L-methionine gamma-lyase from Citrobacter freundii: cloning of the gene and kinetic parameters of the enzyme.
It is shown for the first time for the Enterobacteriaceae family that a gene encoding L-methionine gamma-lyase (MGL) is present in the genome of Citrobacter freundii. Homogeneous enzyme has been purified from C. freundii cells and its N-terminal sequence has been determined. The hybrid plasmid pUCmgl obtained from the C. freundii genomic library contains an EcoRI insert of about 3000 bp, which ensures the appearance of MGL activity when expressed in Escherichia coli TG1 cells. The nucleotide sequence of the EcoRI fragment contains two open reading frames. The first frame (the megL gene) encodes a protein of 398 amino acid residues that has sequence homology with MGLs from different sources. The second frame encodes a protein with sequence homology with proteins belonging to the family of permeases. To overexpress the megL gene it was cloned into pET-15b vector. Recombinant enzyme has been purified and its kinetic parameters have been determined. It is demonstrated that a presence of a hybrid plasmid pUCmgl, containing the megL gene in the E. coli K12 cells, leads to a decrease in efficiency of EcoKI-restriction. It seems likely that decomposition of L-methionine under the action of MGL leads to a decrease in the intracellular content of S-adenosylmethionine. Expression of the megL gene in the C. freundii genome occurs only upon induction by a significant amount of L-methionine.
Manukhov
I V
IV
State Research Institute of Genetics and Selection of Industrial Microorganisms, 117545 Moscow, Russia.
Mamaeva
D V
DV
Morozova
E A
EA
Rastorguev
S M
SM
Faleev
N G
NG
Demidkina
T V
TV
Zavilgelsky
G B
GB
D000595:Amino Acid Sequence; D001483:Base Sequence; D013437:Carbon-Sulfur Lyases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D016971:Citrobacter freundii / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D015246:Deoxyribonuclease EcoRI / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D016680:Genome, Bacterial; D007700:Kinetics; D008969:Molecular Sequence Data; D010802:Phylogeny; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017385:Sequence Homology
D011994:Recombinant Proteins; D015246:Deoxyribonuclease EcoRI; D013437:Carbon-Sulfur Lyases; C021558:L-methionine gamma-lyase