CRYSTAL STRUCTURE OF THERMOSTABLE ASPARTASE AND STRUCTURE-BASED Exploration of Functional Sites In the ASPARTASE family

Yasuo Hata,^a Tomomi Fujii,^a Hisanobu Sakai,^a and Yasushi Kawata^b

^aInstitute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan; ^bDepartment of Biotechnology, Faculty of Engineering, Tottori University, Tottori 680-0945, Japan (hata@scl.kyoto-u.ac.jp)

Aspartase (L-aspartate ammonia-lyase, EC 4.3.1.1) plays an important role in bacterial nitrogen metabolism by catalyzing the reversible conversion of L-aspartate to fumarate and ammonium ion. The crystal structure of the thermostable aspartase from Bacillus sp. YM55-1 has been solved and refined for 2.5 Å resolution data with an R-factor of 22.1% [1]. The present enzyme is a homotetramer with subunits (M_r= 51,627, 468 amino acid residues) composed of three domains: the N-terminal large domain, the central helix domain, and the C-terminal small domain. It exhibits no allosteric effects, in contrast to the E. coli aspartase which is activated by divalent metal cation (Mg²⁺) and L-aspartate, but is four-times more active than the E. coli enzyme. The overall folding of the present enzyme subunit is similar to those of the E. coli aspartase [2] and the E. coli fumarase C [3], both of which belong to the same superfamily with the present enzyme. A local structural comparison of these three enzymes revealed seven structurally different regions. Five of the regions were located around putative functional sites, suggesting the involvement of these regions into the functions characteristic of the enzymes. Of these regions, the region of Gln96–Gly100 is proposed as a part of the recognition site of the a-amino group in L-aspartate for aspartase and the hydroxyl group in L-malate for fumarase. The region of Gln315–Gly323 is a flexible but well sequence(–G₃₁₇–S–S–I–M–P₃₂₂–)-conserved loop that is suggested to be involved in the catalytic reaction. The region of Lys123–Lys128 corresponds to a part of the putative activator-binding site in the E. coli fumarase C. The region in the Bacillus aspartase, however, adopts a main-chain conformation which prevents the activator binding. The regions of Gly228–Glu241 and Val265–Asp272, which form a part of the active-site wall, are suggested to be involved in the allosteric activation of the E. coli aspartase by the binding of the metal ion and the activator L-aspartate. Moreover, an increase in the numbers of intersubunit hydrogen-bonds and salt-bridges is observed in the Bacillus aspartase relative to those of the E. coli enzyme, implying a contribution to the thermostability of the present aspartase.

References

1 Fujii, T., Sakai, H., Kawata, Y. and Hata, Y. (2003) J. Mol. Biol. (accepted on March 3).

2 Shi, W., Dunbar, J., Jayasekera, M. M. K., Viola, R. E. and Farber, G. K. (1997) Biochemistry, 36, 9136–9144.

3 Weaver, T. and Banaszak, L. (1996) Biochemistry, 35, 13955–13965.