Expression and characterization of 1-aminocyclopropane-1-carboxylate deaminase from the rhizobacterium Pseudomonas putida UW4: a key enzyme in bacterial plant growth promotion.
The enzyme 1-aminocyclopropane-1-carboxylate deaminase (ACCD) converts ACC, the precursor of the plant hormone ethylene, to alpha-ketobutyrate and ammonium. This enzyme has been identified in soil bacteria and has been proposed to play a key role in microbe-plant association. A soluble recombinant ACCD from Pseudomonas putida UW4 of molecular weight 41 kDa has been cloned, expressed, and purified. It showed selectivity and high activity towards the substrate ACC: K(M)=3.4+/-0.2 mM and k(cat)=146+/-5 min(-1) at pH 8.0 and 22 degrees C. The enzyme displayed optimal activity at pH 8.0 with a sharp decline to essentially no activity below pH 6.5 and a slightly less severe tapering in activity at higher pH resulting in loss of activity at pH>10. The major component of the enzyme's secondary structure was determined to be alpha-helical by circular dichroism (CD). P. putida UW4 ACCD unfolded at 60 degrees C as determined by its CD temperature profile as well as by differential scanning microcalorimetry (DSC). Enzyme activity was knocked out in the point mutant Gly44Asp. Modeling this mutation into the known yeast ACCD structure shed light on the role this highly conserved residue plays in allowing substrate accessibility to the active site. This enzyme's biochemical and biophysical properties will serve as an important reference point to which newly isolated ACC deaminases from other organisms can be compared.
Hontzeas
Nikos
N
Department of Biology, University of Waterloo, 200 University Avenue West, Waterloo, Ontario, Canada N2L 3G1.
Zoidakis
Jérôme
J
Glick
Bernard R
BR
Abu-Omar
Mahdi M
MM
D000595:Amino Acid Sequence; D002152:Calorimetry, Differential Scanning; D019755:Carbon-Carbon Lyases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017354:Point Mutation; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D016958:Pseudomonas putida / Q000201:enzymology*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012230:Rhizobiaceae / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D012995:Solubility; D013379:Substrate Specificity; D013696:Temperature
D011994:Recombinant Proteins; C033634:1-aminocyclopropane-1-carboxylate deaminase; D019755:Carbon-Carbon Lyases