Structure of 1-aminocyclopropane-1-carboxylate synthase, a key enzyme in the biosynthesis of the plant hormone ethylene

The 2.4 A crystal structure of the vitamin B6-dependent enzyme 1-aminocyclopropane-1-carboxylate (ACC) synthase is described. This enzyme catalyses the committed step in the biosynthesis of ethylene, a plant hormone that is responsible for the initiation of fruit ripening and for regulating many other developmental processes. ACC synthase has 15 % sequence identity with the well-studied aspartate aminotransferase, and a completely different catalytic activity yet the overall folds and the active sites are very similar. The new structure together with available biochemical data enables a comparative mechanistic analysis that largely explains the catalytic roles of the conserved and non-conserved active site residues. An external aldimine reaction intermediate (external aldimine with ACC, i.e. with the product) has been modeled. The new structure provides a basis for the rational design of inhibitors with broad agricultural applications.     

Aminotransferase activity and bioinformatic analysis of 1-aminocyclopropane-1-carboxylate synthase

The mechanistic fate of pyridoxal phosphate (PLP)-dependent enzymes diverges after the quinonoid intermediate. 1-Aminocyclopropane-1-carboxylate (ACC) synthase, a member of the alpha family of PLP-dependent enzymes, is optimized to direct electrons from the quinonoid intermediate to the gamma-carbon of its substrate, S-adenosyl-L-methionine (SAM), to yield ACC and 5'-methylthioadenosine. The data presented show that this quinonoid may also accept a proton at C(4)' of the cofactor to yield alpha-keto acids and the pyridoxamine phosphate (PMP) form of the enzyme when other amino acids are presented as alternative substrates. Addition of excess pyruvate converts the PMP form of the enzyme back to the PLP form. C(alpha)-deprotonation from L-Ala is shown by NMR-monitored solvent exchange to be reversible with a rate that is less than 25-fold slower than that of deprotonation of SAM. The rate-determining step for transamination follows the formation of the quinonoid intermediate. The rate-determining step for alpha, gamma-elimination from enzyme-bound SAM is likewise shown to occur after C(alpha)-deprotonation, and the quinonoid intermediate accumulates during this reaction. BLAST searches, sequence alignments, and structural comparisons indicate that ACC synthases are evolutionarily related to the aminotransferases. In agreement with previously published reports, an absence of homology was found between the alpha and beta families of the PLP-dependent enzyme superfamily.     

Structural basis for reduced activity of 1-aminocyclopropane-1-carboxylate synthase affected by a mutation linked to andromonoecy

1-aminocyclopropane-1-carboxylate synthase (ACS) is a key enzyme in the biosynthesis of the plant hormone ethylene. Recently, a new biological role for ACS has been found in Cucumis melo where a single point mutation (A57V) of one isoform of the enzyme, causing reduced activity, results in andromonoecious plants. We present here a straightforward structural basis for the reduced activity of the A57V mutant, based on our work on Malus domestica ACS, including a new structure of the unliganded apple enzyme at 1.35 Å resolution.     

Cloning of a cDNA encoding 1-aminocyclopropane-1-carboxylate synthase and expression of its mRNA in ripening apple fruit

1-Aminocyclopropane-1-carboxylate (ACC) synthase (EC 4.4.1.14) purified from apple (Malus sylvestris Mill.) fruit was subjected to trypsin digestion. Following separation by reversed-phase high-pressure liquid chromatography, ten tryptic peptides were sequenced. Based on the sequences of three tryptic peptides, three sets of mixed oligonucleotide probes were synthesized and used to screen a plasmid cDNA library prepared from poly(A)⁺ RNA of ripe apple fruit. A 1.5-kb (kilobase) cDNA clone which hybridized to all three probes were isolated. The clone contained an open reading frame of 1214 base pairs (bp) encoding a sequence of 404 amino acids. While the polyadenine tail at the 3-end was intact, it lacked a portion of sequence at the 5-end. Using the RNA-based polymerase chain reaction, an additional sequence of 148 bp was obtained at the 5-end. Thus, 1362 bp were sequenced and they encode 454 amino acids. The deduced amino-acid sequence contained peptide sequences corresponding to all ten tryptic fragments, confirming the identity of the cDNA clone. Comparison of the deduced amino-acid sequence between ACC synthase from apple fruit and those from tomato (Lycopersicon esculentum Mill.) and winter squash (Cucurbita maxima Duch.) fruits demonstrated the presence of seven highly conserved regions, including the previously identified region for the active site. The size of the translation product of ACC-synthase mRNA was similar to that of the mature protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), indicating that apple ACC-synthase undergoes only minor, if any, post-translational proteolytic processing. Analysis of ACC-synthase mRNA by in-vitro translation-immunoprecipitation, and by Northern blotting indicates that the ACC-synthase mRNA was undetectable in unripe fruit, but was accumulated massively during the ripening proccess. These data demonstrate that the expression of the ACC-synthase gene is developmentally regulated.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AdoMet S-adenosyl-l-methionine - HPLC high-pressure liquid chromatography - kDa kilodalton - kb kilobase - mAb monoclonal antibody - Met methionine - PCR polymerase chain reaction - poly(A)⁺ RNA polyadenylated RNA - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis This work was supported by grants DCB-9004129 and INT-8915155 from the National Science Foundation.     

Reaction mechanisms of the bacterial enzyme 1-aminocyclopropane-1-carboxylate deaminase

The enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase promotes plant growth by sequestering and cleaving plant-produced ACC thereby lowering the level of ethylene in the plant. Decreased ethylene levels allow the plant to be more resistant to a wide variety of environmental stresses. Here the biochemical reaction mechanisms involved in ACC deaminase activity are critically reviewed.     

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.     

Glutamate 47 in 1-aminocyclopropane-1-carboxylate synthase is a major specificity determinant

Glutamate 47 is conserved in 1-aminocyclopropane-1-carboxylate (ACC) synthases and is positioned near the sulfonium pole of (S,S)-S-adenosyl-L-methionine (SAM) in the modeled pyridoxal phosphate quinonoid complex with SAM. E47Q and E47D constructs of ACC synthase were made to investigate a putative ionic interaction between Glu47 and SAM. The k(cat)/K(m) values for the conversion of (S,S)-SAM to ACC and methylthioadenosine (MTA) are depressed 630- and 25-fold for the E47Q and E47D enzymes, respectively. The decreases in the specificity constants are due to reductions in k(cat) for both mutant enzymes, and a 5-fold increase in K(m) for the E47Q enzyme. Importantly, much smaller effects were observed for the kinetic parameters of reactions with the alternate substrates L-vinylglycine (L-VG) (deamination to form alpha-ketobutyrate and ammonia) and L-alanine (transamination to form pyruvate), which have uncharged side chains. L-VG is both a substrate and a mechanism-based inactivator of the enzyme [Feng, L., and Kirsch, J. F. (2000) Biochemistry 39, 2436-2444], but the partition ratio, k(cat)/k(inact), is unaffected by the Glu47 mutations. ACC synthase primarily catalyzes the beta,gamma-elimination of MTA from the (R,S) diastereomer of SAM to produce L-VG [Satoh, S., and Yang, S. F. (1989) Arch.Biochem. Biophys. 271, 107-112], but catalyzes the formation of ACC to a lesser extent via alpha,gamma-elimination of MTA. The partition ratios for (alpha,gamma/beta,gamma)-elimination on (R,S)-SAM are 0.4, < or =0.014, and < or =0.08 for the wild-type, E47Q, and E47D enzymes, respectively. The results of these experiments strongly support a role for Glu47 as an anchor for the sulfonium pole of (S,S)-SAM, and consequently a role as an active site determinant of reaction specificity.     

Tyr152 plays a central role in the catalysis of 1-aminocyclopropane-1-carboxylate synthase

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.     

S-methylmethionine is both a substrate and an inactivator of 1-aminocyclopropane-1-carboxylate synthase

S-methyl-l-methionine (SMM) is ubiquitous in the tissues of flowering plants, but its precise function remains unknown. It is both a substrate and an inhibitor of the pyridoxal 5^′-phosphate-dependent enzyme 1-aminocyclopropane-1-carboxylate (ACC) synthase, due to its structural similarity to the natural substrate of this enzyme, S-adenosyl-l-methionine. In the reaction with ACC synthase, SMM can either be transaminated to yield 4-dimethylsulfonium-2-oxobutyrate; converted to α-ketobutyrate, ammonia, and dimethylsulfide; or inactivate the enzyme covalently after elimination of dimethylsulfide. These results suggest a previously unrecognized role for SMM in the regulation of ACC synthase activity in plants.     

Purification and characterization of 1-aminocyclopropane-1-carboxylate synthase from apple fruits

1-Aminocyclopropane-1-carboxylate (ACC) synthase, a key enzyme in ethylene biosynthesis, was isolated and partially purified from apple (Malus sylvestris Mill.) fruits. Unlike ACC synthase isolated from other sources, apple ACC synthase is associated with the pellet fraction and can be solubilized in active form with Triton X-100. Following five purification steps, the solubilized enzyme was purified over 5000-fold to a specific activity of 100 micromoles per milligram protein per hour, and its purity was estimated to be 20 to 30%. Using this preparation, specific monoclonal antibodies were raised. Monoclonal antibodies against ACC synthase immunoglobulin were coupled to protein-A agarose to make an immunoaffinity column, which effectively purified the enzyme from a relatively crude enzyme preparation (100 units per milligram protein). As with the tomato enzyme, apple ACC synthase was inactivated and radiolabeled by its substrate S-adenosyl-l-methionine. Apple ACC synthase was identified to be a 48-kilodalton protein based on the observation that it was specifically bound to immunoaffinity column and it was specifically radiolabeled by its substrate S-adenosyl-l-methionine.     

A human placental cDNA clone that encodes nonhistone chromosomal protein HMG-1.

From a human placental lambda gt11 cDNA library, we have isolated a cDNA clone that encodes the entire 215-residue amino acid sequence of HMG-1. Analysis of an internal sequence similarity suggests that the DNA-binding domains of HMG-1 are separated by a rather long and flexible linker segment. Southern blotting of DNA digested with BamHI indicated a highly variable number of genes (or pseudogenes) for HMG-1 in different species. Characterization of HMG-1 mRNA expression by Northern blotting showed that three mRNA species of approximately 1.0, 1.4 and 2.4 kb were expressed in all mammalian organs and cell lines examined. These included several rat organs at different stages of development. Northern analysis also suggested the occurrence of HMG-1 mRNA in an invertebrate and a plant species.     

Structural and enzymatic properties of 1-aminocyclopropane-1-carboxylate deaminase homologue from Pyrococcus horikoshii

1-Aminocyclopropane-l-carboxylate deaminase (ACCD) is a pyridoxal 5/-phosphate dependent enzyme that shows deaminase activity toward ACC, a precursor of plant hormone ethylene. ACCD from some soil bacteria has been reported to be able to break the cyclopropane ring of ACC to yield a-ketobutyrate and ammonia. We reported the crystal structure of ACCD from the yeast Hansenula saturnus in the absence/presence of substrate ACC, and proposed its ingenious reaction mechanisms. In order to study the enzyme further, we overexpressed the ACCD homologue protein (phAHP) from the fully decoded hyperthermophilic archearon, Pyrococcus horikoshii OT3. However, phAHP does not show ACCD activity at high temperature as well as at room temperature, though it has significant sequence similarity. Instead of ACCD activity, the GC-MS analysis and enzymatic method show that phAHP has deaminase activity toward L and D-serine. Here, we present the crystal structures of the native and ACC-complexed phAHP. The overall topology of the phAHP structure is very similar to that of ACCD; however, critical differences were observed around the active site. Here, the differences of enzymatic activity between phAHP and ACCD are discussed based on the structural differences of these two proteins. We suggest that the catalytic disagreement between these two enzymes comes from the difference of the residues near the pyridine ring of pyridoxal 5'-phosphate (PLP), not the difference of the catalytic residues themselves. We also propose a condition necessary in the primary sequence to have ACCD activity.     

Lys296 and Arg299 residues in the C-terminus of MD-ACO1 are essential for a 1-aminocyclopropane-1-carboxylate oxidase enzyme activity

The 1-aminocyclopropane-1-carboxylate (ACC) oxidase catalyzes the last step in the biosynthesis of ethylene from ACC in higher plants. The complex structure of ACC oxidase/Fe(2+)/H(2)O derived from Petunia hybrida has recently been established by X-ray crystallography and it provides a vast structural information for ACC oxidase. Our mutagenesis study shows that both Lys296 and Arg299 residues in the C-terminal helix play important roles in enzyme activity. Both K296R and R299K mutant proteins retain only 30-15% of their enzyme activities with respect to that of the wild-type, implying that the positive charges of C-terminal residues are involved in enzymatic reaction. Furthermore, the sequence alignment of ACC oxidases from 24 different species indicates an existence of the exclusively conserved motif (Lys296-Glu301) especially in the C-terminus. The structure model based on our findings suggests that the positive-charged surface in the C-terminal helix of the ACC oxidase could be a major stabilizer in the spatial arrangement of reactants and that the positive-charge network between the active site and C-terminus is critical for ACC oxidase activity.     

Apple 1-aminocyclopropane-1-carboxylate synthase in complex with the inhibitor L-aminoethoxyvinylglycine. Evidence for a ketimine intermediate

The 1.6-A crystal structure of the covalent ketimine complex of apple 1-aminocyclopropane-1-carboxylate (ACC) synthase with the potent inhibitor l-aminoethoxyvinylglycine (AVG) is described. ACC synthase catalyzes the committed step in the biosynthesis of ethylene, a plant hormone that is responsible for the initiation of fruit ripening and for regulating many other developmental processes. AVG is widely used in plant physiology studies to inhibit the activity of ACC synthase. The structural assignment is supported by the fact that the complex absorbs maximally at 341 nm. These results are not in accord with the recently reported crystal structure of the tomato ACC synthase AVG complex, which claims that the inhibitor only associates noncovalently. The rate constant for the association of AVG with apple ACC synthase was determined by stopped-flow spectrophotometry (2.1 x 10(5) m(-1) s(-1)) and by the rate of loss of enzyme activity (1.1 x 10(5) m(-1) s(-1)). The dissociation rate constant determined by activity recovery is 2.4 x 10(-6) s(-1). Thus, the calculated K(d) value is 10-20 pm.     

Purification and partial characterization of 1-aminocyclopropane-1-carboxylate synthase from tomato pericarp

1-Aminocyclopropane-1-carboxylate synthase was purified 5000-fold from LiCl-induced tomato fruit slices by conventional and high-performance liquid chromatography. The final preparation was estimated to be between 25% and 50% pure. Two-dimensional gel electrophoresis indicates that 1-aminocyclopropane-1-carboxylate synthase activity is associated with a 45-kDa polypeptide, with a pI of 5.8 +/- 0.2. The enzyme is inactivated both by its substrate, S-adenosyl-L-methionine (AdoMet) and by one of its products, 1-aminocyclopropane-1-carboxylate. Due to the extremely low abundance of the protein it was necessary to scale up the extraction in order to obtain reasonable amounts for sequence analysis. Therefore, 200 kg tomatoes were extracted on semi-industrial scale and 1-aminocyclopropane-1-carboxylate synthase purified. This yielded approximately 150 micrograms enzyme.     

A comparison of 1-aminocyclopropane-1-carboxylate synthase in vitro translation product and in-vivo-labeled protein in ripening tomatoes

We determined the time course of increases in 1-aminocyclopropane-1-carboxylate (ACC) synthase activity in ripening tomato (Lycopersicon esculentum (L.) Mill.) pericarp discs following wounding and treatment with 75 mM LiCl. Over the course of 24 h, we detected oscillations in the amount of enzyme activity from an initial peak at 6 h to a subsequent, even higher level at 18 h. In-vitro translation products derived from poly(A)⁺ RNAs isolated at various times of treatment and in-vivo-labeled proteins were immunoprecipitated using antibodies specific for ACC synthase. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography showed that wounding and treatment with LiCl induced an accumulation of translatable ACC-synthase-specific mRNAs. In addition, single, prominent bands were apparent for both in-vivo and in-vitro samples but their molecular masses differed. It appears that the in-vitro translation product is a polypeptide of 56 kDa while the in-vivo-labeled enzyme has a molecular mass of 47 kDa. The authors greatly appreciate the skilled technical assistance of Renate deZacks and Gail Robinson. This research was supported by the National Science Foundation through Grant No. DCB-8718873 and by the Department of Energy through Contract No. DE-AC02-76ER-01338.