chain through a pi-pi interaction. The ribose group of SAM interacts with Ala154 and Arg157 of the same monomer, and may also interact with Tyr92 from the neighbouring monomer. The guanidine group of Arg157 may form a hydrogen bond with the ribose oxygen. The methionine portion of SAM contacts residues Leu53, Ala54, Tyr152, Lys278, and Arg412 as well as PLP. These interactions of SAM with LeACS2 are similar to the earlier models proposed for SAM-binding and SAM is able to form the external aldimine intermediate (Huai et al., 2001; McCarthy et al., 2001; Capitani et al., 2002). Our model showed that the oxygen of the OH group of Tyr152 is positioned 
3.7 Å from the C of SAM, suggesting a potential interaction between Tyr152 and the C of SAM. In addition, the amine group of the methionine portion of SAM is close to PLP so as potentially to form an external aldimine intermediate.
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Multiple sequence alignment has proved to be effective for predicting the essential residues of proteins that functionally and structurally resemble one another. In the tomato plant, there are at least nine ACC synthase genes (LeACS1A, LeACS1B, and LeACS2-8) identified so far (Van der Straeten et al., 1990
; Zarembinski and Theologis, 1993; Oetiker et al., 1997; Shiu et al., 1998). The alignment of ACC synthases from tomato and other plant species showed that both Tyr151 and Tyr152 of LeACS2 are two highly conserved residues among various ACC synthase isozymes from both monocotyledons and dicotyledons (Fig. 1B). Two tyrosines are found at these positions in those reported functional ACC synthases (see website http://www.ncbi.nlm.nih.gov/ for references). One exception is AtACS10, which is an ACC synthase isozyme from Arabidopsis newly defined by Yamagami et al. (2003). In this defined ACC synthase, a cysteine and a serine replace the two corresponding tyrosine residues. However, their experimental data showed that AtACS10 functions as an aminotransferase instead of an ACC synthase (Yamagami et al., 2003). Thus, the high conservation of these two tyrosines among all the functional ACC synthases tested thus far suggests potentially important roles for these residues in either the catalysis of ACC formation or in enzyme folding.
Mutagenesis of Tyr152 and determination of kinetics
Tyr151 and Tyr152 were replaced individually by either a glycine or a phenylalanine through site-directed mutagenesis. These substitutions presumably generated either a maximal or a minimal conformational change in ACC synthase. In addition, both tyrosines were concurrently mutated to phenylalanines. The point mutations were confirmed by DNA sequencing of the full-length mutant LeACS2 gene (mACS). The resultant wild-type and five mutant LeACS2 enzymes were overproduced in E. coli as GST fusion proteins (Fig. 2A) to facilitate the purification of these enzymes. The expression levels of wild-type and mutant fusion proteins were determined to be similar (Fig. 2). All enzymes were purified using the methods described in the Materials and methods. Removal of the GST tag was thought to eliminate the potential interference from the GST tag in the determination of the enzyme kinetic parameters. SDS–PAGE and immunoblotting analysis with anti-GST antibodies revealed an 83 kDa ACS-GST fusion protein and a 55 kDa LeACS2 enzyme (Fig. 2B, C). These molecular weights are consistent with the sizes deduced from their amino acid sequences.
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The enzyme kinetic curves and the biochemical parameters of the wild-type and mutant LeACS2 are presented in Fig. 3 and Table 1, respectively. The wild-type LeACS2 exhibited a Km of 31.5 µM for SAM and a Kcat of 1.41 s–1 (Table 1). This result is closely in accord with that previously reported (Li and Mattoo, 1994
).
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The Kcat/Km ratio of LeACS2 mutants, Y151F and Y151G, were determined to be 73% and 17% (Table 1) of the wild-type enzyme, respectively, indicating that removal of the OH group from Tyr151 reduced the catalytic efficiency but retained a similar turnover number (Table 1). The substitution of an aromatic ring and an OH group of Tyr151 generated mutant ACC synthases similar to Class II mutant ACC synthases, which were defined to maintain 5–50% of the wild-type ACC synthase activity (Tarun et al., 1998
). Based on random mutagensis of ACC synthase (Tarun et al., 1998), a total of 125 Class II ACC synthase mutants were found. Although those Class II point mutations were identified as located within both the highly conserved and non-conserved regions, none of those point mutations was found at Tyr151. This may be due to the fact that the random substitution of amino acid in the LeACS2 enzyme was not saturated. Given that amino acid substitutions in some cases are unable to inactivate the enzyme activity (Warren et al., 1996), it has been proposed that these residues might not play important roles in catalysis. In addition, because removal of the aromatic ring of Tyr151 in the Y151G mutant increases Km of ACC synthase 5.3-fold while its Kcat remains nearly the same (Table 1), it is thus believed that Tyr151 may play a role in the formation of the active site rather than in catalysis. Furthermore, because the OH group of Tyr151 orients away from SAM, it is therefore less likely that the OH group of Tyr151 has a direct interaction with SAM and participates in catalysis. In conclusion, Tyr151 should act as a scaffold to form the active site and place the Tyr152 close to SAM.
By contrast with Tyr151, when the OH group of Tyr152 was removed by substituting Tyr152 with Phe152 in the Y152F mutant, this substitution increased Km of ACC synthase 16.3-fold and reduced Kcat to about one-third of that of the wild type (Table 1). Therefore, removal of a hydroxyl group from ACC synthase at Tyr152 decreased its catalytic efficiency (Kcat/Km) more than 98% (Table 1). More significantly, when Tyr152 was substituted with Gly, no measurable ACC synthase activity was detected. Even when a larger amount of Y152G mutant enzyme (0.6 µg) was used in the ACC synthase activity assay, no reliable biochemical parameters were determined. The sensitivity of Tyr152 to conservative substitution, such as Y152F mutation, strongly suggests the importance of this residue in ACC synthase catalysis because the conservative substitutions are generally believed to be less disruptive to the protein function (Terwilliger et al., 1994). It is therefore indicative that the hydroxyl group of Tyr152 should be involved in both the substrate-binding and catalysis of SAM to ACC.
Double mutations in mACS, Y151FY152F, further decreased ACC synthase turnover number and catalytic efficiency to 0.176% and 1% of the wild type, respectively (Table 1). These results confirmed the role of Tyr152 in ACC synthase catalysis.
Putative mechanism of the ACC synthase catalysis
A couple of mechanisms have been reported for the ACS catalysis (Huai et al., 2001; McCarthy et al., 2001). The -elimination in general requires a nucleophilic attack on C of SAM. One proposal is that the electron pair of the C-N double bond in the external aldimine intermediate of SAM-PLP interacts with C of SAM to facilitate the break of the C-S bond (McCarthy et al., 2001). However, this argument was challenged by the difficulty of propagation of the electron pair through two single C-C bonds. Alternatively, the nucleophilic attack was initiated by the protein residue Tyr152 (Huai et al., 2001). This mechanism is consistent with the pathway of transmethylations involving SAM. As it was extensively reviewed previously, the bond breaks around the sulphur of SAM and starts with the nucleophilic attack on the carbon, and the electron pair in atoms of sulphur, oxygen, nitrogen, or carbon-carbon double bonds will serve as the nucleophile (Walsh, 1979). Thus, Tyr152 is a strong candidate as the nucleophile because of its spatial proximity to C of SAM. The mutagenesis experiment that the Y152F mutation, resulting in a loss of the OH group from Y152, lost 98.3% activity supports the argument. The residual amount of activity in the Y152F mutant may come from the role of the electron pair in the carbon-carbon double bonds (Walsh, 1979) of the phenyl residue, in which removal of the Tyr152 phenyl group in the Y152G mutant fully abolishes enzymatic activity.
In conclusion, site-directed mutagenesis helped to pinpoint the catalytic significance of the active site Tyr152 residue in LeACS2 function. Notably, the absence of a single OH group from a 55 kDa LeACS2 enzyme resulting in the loss of 98.3% enzymatic activity strongly suggests that this hydroxyl group of Tyr152 is crucial in both SAM binding and catalysis. A putative Tyr152-mediated catalytic mechanism is presented here to demonstrate a catalysis model (Fig. 4). In this model, the hydroxyl group of Tyr152 would perform a pivotal role to promote the , -elimination of the substrate and the sequential formation of ACC. This catalytic mechanism may be applicable in functional ACC synthase identified thus far.
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Acknowledgements |
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This work was supported by grants from the Hong Kong Research Grant Council (HKUST6276/03M, HKUST6102/02M, HKUST6105/01M) and the China National Science Foundation (No. 30129001) awarded to Dr Ning Li. We thank Dr H Ke for his contribution in editing and proofreading this manuscript.
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Footnotes |
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Abbreviations: SAM, S-adenosylmethionine; ACC, 1-aminocyclopropane-1-carboxylic acid; ACS, ACC synthase; PLP, pyridoxal 5'-phosphate; GST, glutathione S-transferase; AVG, aminoethoxyvinylglycine.
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