title: 1-Aminocyclopropane-1-carboxylate (ACC) deaminases from Methylobacterium radiotolerans and Methylobacterium nodulans with higher specificity for ACC.
abstract: The 1-aminocyclopropane-1-carboxylate (ACC) deaminases (EC 3.4.99.7), the key enzymes of degradation of the precursor of the phytohormone ethylene, have not been well studied despite their great importance for plant-bacterial interactions. Using blast, the open reading frames encoding ACC deaminases were found in the genomes of epiphytic methylotroph Methylobacterium radiotolerans JCM2831 and nodule-forming endosymbiont Methylobacterium nodulans ORS2060. These genes were named acdS and cloned; recombinant proteins were expressed and purified from Escherichia coli. The enzyme from M. nodulans displayed the highest substrate specificity among all of the characterized ACC deaminases (Km 0.80 ± 0.04 mM), whereas the enzyme from M. radiotolerans had Km 1.8 ± 0.3 mM. The kcat values were 111.8 ± 0.2 and 65.8 ± 2.8 min(-1) for the enzymes of M. nodulans and M. radiotolerans, respectively. Both enzymes are homotetramers with a molecular mass of 144 kDa, as was demonstrated by size exclusion chromatography and native PAGE. The purified enzymes displayed the maximum activity at 45-50 °C and pH 8.0. Thus, the priority data have been obtained, extending the knowledge of biochemical properties of bacterial ACC deaminases.
authors.affiliation: G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms RAS, Pushchino, Moscow region, Russia.
authors.forename: Dmitry N
authors.initials: DN
authors.lastname: Fedorov
authors.forename: Galina A
authors.initials: GA
authors.lastname: Ekimova
authors.forename: Nina V
authors.initials: NV
authors.lastname: Doronina
authors.forename: Yuri A
authors.initials: YA
authors.lastname: Trotsenko
mesh_terms: D019755:Carbon-Carbon Lyases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D019295:Computational Biology; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D020580:Methylobacterium / Q000201:enzymology* / Q000235:genetics; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017385:Sequence Homology; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D011994:Recombinant Proteins; C033634:1-aminocyclopropane-1-carboxylate deaminase; D019755:Carbon-Carbon Lyases