Manukhov I V, Mamaeva D V, Morozova E A, Rastorguev S M, Faleev N G, Demidkina T V, Zavilgelsky G B
State Research Institute of Genetics and Selection of Industrial Microorganisms, 117545 Moscow, Russia.
Biochemistry (Mosc). 2006 Apr;71(4):361-9. doi: 10.1134/s0006297906040031.
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.
首次在肠杆菌科中发现，弗氏柠檬酸杆菌基因组中存在编码L-蛋氨酸γ-裂解酶（MGL）的基因。已从弗氏柠檬酸杆菌细胞中纯化出均一的酶，并测定了其N端序列。从弗氏柠檬酸杆菌基因组文库获得的杂交质粒pUCmgl含有一个约3000 bp的EcoRI插入片段，该片段在大肠杆菌TG1细胞中表达时可确保MGL活性的出现。EcoRI片段的核苷酸序列包含两个开放阅读框。第一个框（megL基因）编码一个由398个氨基酸残基组成的蛋白质，该蛋白质与来自不同来源的MGL具有序列同源性。第二个框编码一个与属于通透酶家族的蛋白质具有序列同源性的蛋白质。为了过量表达megL基因，将其克隆到pET-15b载体中。已纯化重组酶并测定了其动力学参数。结果表明，在大肠杆菌K12细胞中存在含有megL基因的杂交质粒pUCmgl会导致EcoKI限制效率降低。似乎在MGL的作用下L-蛋氨酸的分解会导致细胞内S-腺苷甲硫氨酸含量的降低。megL基因在弗氏柠檬酸杆菌基因组中的表达仅在大量L-蛋氨酸诱导时发生。
