Isolation, sequence, and expression in Escherichia coli of the Pseudomonas sp. strain ACP gene encoding 1-aminocyclopropane-1-carboxylate deaminase

Abstract

Pseudomonas sp. strain ACP is capable of growth on l-aminocyclopropane-l-carboxylate (ACC) as a nitrogen source owing to induction of the enzyme ACC deaminase and the subsequent conversion of ACC to a-ketobutyrate and ammonia (M. Honma, Agric. Biol. Chem. 49:567-571, 1985). The Pseudomonas sp. strain ACP and the yeast Hansenula saturnus are capable of utilizing the cyclopropanoid amino acid 1-aminocyclopropane-1-carboxylate (ACC) as a nitrogen source owing to induction of the enzyme ACC deaminase (EC 4.1.99.4) in these organisms (8). Pseudomonas ACC deaminase has an estimated molecular mass of 110 kDa and is composed of three identical subunits, each with a molecular mass estimated to be 36.5 kDa (7,8). ACC deaminase utilizes pyridoxal 5'-phosphate as a cofactor in catalyzing the cleavage of ACC to a-ketobutyrate and ammonia (7,22).ACC was originally identified as a natural product in the juices of several fruits (2) and is now regarded as a key intermediate in the biosynthesis of the plant hormone ethylene (23). Ethylene is biosynthesized in higher plants from methionine via S-adenosylmethionine and ACC (1, 24). The rate-limiting step in ethylene biosynthesis is the production of ACC by a ring-closing displacement of methylthioadenosine from S-adenosylmethionine in a reaction catalyzed by ACC synthase (1). The reverse of this reaction can be viewed as analogous to the reaction catalyzed by ACC deaminase (22).Ethylene influences many aspects of plant development, including leaf and flower senescence and fruit ripening (23). Techniques for the integration and expression of bacterial genes in plants utilizing Agrobacterium tumefaciens are available (14), and the expression of an ACC deaminase gene in plants may provide a means to perturb ACC levels and ethylene biosynthesis, thus leading to a better understanding of the role of this plant hormone. As an initial step in this process, the ACC deaminase gene from Pseudomonas sp. strain ACP was isolated and characterized. The amino acid * Corresponding author. sequence of purified ACC deaminase was determined and used to design oligonucleotide primers. The primers were used in polymerase chain reactions (PCR) to isolate a portion of the gene, which was used to screen a Pseudomonas genomic library. The DNA sequence of the ACC deaminase gene is presented, and its authenticity is demonstrated by expression of the gene in Escherichia coli. MATERIALS AND METHODSBacterial strains and growth media. Lambda Zap II and E. coli XL1-Blue and SURE were obtained from Stratagene (La Jolla, Calif.). Bluescript plasmids pBCSK+, pBSK-, and pBCKS+ (Stratagene) in E. coli SURE or DH5a (5) were used for DNA sequencing. pUC19 (25) was used for subcloning in E. coli SURE. E. coli XL1-Blue and SURE strains were maintained on Luria-Bertani (LB) agar plates supplemented with 20 jig of tetracycline per ml and routinely grown at 37°C in LB medium or Terrific broth (T-broth) (13). Pseudomonas sp. strain ACP was grown and maintained in T-broth or 2% glucose4.5% Bacto-Peptone (Difco)-0.3% yeast e...

Crystal Structure of 1-Aminocyclopropane-1-carboxylate Deaminase from Hansenula saturnus

“…Except for ␣-helices 2 and 10, all ␣-helices of the two domains have a chain direction toward the molecular surface, and all ␤-strands are directed toward the molecular center. The 3 10 -helix 12 at the C terminus consists of 11 residues with a kink in the middle. The 3 10 -helix is energetically unfavorable, and only short pieces are found in protein structures.…”

“…The 3 10 -helix is energetically unfavorable, and only short pieces are found in protein structures. The kinked 3 10 -helix of yACCD is at the interface region between two domains. The two domains are connected by two linkers (residues 56 -58 and 162-173).…”

“…The ACC deaminases from the bacterium Pseudomonas sp. (bACCD; EC 4.1.99.4) and the yeast Hansenula saturnus (yACCD; EC 4.1.99.2) have been well characterized (1,10). These two kinds of ACCD perform the same activities and have amino acid sequences that are 60% identical.…”

ACC (1-Aminocyclopropane-1-Carboxylate) Deaminase Activity, a Widespread Trait in Burkholderia Species, and Its Growth-Promoting Effect on Tomato Plants

“…Analysis of the sequence of 2.2-kb pUX-UD revealed two putative open reading frames, one of which is 1,017 bp long and encodes an ACC deaminase with high levels of identity (69 to 87%) with other complete ACC deaminases (8,66,67). The second open reading frame (501 bp), transcribed in the opposite direction from acdS, encodes a putative polypeptide that showed 74% identity to Lrp (leucine-responsive regulatory protein) of Pseudomonas putida UW4.…”

Substitutions of Alanine for Cysteine at a Reactive Thiol Site and for Lysine at a Pyridoxal Phosphate Binding Site of 1-Aminocyclopropane-1-carboxylate Deaminase

“…The construction of a ACC deaminase gene containing plasmid pCGN I 472 was described previously. 8) Plasmid pUC18 was purchased from Takara ShuZQ Co., pTrc99A from Pharmacia Biotech, and pBluescript, M13mpI8, and M13mpl9 from Toyobo Co., Ltd., Japan. All the restriction enzymes used were from Takara Shuzo Co.…”