Exploring methionine γ-lyase structure-function relationship via microspectrophotometry and X-ray crystallography.
Pyridoxal 5'-phosphate (PLP) dependent methionine γ-lyase catalyzes the breakdown of L-methionine to α-ketobutyric acid, methanethiol and ammonia. This enzyme, present in anaerobic microorganisms, has biomedical interest both for its activity as antitumor agent, depleting methionine supply in methionine-dependent cancers, and as target in the treatment of human pathogen infections, activating the pro-drug trifluoromethionine. To validate the structure of the enzyme from Citrobacter freundii, crystallized from monomethyl ether polyethylene glycol 2000, for the development of lead compounds, the reactivity of the crystalline enzyme towards L-methionine, substrate analogs and inhibitors was determined by polarized absorption microspectrophotometry. Spectral data were also collected for enzyme crystals, grown in monomethyl ether polyethylene glycol 2000 in the presence of ammonium sulfate. The three-dimensional structure of these enzyme crystals, solved at 1.65Å resolution with R(free) 23.2%, revealed the surprising absence of the aldimine bond between the active site Lys210 and PLP. Different hypothesis are proposed and discussed in the light of spectral and structural data, pointing out to the relevance of the complementarity between X-ray crystallography and single crystal spectroscopy for the understanding of biological mechanisms at molecular level. This article is part of a Special Issue entitled: Protein Structure and Function in the Crystalline State.
Ronda
Luca
L
Department of Biochemistry and Molecular Biology, University of Parma, Parma, Italy.
Bazhulina
Natalia P
NP
Morozova
Elena A
EA
Revtovich
Svetlana V
SV
Chekhov
Vladimir O
VO
Nikulin
Alexei D
AD
Demidkina
Tatyana V
TV
Mozzarelli
Andrea
A
D000596:Amino Acids; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D013437:Carbon-Sulfur Lyases / Q000737:chemistry* / Q000378:metabolism; D016971:Citrobacter freundii / Q000201:enzymology; D018360:Crystallography, X-Ray; D015591:Microspectrophotometry; D008958:Models, Molecular; D011732:Pyridoxal Phosphate / Q000378:metabolism; D013329:Structure-Activity Relationship
D000596:Amino Acids; D001426:Bacterial Proteins; D011732:Pyridoxal Phosphate; D013437:Carbon-Sulfur Lyases; C021558:L-methionine gamma-lyase