Research Project
Grant-in-Aid for Scientific Research (C)
A highly potent recombinant L-methionine γ-lyase from Pseudomonas putida (MGL_Pp. EC 4.4.1.11) has been characterized physicochemically and pharmacokinetically in vivo and in vitro as a potent anticancer agent that can deplete L-methionine from plasma. The detailed structure of MGL_Pp and the function of the active site residue have so far not been cleared to improve as a next generation antitumor enzyme. We demonstrated that the three-dimensional structure of MGL_Pp has been completely solved by the molecular replacement method at 1.8A resolution. Detailed information of the overall structure of MGL_Pp supplied clear pictures of the N-terminal domain and the substrate-and PLP-binding pockets. It was found that a hydrogen bond network was formed around a cofactor pyridoxal 5'-phosphate in the MGL active site, which is specific for MGLs. The detailed structure will facilitate the development of MGL_Pp as an anticancer drug. The 3D structure of MGL_Pp suggested that Cys116 might be involved in the hydrogen bond network at the active site. We found that Cys116 plays an important role in the γ-elimination reaction of L-methionine and for the substrate recognition in the MGLs. We also discovered that the substitution of Cys116 for His led to a marked increase in activity toward L-cysteine and a change of the substrate specificity, suggesting that the His residue may be directly involved in the γ-elimination reaction. With a low purity, it was found that MGL_Pp was degraded between Cys49-Phe50 and the degraded enzyme lost the activity. Conjugation of MGL_Pp with polyethylene glycol (PEG) will importantly reduce proteolysis. Recently, the complete nucleotide sequence of the linear chromosome of S. avermitilis has been determined. The gene encoding L-methionine γ-lyase from Streptomyces avermitilis was cloned and expressed in Escherichia coli to characterize the enzymological properties of the gene product. MGL_Sa. for the first time over the world.
All 2008 2007 2006
All Journal Article (19 results) (of which Peer Reviewed: 7 results) Presentation (8 results) Book (2 results)
Proteins 70
Pages: 63-71
Biosci. Biotech. Biochem. 72(印刷中)
10027529566
J. Biochem 141
Pages: 535-54
10020100176
J. Biochem. 141
Pages: 535-544
生化学 79
Pages: 682-686
10019730174
ビタミン 81
Pages: 497-499
110006449220
Biosci. Biotech. Biochem 72(in press)
SEIKAGAKU 79
Vitamines 81
Structural Biology(S. Kuramitsu & M. Sugiyama Eds.)(Kyoritsu Pub. Tokyo)
Appl.Microbiol.Biotechnol 70
Pages: 183-192
Cancer Res 66
Pages: 2807-2814
J.Biosci.Bioeng 101
Pages: 315-321
110004736889
Biosci.Biotechnol.Biochem 70
Pages: 1154-1159
PROTEINS : Structure,Function,and Bioinformatics 64
Pages: 552-557
Sensor and Actuators B 119
Pages: 570-576
