Purification and characterization of a novel lactonohydrolase from Agrobacterium tumefaciens.
A novel lactonohydrolase, catalyzing the stereospecific hydrolysis of L-pantoyl lactone to L-pantoic acid, was purified 2,400-fold to apparent homogeneity with a 1.96% overall recovery from Agrobacterium tumefaciens AKU 316 through a purification procedure including ammonium sulfate fractionation, and column chromatographies on DEAE-Sephacel, phenyl-Sepharose CL-4B, Sephacryl S-200, Mono-Q and alkyl-Superose. The relative molecular mass of the native enzyme estimated on high-pressure gel permeation chromatography was 62,000 Da, and the subunit molecular mass was estimated to 26,500 Da on SDS-polyacrylamide gel electrophoresis. The enzyme hydrolyzes several aromatic lactones, such as 3,4-dihydrocoumarin and homogentisic acid lactone, other than L-pantoyl lactone. The Km and Vmax for L-pantoyl lactone were 3.59 mM and 13.7micromol/min/mg, respectively. The enzymatic activity was inhibited by several chelating reagents, Fe2+, Sn2+, Pb2+, and Fe3+.
Kataoka
M
M
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kitashirakawa-Oiwakecho, Japan. kataoka@kais.kyoto-u.ac.jp
Nomura
J I
JI
Shinohara
M
M
Nose
K
K
Sakamoto
K
K
Shimizu
S
S
D016960:Agrobacterium tumefaciens / Q000201:enzymology* / Q000235:genetics; D000595:Amino Acid Sequence; D002265:Carboxylic Ester Hydrolases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007783:Lactones; D008969:Molecular Sequence Data; D008970:Molecular Weight; D021122:Protein Subunits; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature
D007783:Lactones; D021122:Protein Subunits; D002265:Carboxylic Ester Hydrolases; C416845:pantoyl lactone hydrolase