Characterization, gene cloning and expression of isocitrate lyase involved in the assimilation of one-carbon compounds in Hyphomicrobium methylovorum GM2.
Isocitrate lyase of an obligate methylotrophic bacterium, Hyphomicrobium methylovorum GM2, was purified to homogeneity and characterized. The enzyme is a homotetramer of identical 62-kDa subunits. After the enzyme had been incubated at temperatures up to 25 degrees C for 30 min, no loss of activity was observed. The enzyme was stable in the pH range of 7.5-9.0. Maximum activity was observed at pH 7.5 and around 45 degrees C. The Km value for Ds-isocitrate was 0.51 mM. The activity required Mg2+ and was inhibited by oxalate, succinate and glycolate. The gene encoding the isocitrate lyase and its flanking regions were isolated from H. methylovorum GM2. Nucleotide sequencing of recombinant plasmids revealed that the isocitrate lyase gene codes for a 540-amino-acid protein. The amino acid sequence of the enzyme is similar to those of the enzymes from Escherichia coli (40% identity) and cucumber (37% identity). The recombinant plasmid, which was constructed by ligation of the cloned gene and an expression vector, pKK223-3, was introduced into E. coli HB101. The transformed E. coli cells expressed isocitrate lyase, which was indistinguishable from the purified H. methylovorum GM2 isocitrate lyase on analysis by SDS/PAGE.
Tanaka
Y
Y
Department of Food and Nutrition, Faculty of Agriculture, Kinki University, Japan.
Yoshida
T
T
Watanabe
K
K
Izumi
Y
Y
Mitsunaga
T
T
D000595:Amino Acid Sequence; D001419:Bacteria / Q000201:enzymology*; D001483:Base Sequence; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007522:Isocitrate Lyase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA; D013696:Temperature
D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D007522:Isocitrate Lyase