Protein crystallography as a perfect tool for study of enzyme working mechanism
A. Nikulin, M. Garber, S. Nikonov
Institute of Protein Research RAS, Pushchino, Moscow region, Russia
S. Revtovich, E. Morozova, T. Demidkina
Engelhardt Institute of Molecular Biology RAS, Moscow, Russia.
The X-ray crystallography is a perfect and a widespread method to study conformations of enzymes active centers and to determine their working mechanism. This method is commonly used in the world for drug design of novel medicine.
Institute of Protein Research RAS and Engelhardt Institute of Molecular Biology RAS are leading Russian institutes in the field of molecular biology. They possess modern equipment, software and experienced staff to conduct crystallographic studies of macromolecular objects of any complexity. Determination of the L-methionine γ-lyase crystal structure is a good example of the fruitful collaborative work in this field.
For the last 50 years protein crystallography has matured from a very laborious method to commonly used technique for determination and analysis of macromolecular spatial structures. Recent success in developing of crystallographic instruments and methods has been allowed scientists to study fine structural details of such complicated biological process as protein biosynthesis and photosynthesis. Determination the structures of macromolecules is not only fundamental significance but also a great practical importance. Now the X-ray crystallography is widely used to construct and to modify small molecules for the medical purpose. In fact it is one of the nanotechnology methods.
The importance of the protein crystallography produced very intensive growing of crystallographic laboratories in the world. Unfortunately there are a very few such laboratories in Russia. Institute of Protein Research RAS is the unique research institute in Russia which has both modern experimental equipment for X-ray crystallography and qualified scientific staff. We have modern X-ray Image Plate detector MAR Research IP345 in complex with crystal cryocooling system by Oxford Instruments. A new high-brilliant microfocus X-ray generator Micro Star BRUKER-AXS will be installed this year. The laboratory is equipped with modern biochemical apparatus for producing, purifying and crystallization of proteins and nucleic acids. Several PC workstations with stereo graphics are used to determination 3D structures of macromolecules and analyzing the results of the investigations. The collective of the laboratory consists of many young but very experienced researches. As a result we have an impressive list of the solved macromolecular structures for the last 10 years. It includes more then 15 non-homological proteins and 7 RNA-protein complexes.
Our Institute has very fruitful collaboration with the Engelhardt Institute of Molecular Biology RAS which is another leading institute in the field of molecular biology. Structural studies of the L-methionine γ-lyase are a good example of the fruitful collaborative work in this field.
L-methionine γ -lyase (MGL, EC 4.4.1.11) is a pyridoxal 5'-phosphate (PLP) dependent enzyme that catalyzes γ-elimination and γ-replacement of L-methionine and its derivatives as well as β-elimination and β-replacement reactions of L-cysteine and S-substituted L-cysteines [1,2]. There has been an increasing interest in this enzyme, as methionine dependency has been reported in cancer cell lines and primary tumors. The enzyme has been found to be an effective anti-tumor agent in vitro and in vivo [3] and it is of potential value to treat Parkinson's disease, arteriosclerosis, aging, and obesity. Sulfur amino acid metabolism in bacteria is not fully understood yet, and it is likely that many of them possess MGL. Thus, inhibitors of this enzyme could ultimately prove to be effective against pathogens. Moreover, since mammals apparently do not have MGL [3] the enzyme is a promising target for anti-trichomonad and anti-entamoeba chemotherapy.
In 2004 we determined the crystal structure of MGL from Citrobacter freundii at 2.0Å resolution used data obtained at the EMBL (Hamburg, Germany) PX beamline BW7A [4]. MGL exists as homotetramers. Each subunit comprises three different domains, which are known as N-terminal domain, PLP binding domain and C-terminal domain. The tetrameric MGL molecule can be subdivided into two so called catalytic dimers in which two active sites contain residues belonging to both subunits. Two catalytic dimers build the tetrameric molecule of the enzyme. PLP is covalently attached to Lys210 which is located in the PLP-binding domain.
In 2005 we have improved the resolution of the enzyme crystal structure up to 1.35Å and obtained crystals of several complexes of MGL with substrates and inhibitors. Diffraction data were collected from these crystals and 3D structures were solved with resolution from 1.45Å to 1.80Å. A large number of the high quality structural data make possible to investigate of MGL working mechanism with very fine details. The obtained results is not only valuable for study of the enzyme action, furthermore, they could be a nice starting point to construct a very specific inhibitor of the enzyme.
This work was supported by the Russian Academy of Sciences and the Russian Foundation for Basic Research (grant 05-04-48010), the Council at the RF President and the Program for Molecular and Cellular Biology RAS. The research of MG was supported in part by an International Research Scholar's award from the Howard Hughes Medical Institute (grant #55005609). Partial support for TD was provided by International Fogarty Foundation (grant # 1 R03 TW006045-01A2).
[1] H. Tanaka, N. Esaki & K. Soda, Biochemistry 16, 100-106 (1977).
[2] H. Tanaka, N. Esaki & K. Soda, Enzyme Microb. Technol. 7, 530-537 (1985).
[3] R.M. Hoffman, Human Cell 10, 69-80 (1997).
[4] D.V. Mamaeva, E.A. Morozova, A.D. Nikulin, S.V. Revtovich, S.V. Nikonov, M.B. Garber and T.V. Demidkina, Acta Crystallographica F61, 546-549 (2005).