Structure , catalytic activity and evolutionary relationships of 1-aminocyclopropane-1-carboxylate synthase , the key enzyme of ethylene synthesis in higher plants

Both ethylene and the enzymes of ethylene synthesis are subjects of intensive scientific investigation. The present review discusses structure, catalytic activity and evolutionary relationships of 1-aminocyclopropane-1-carboxylate synthase, identified for the first time in ripening tomato in 1979. This enzyme is responsible for the conversion of S-adenosyl-L-methionine to 1-aminocyclopropane-1-carboxylic acid, which is the key step of ethylene synthesis in higher plants. The role of this enzyme (especially in the fruit ripening) was demonstrated in 1991 in transgenic tomato plants, expressing 1-aminocyclopropane-1-carboxylate synthase antisense RNA. On the basis of mutagenesis and crystallization of the enzyme, new data were provided on the three-dimensional structure and amino-acid residues which are critical for catalysis. The control of ethylene production is of great interest for plant biotechnology because it can delay senescence and overmaturation. These processes are responsible for large loss of vegetables and fruit on storage. Detailed structural and biochemical data are necessary to help design 1-aminocyclopropane-1-carboxylate synthase inhibitors, whose application is expected to have immense agricultural effects. Vol. 49 No. 3/2002

ACC synthase is the key regulatory enzyme in ethylene synthesis in higher plants (step 2).
The regulatory effect of steps 1 and 3 is not so important because SAM is constantly synthesized and also utilized by other reactions, such as methylation and polyamine synthesis, whereas ACC oxidase activity is most often constitutive (Botha & Whitehead, 1992;Li et al., 1992).
Ethylene controls its own synthesis and in higher plants two systems of ethylene production operate.System 1 operates in all vegetative tissues and in the fruit before its ripening, system 2 during the ripening of climacteric fruit and during petal senescence.Ethylene shows auto-inhibitory effect in system 1 but auto-stimulatory effect in system 2 (Barry et al., 2000).
Because of large loss of fruit, flowers and vegetables due to ethylene's effects on ripening and senescence, attempts have been made to prevent or delay fruit ripening in a reversible manner.ACC synthase activity has been demonstrated by antisense RNA experiments in to- matoes as the rate-limiting step in ethylene synthesis.Transgenic tomatoes expressing ACC synthase antisense RNA do not ripen unless exposed to exogenous ethylene (Rottmann et al., 1991;Theologis et al., 1993).Transgenic tomatoes with introduced ACC deaminase gene are characterized by a reduced ACC level which results in delayed fruit ripening (Klee et al., 1991;Theologis et al., 1992).
Microorganisms liberate ethylene as a product of metabolism of: a) 2-oxoglutarate, b) 2-oxo-4-methylthiobutyrate.These pathways do not involve ACC as the intermediate.The fungus Penicillium citrinum is the first reported microorganism which is able to synthesize ACC from SAM and to degrade it into ammonia and 2-oxobutyrate by the action of ACC deaminase (Jia et al., 1999;Kakuta et al., 2001).

REACTION MECHANISM OF THE CONVERSION OF SAM TO ACC AND MTA CATALYZED BY ACC SYNTHASE
ACC synthase belongs to the family of pyridoxal 5¢-phosphate-dependent enzymes.In PLP-dependent enzymes, the formyl group at position 4 of pyridoxal phosphate forms an aldimine bond with the e-amino group of a specific lysine residue, forming a Schiffs base within the active site of the enzyme.ACC synthases have absorption maxima between 426 and 431 nm, which is probably due to the internal Schiff base (Satoh et al., 1993).A proposed reaction mechanism -the conversion of SAM to ACC by ACC synthase -involves the following steps: 1.The active site lysine of ACC synthase forms a Schiff base (internal aldimine) with the bound PLP in unligated enzyme (Fig. 1a).
2. The amino group of the substrate SAM replaces that of active site lysine as the Schiff base partner of the cofactor (external aldimine), (Fig. 1a).
3. The C-a proton of SAM is abstracted by the e-NH 2 function of active site lysine to form a resonance-stabilized carbanion (Fig. 1a), that in turn attacs the C-g atom (Fig. 1b) to expel 5¢-methylthioadenosine (MTA), with the formation of the imine of ACC (Fig. 1b, 1c).
4. The product is released upon transimination by active site lysine to complete the catalytic sequence (Fig. 1d).
The scheme of ethylene synthesis with the atoms of methionine which are the precursors of ethylene, marked by bold type set, is presented in Fig. 1e.

THE MECHANISM-BASED INACTIVATION OF ACC SYNTHASE BY THE SUBSTRATE SAM, AND OTHER ACTIVITIES OF THIS ENZYME
ACS is inactivated by its substrate SAM in an irreversible manner (mechanism-based inactivation).ACC synthase is optimized to direct electrons from the quinonoid intermediate to the g-carbon of its substrate -SAM (Fig. 1b), to yield ACC and MTA.SAM exists as an S,S or R,S diastereomeric mixture (isomers with respect to its sulfonium centre); only the S,S diastereomer yields ACC.The R,S form inactivates the enzyme.The inactivation of ACC synthase is a result of a nucleophilic attack on residual enzyme-bound L-vinylglycine (L-VG) originating from the b, g-elimination of MTA from the internal SAM aldimine (Satoh & Esashi, 1986;Casas et al., 1993;White et al., 1994;Feng & Kirsch, 2000).A scheme of the proposed mechanism-based inactivation of ACS is shown in Fig. 2.
In vivo SAM leads to the formation of L-vinylglycine, causing alkylation and inactivation of the enzyme about once in 30000 turnovers (Oetiker et al., 1997).The peptide Ser-Leu-Ser-X-Asp-Leu-Gly-Leu-Pro-Gly-Phe-Arg from SAM-inactivated ACC synthase from apple was isolated.X was a modified amino acid (alkylation product) coincident with the position of active-site Lys273 (Satoh et al., 1993;Feng & Kirsch, 2000).
The half-life of ACS was found to be 25 min in mung bean hypocotyl segments treated with auxin but 30-40 minutes in wounded green tomato pericarp, and 114 min in ripening tomato pericarp.The mechanism-based inactivation of ACC synthase plays a pivotal role in the rapid decrease in the enzymatic activity that was observed in plant tissue (Satoh & Esashi, 1986;Satoh et al., 1993).ACC synthase reveals some additional catalytic activity apart from the preferred physiological role.L-Vinylglycine is an alternative substrate as well as a mechanism-based inhibitor of ACS.The high molar ratio of L-VG/ACS (over 5000) is necessary for a complete inactivation of ACS.

L-vinylglycine + ACS-PLP ® vinylglycine-PLP-ACS ® alkylated inactive ACS + PLP
The majority of L-VG (98.2%) is converted to a product other than the inactive enzyme.The primary reaction is the conversion of L-VG to a-ketobutyrate and ammonia: The L-VG deaminase activity of ACC synthase yields the products a-ketobutyrate and ammonia with a k cat value of 1.8 s -1 and a K m value of 1.4 mM (Feng & Kirsch, 2000).Feng et al. (2000) investigated the aminotransferase activity of ACC synthase from apple and characterized it as non-a-ketoglutaratecoupled.PLP is reductively aminated to PMP (pyridoxalamine) by transamination of an amino acid to its corresponding a-keto acid: The enzyme exhibits substrate specifity in transamination with the preference of L-Ala > L-Arg > L-Phe > L-Asp.Transamination of L-Asp and L-Phe by ACC synthase is very slow.Addition of excess pyruvate converts the PMP form of the enzyme back to the PLP form.The PMP form of ACC synthase is quite unstable.This unstability would generate apoenzyme which would capture PLP to restore the physiologically active form of ACS.
ACC synthase activity may be reversibly regulated by various substances associated with the methionine-recycling pathway, SAM metabolism, polyamine synthesis and natural and chemical analogs of SAM or inhibitors of pyridoxal phosphate-linked enzymes.MTA and KMB, intermediates of the methionine-recycling pathway inhibit the enzyme activity in an uncompetitive and noncompetitive fashion, respectively (Hyodo & Tanaka, 1986).
Sinefungin (a naturally occuring antifungal antibiotic isolated from Streptomyces griseus), SAHC, 3dz-SAH, SIBA, 1dz-SIBA, 3dz-SIBA, 7dz-SIBA are analogs to SAM (Fig. 3).When tested with ACC synthase from tomatoes the order of decreasing inhibitory activity of the SAM analogs was as follows: sinefungin > SAHC and SAHC derivates > S-n-butyladenosine and SIBA.The inhibitors tested can be divided into two groups, those modified in the adenine moiety and those modified in the 5¢ substituent.The 7-nitrogen of adenine is apparently necessary for the inhibitory activity, judging from the inactivity of 7dz-SIBA.Although 3dz-SIBA was inactive, the inhibitory activity of 3dz-SAHC suggests that the 5¢ substituent containing an amino group may be important for inhibition (Icekson & Apelbaum, 1983;Miura & Chiang, 1985).SAM serves as an important common precursor for the pathway of polyamine and ethylene synthesis.The interactions between ethylene and polyamine metabolism are discussed and  (Hyodo & Tanaka, 1986;Botha & Whitehead, 1992).
The hydroxylamine or vinylglycine analogs are potent inhibitors of PLP-dependent enzymes activity in vivo and in vitro.The hydroxylamine analogs inactivate the enzyme forming stable oximes with PLP.
Several vinylglycine analogs are shown below.
The bacterial toxin induces foliar chlorosis of the host plant but the role of the toxin in symbiosis and pathogenicity has not been resolved.It was first isolated from root nodules produced by Rhizobium japonicum in soybean.
Rhizobitoxine inhibits b-cystathionase and ACC synthase in the methionine and ethylene biosynthetic pathway, respectively.Aminoethoxyvinylglycine (AVG) was isolated from a Streptomyces strain, the methoxy analog (MVG) was discovered in fermentation broth of Pseudomonas aeruginosa (Mattoo et al., 1979).These unsaturated enol ether amino acids are potent inhibitors of ethylene synthesis in higher plants.It is interesting that both symbiotic and pathogenic bacteria produce ACC synthase inhibitors structurally and enzymologically similar to rhizobitoxine so as to control the ethylene-induced plant response that would prevent a successful infection.
The crystal structures of tomato ACS in complex with AVG and PLP imply that the competitive inhibitor AVG binds close to PLP.It is a good position for the formation the external aldimine with PLP.The a-amino group of AVG is located 4 Å away from C4¢ of the pyridine ring of PLP.The a-carboxylate group of AVG forms the three following hydrogen bonds -with the backbone nitrogen of alanine, the guanidine nitrogen of arginine (in tomato ACS Ala54, Arg412) and water.The amine group at the other end of AVG forms a van der Waals contact with a specific tyrosine residue (in tomato ACS Tyr152).Probably the a-amino and a-carboxylate groups of AVG simulate SAM binding in ACS (Huai et al., 2001).

THE STRUCTURE OF ACC SYNTHASE AND AMINO-ACID RESIDUES CRITICAL FOR ITS FUNCTION
ACC synthase, an enzyme located in the cytoplasm, is encoded by a highly divergent multigene family but the low content of ACS polypeptides (0.0001%-0.001% of plant soluble proteins) has strongly limited characterisation of these proteins.The expression of functional ACS cDNAs in heterologous systems provides an opportunity to explore these enzymes.The first ACS cDNA was cloned from zucchini fruit tissue and overexpressed in E. coli and yeast (Zarembinski & Theologis, 1994;Abel et al., 1995).
Despite the polymorphic character of the multigene family of ACS isoenzymes their primary structures show similar molecular size (441aa to 496aa) and seven strongly conserved regions (Wong et al., 1999).ACC synthase from carnation is the only exception characterised to date.It is larger than others (516aa) because of the presence of a stretch of 18 threonine residues (ACA) at position 452-469aa encoded by repeats of the CAA sequence.The stretch of (CAA) 18 probably represents a highly repeated element within the carnation genome and is not unique to the ACC synthase gene (Park et al., 1992).A necessary feature of the polypeptide during its attachment to the ACS family is the PLP-binding site (region 5): -Ser-Leu-Ser-Lys-Asp-Leu/Met-Gly-Leu-Pro-Gly-Phe-Arg-.The highly conserved regions (regions 1 to 7) among all plant ACS are shown below (the position of residues numbered according to the 485aa long polypeptide of LE-ACS2 from tomato).
1 -49 Ile-Gln-Met-Gly-Leu-Ala-Glu-Asn-Gln-Leu 58-2 -89 Phe-Gln-Asp-Tyr-His-Gly-Leu-Pro 97- The roles of amino-acid residues critical for ACC synthase activity have been elucidated by site-directed or random mutagenesis investigations performed on ACC synthases from apple and tomato (White et al., 1994;Li et al., 1997;Tarun et al., 1998;Capitani et al., 1999;Zhou et al., 1999;Huai et al., 2001;McCarthy et al., 2001).The residues important for catalysis and structure of ACC synthase from tomato are shown in Fig. 4 and are listed below (the numbers in brackets indicate equivalent residue in ACS from apple according to Capitani et al., 1999).Lys278 (273): active site lysine forming the Schiff base with the bound PLP in the unligated enzyme.Mutated enzymes from apple and tomato with the active-site lysine replaced by alanine have no detectable activity.Asn209 (202), Tyr240 (233): O3 of the pyridine ring of PLP forms two hydrogen bonds with these amino-acid residues, Tyr240 attracts the proton of Lys278 to promote the formation of the external aldimine.
Tyr152 (145): stabilization of the pyridine ring of PLP and catalytic function.Tyr152 performs a nucleophilic attack to break the C-g-S bond what leads to the formation of the key intermediate -ACC-aldimine (the OH group of Tyr152 is positioned 3.7 and 3.5 Å from C-g and S of SAM, respectively).
Glu55 (47): putative ionic interaction between SAM and this residue positioned near the sulfonium pole of the (S,S)-diastereomer of SAM.
Ala54 (46), Arg412 (407): a-carboxylate group of SAM forms hydrogen bonds with the nitrogen of alanine and the guanidino moiety of arginine.
Arg157 (150): O2¢ and O3¢ atoms of the sugar of SAM form hydrogen bonds with the guanidine group of arginine.
Tyr92 (85): the complete conservation of this tyrosine residue among all known ACS suggests that it may be involved in substrate recognition.In the apple, Tyr85 to Ala85 mutation leads to an inactive ACC synthase, whereas in the tomato Tyr92 to Phe92 or Trp92 mutations produce a partially active enzyme, while substitutions Tyr92 to His92 or Leu92 inactivate the enzyme.In contrast, according to Tarun et al. (1998), Tyr92 residue from tomato ACS is not essential for the activity of the enzyme but it may play a role in binding the PLP cofactor and in making intersubunit contacts in the dimeric form of ACS (Tarun et al., 1998;Capitani et al., 1999).
Replacement of Tyr233 with Phe233 in ACS from apple caused a 24-fold increase in K m for SAM (White et al., 1994).This tyrosine residue is not absolutely conserved in all ACS one of the ACC synthases from carnation shows a Phe residue at this position (Tarun et al., 1998).
Substitution of Arg286 with Leu286 in tomato ACS results in the mutant's lower affinity for both PLP and SAM.The loss of the correct orientation of PLP at the active site of the mutant with Leu286 decreases the binding affinity of the enzyme for SAM.Moreover, the Leu286 mutant shows the reduction in k cat which suggests that Arg286 participates in the substrate catalysis.The replacement of Arg286 with Val, Thr, Ile, and Ala converts tomato ACS into mutants losing 95% to 100% of the original catalytic activity (Tarun et al., 1998).
The mutation of Arg407 (tomato Arg412) to Lys407 in ACS from apple results in at least 20-fold increase in K m for SAM (White et al., 1994).
The N-termini of ACS are characterised by two highly conserved leucine and serine residues (Leu12 and Ser13 in tomato ACS, Leu4 and Ser5 in apple ACS).The removal of ten residues from the N-terminus of tomato ACS leads to a slight increase in ACC synthase activity but a deletion of any more residues would result in a substantial reduction in the activity.Addition of several amino-acid residues to the N-terminus does not affect the formation of the dimeric protein (Satoh et al., 1993;Li et al., 1996;Tarun et al., 1998).
The carboxyl termini of all known ACC synthases are quite divergent and consist of a hypervariable region of 18-85 C-terminal residues (Wong et al., 1999).Mutations in this hypervariable region (for tomato LE-ACS2 in the region of the last 56 residues) do not lead to any decrease in the enzyme activity.ACS polypeptides prepared from plant tissues or overexpression systems are mixtures of proteins of very similar molecular masses which differ from each other in the length of the C-truncated fragments.These values for winter squash and tomato ACS are lower by 6-8 kDa than those calculated from the corresponding cDNA.Purified wound-inducible tomato ACC synthase appears as two distinct processed polypeptides, despite the presence of protease inhibitors in the purification procedure.This suggests that the nature of the C-terminus of ACC synthase makes it susceptible to cleavage.The processing seems to occur invariably when the tissue is wounded or when cells are homogenized.The monomeric deletion mutant (tomato ACS) of the last 52aa C-terminal residues shows a dramatic increase in activity.Further deletion of the C-terminus through Arg429 results in a complete inactivation of the enzyme.Limited C-terminal proteolysis of ACC synthase may occur naturally and certain endogenous or exogenous stimuli cause the activation of ACC synthase by forming the enzymes with the higher specific activity.The phenomenon may be attributed to the release of proteases upon wounding and during tissue senescence (Kende, 1993;Satoh et al., 1993;Li & Mattoo, 1994;Zarembinski & Theologis, 1994;Tarun et al., 1998).
The subunit structure of ACS is controversial (Nakajima et al., 1988;Rottmann et al., 1991;Lincoln et al., 1993;Oetiker et al., 1997; Denotations: open blocks, a-helical regions (H1-H14), filled blocks, b-strand (B1-B12) and helix 3 10 regions.Huxtable et al., 1998).ACS enzymes from mung bean hypocotyls and a wound-inducible ACC synthase from zucchini have dimeric forms.Satoh et al. (1993) report that enzymes from ripe tomato are monomers, whereas the same enzymes overexpressed from tomato ACS cDNAs occur in dimeric forms.In contrast, Li & Mattoo (1995) report that tomato LE-ACS2 isoenzyme from fruit is a dimer whereas its carboxyl terminal deletion mutants (with last 46 and 52 residues deleted) function as highly active monomers.According to Tarun & Theologis (1998) the highly active carboxyl terminal deletion mutant LE-ACS2 with last 46 residues deleted is able to dimerize.
The catalytically inactive tomato and apple ACS mutants with substitutions of the highly conserved tyrosine or lysine residues and the hyperactive mutant with 46 C-terminal amino-acid residues deleted were coexpressed in E. coli (Li et al., 1997;Tarun & Theologis, 1998).The catalytic activity of ACS was measured.The conclusions are the following: 1) an active dimer may be formed from two different properly folded defective monomer subunits.
2) the hyperactive ACS monomer is able to dimerize with inactive monomer, which results in a reduced activity.
3) two inactive monomers of different tomato isoenzymes are able to undergo heterodimerization and show limited restoration of activity.
The question is whether ACS may be active both as a monomer and as a dimer.On the basis of the experimental data, we cannot distinguish these two possibilities.
The AT-ACS1 polypeptide from Arabidopsis thaliana is missing the highly conserved tripeptide Thr-Asn-Pro (region 4) and has no ACS activity.It is possible that AT-ACS1 may act as a dominant negative regulator of other ACS by forming heterodimers (Liang et al., 1995).Satoh et al. (1993) suggest that the dimeric forms of tomato ACS isoenzymes (LE-ACS2 and LE-ACS4) are more sensitive to mechanism-based inactivation than their monomers because of minor differences in the parameters of the active sites of these two forms.The concentration of SAM localized in the protoplasm in ripe tomato pericarp (higher than 100 micromolar) should almost saturate the enzyme's active site.Changes in the sensitivity of the enzyme to mechanism-based inactivation probably take part in the regulation of ethylene synthesis in plant tissue.
Despite the quite similar molecular masses of ACS isoenzymes their isoelectric points differ considerably, as shown in Table 1.
Penicillium citrinum synthesizes ACC from SAM and metabolizes ACC to ammonia and 2-oxobutyrate, not to ethylene (step 3, ACC deaminase).
The ACC synthase from Penicillium citrinum is a 430aa long polypeptide (a shorter C-terminus than that in plant enzymes) with molecular mass 48 kDa.The native form of the enzyme is a dimer.ACS from P. citrinum has K m for SAM about 100-fold higher than plant ACS (Jia et al., 1999).Five of the seven highly conserved regions (regions 1, 3, 4, 5, 7) found in plant ACS occur in ACS from P. citrinum and have 45-50% of residues identical with their plant counterparts.P. citrinum ACS contains six highly conserved residues responsible for the binding of PLP and SAM (Lys268, Tyr225, Asn194, Asp222, Arg276, Arg408) and Arg424 corresponding to the conserved Arg429 of the tomato enzyme.The highly conserved tyrosine residue -Tyr85 or Tyr92 (in apple or tomato ACS) is not conserved and Gly74 is at the equivalent position in P. citrinum ACS (Kakuta et al., 2001).An artificial bifuncional enzyme -ACSO (90 kDa) was created by in-frame fusion of a C-terminus truncated ACC synthase from soybean and an N-terminus truncated ACC oxidase from tomato.ACSO was able to con- vert SAM to ethylene.Native ACSO is oligomeric, about 360 kDa, suggesting that this fusion enzyme has a tetrameric form (Li et al. 1996).

THE CRYSTAL STRUCTURE OF ACC SYNTHASE
The crystal structure of ACC synthase from apple without the PLP cofactor and two crystal structures of tomato ACS: with PLP and with PLP and the inhibitor AVG were described (Hohenester et al., 1994;Capitani et al., 1999;Huai et al., 2001).The structures with and without PLP represent dimeric forms but the structure that with PLP and AVG is a monomer.The residues involved in catalysis and PLP and AVG interaction are located within the active site of the monomer.The monomeric form of tomato ACS implies should be sufficient to catalysis.A schematic presentation of the secondary structures building the large and the small domain in the three-dimensional structure of ACS from tomato is shown in Fig. 4. The large domain (a/b/a sandwich domain) contains a central several strand b-sheet flanked by nine a-helices (residues 82-322).The small domain (a/b domain) comprises a five b strands and five a-helices (residues 11-19, 25-77, 323-438), (Huai et al., 2001).
The full-length polypeptide from apple (52 kDa) is C-terminally truncated to a shorter form (48 kDa) by unknown proteolytic processes upon storage.Only the truncated form has been crystallized to date.ACC synthase from apple is a two-domain protein with each domain composed of a central sheet of b-strands connected by a-helices packed on both sides.The large domain of this ACC synthase contains a seven-stranded b-sheet, the central part of the small domain consists of a four-stranded antiparallel and a twostranded parallel sheet.In the dimeric form the active site is localized close to the subunit interface, in a cleft between the domains.
Most of the strictly conserved amino-acid residues are located on the dimer surface and nearly all are clustered close to the active site (Capitani et al., 1999).The crystallographic data imply that the large domain distinguished in the three-dimensional structure of ACS from apple and tomato contains the majority of the conserved regions (region 2-6).The crystal structures of ACC synthase from tomato and apple are very similar.

EVOLUTIONARY RELATIONSHIPS OF ACC SYNTHASE
The lysine residue forming the internal aldimine with C4¢ of PLP is absolutely conserved among all members of the PLP-dependent superfamily.The enzymes which use the pyridoxal moiety of PLP for catalysis have been classified into four structural fold types (29 enzyme structures deposited in the Protein Data Bank).The four types of PLP-dependent enzymes do not have a common structure fold but a limited conservation of the PLP binding residues.The type I fold consists of a large aba sandwich domain and a small ab domain.Type I has been divided into subgroups on the basis of conformational changes upon the binding of substrate or inhibitors: subgroup Ia -large conformational changes, subgroup Ib -absence of conformational changes.The crystal structure of LE-ACS2 from tomato suggests that this enzyme belongs to subgroup Ib rather than to Ia (Huai et al., 2001).
The PLP-dependent enzymes are found in four out of the six EC classes of enzymes but sequence and structural alignments indicate that ACC synthases are most closely evolutionarily related to the subgroup I of aminotransferases which includes aspartate, alanine, histidinolphosphate, tyrosine and phenylalanine aminotransferases (Alexander et al., 1994;Mehta & Christen, 1994;Feng et al., 2000).
There are only 12 amino-acid residues completely conserved among various amino-transferases.All but one of them are present in appropriate places in all ACC synthases (Rottmann et al., 1991;Theologis, 1992).Dimerization is necessary for the catalytic activity of aminotransferases.Aspartate aminotransferase has two active sites and each of them is formed by the juxtaposition of essential residues from both subunits (Feng et al., 2000).
The closest three-dimensional structural homologues of apple ACS are Thermus thermophilus aspartate and Trypanosoma cruzi tyrosine aminotransferases, but Bacillus sp.aspartate and rat tyrosine aminotransferases are the most similar ones to apple ACS in the primary structure (Feng et al., 2000;Capitani et al., 1999).Other structures of the PLP-dependent enzymes that align well with apple ACS three-dimensional structure include pig cytosolic aspartate aminotransferase, and E. coli b-cystathionase and 8-amino-7-oxonanoate synthase.ACC synthase has been found and its function is known only in higher plants and in the fungus Penicillium citrinum.Possible homologues of plant ACS were found in the human and pufferfish (Fugu rubripes) genomes.Transcripts of these genes were detected in human and F. rubripes cDNA libraries.The function of the encoded proteins have not been resolved yet.The human homologue of ACS has bound PLP but does not convert SAM to ACC.This homologue is capable of deamination of L-VG (Feng et al., 2000;Feng & Kirsch, 2000;Peixoto et al., 2000;Koch et al., 2001).
Members of the PLP-dependent family are characterized by significant conservation of the PLP binding residues.It is not known whether they represent convergent evolution or evolved divergently from a common origin.A phylogenetic tree of ACS isoenzymes from various plant species constructed on the basis of amino-acid sequence identity implies that the ACS gene divergence had taken place before monocots and dicots separation.ACS genes were then divided into three main groups (Liang et al., 1992;Destefano-Beltran et al., 1995;Subramaniam et al., 1996;Trebitsh et al., 1997).Expression of each of these groups is induced by similar stimuli.The multigene family of ACS reflects the evolution of a set of polypeptides which catalyse the rate-limiting step in ethylene synthesis in various plant tissues during plant growth, development or stress.

Figure 3 .
Figure 3. Natural and synthetic analogs of SAM.

Figure 4 .
Figure 4. Schematic presentation of tomato ACS polypeptide with marked a-helical and b-stranded secondary structure regions, residues critical for catalysis and fragments of the polypeptide representing the large and the small domain in the three-dimensional structure of enzyme.