THE FUMARASE FAMILY OF LYASES: STRUCTURE-FUNCTION AND COMPARATIVE STUDIES

V.Zaitsev1, I.Zaitseva1, E.Duke1,J.Keruchenko2,I.Keruchenko2 , A.Svensson3, P.Lindley4, T.Weaver5 M.Lees5, L.Banaszak5

1CCLRC Daresbury Laboratory, Warrington WA4 4AD, UK ,e-mail: I.Zaitseva@dl.ac.uk
2Institute of Biochemistry RAS, 33 Leninsky pr 117071- Moscow, Russia
3Molecular Biophysics, Chemical Center, Lund University, P.O.B. 124 ,Lund, Sweden
4ESRF, BF-220, 38043 Grenoble-CEDEX, France
5Department of Biochemistry, University of Minnesota, 4-225 Millard Hall, Minneapolis, MN 55455-0347, USA

 

The evolutionary family of homologous fumarase enzymes is the main group of non-metal cofactor independent lyases that catalyze reactions of nucleophile addition to activated carbon-carbon double bonds. The enzymes catalyses reactions in biochemical processses such as Krebs cycle, the urea cycle, purine biosynthesis and amino acid metabolism (1,2).

Crystal structures for both native and recombinant forms of yeast fumarase from Saccharomices cerevisiae have been completed to moderate resolution by two separate laboratories. The crystal structure of the native form, NY-fumarase, emcompasses residues R22 through M484, while recombinant form , RY-fumarase, consists of residues S27 through L485. Both crystals structures lack the N-terminal translocation domains. The monomer of fumarase consists of three highly a-helical domains with the conformation similar to that of fumarase E.coli (E-fumarase) and d-crystallin (DCRY) monomers. The packing of four subunits in a tetramer with point symmetry 222 is essentially the same as in E-fumarase (3) and DCRY structures (4). NY-fumarase was crystallized in presence of the competitive inhibitor, meso-tartrate. It occupies a position close to that of the bound citrate molecule found in the active site of E-fumarase (5). Meso-tartrate participates in hydrogen bonding to an active site water molecule. Both RY- and NY-fumarase structures provided a further evidence that the active site water participates in the catalysis.

A detailed comparison of RY-, NY- and E-fumarases with the aspartase (6) and DCRY structures has been carried out (7). It shows that the positions of the catalytically important residues are roughly similar in all the structures, even in the structure of the catalytically inactive DCRY. However, the structural discrepancies observed are difficult to rationalize and this will have to await further structural studies upon a high resolution structures of the other members of the superfamily. The X-ray studies of yeast adenylosuccinate lyase (ADSL) have recently been undertaken. A 2.6 A data have been collected for the monoclinic crystals at the SRS, Daresbury Laboratory. The structure determination of ADSL using MR and MIR techniques is in progress.

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