Crystal Structures of Aspartate Aminotransferase Reconstituted with 1-Deazapyridoxal 5′-Phosphate: Internal Aldimine and Stable l-Aspartate External Aldimine

摘要

The 1.8 Å resolution crystal structures of Escherichiancoli aspartate aminotransferase reconstituted with 1-deazapyr-nidoxal 50n-phosphate (deazaPLP; 2-formyl-3-hydroxy-4-methyl-nbenzyl phosphate) in the internal aldimine and L-aspartatenexternal aldimine forms are reported. The L-aspartate 3ndea-nzaPLP external aldimine is extraordinarily stable (half-life ofn>20 days), allowing crystals of this intermediate to be grown by cocrystallization with L-aspartate. This structure is compared to thatnof the R-methyl-L-aspartate 3nPLP external aldimine. Overlays with the corresponding pyridoxal 50n-phosphate (PLP) aldimines shownvery similar orientations of deazaPLP with respect to PLP. The lack of a hydrogen bond between Asp222 and deazaPLP, whichnserves to “anchor” PLP in the active site, releases strain in the deazaPLP internal aldimine that is enforced in the PLP internalnaldimine [Hayashi, H., Mizuguchi, H., Miyahara, I., Islam, M. M., Ikushiro, H., Nakajima, Y., Hirotsu, K., and Kagamiyama, H.n(2003) Biochim. Biophys. Acta 1647, 103] as evidenced by the planarity of the pyridine ring and the Schiff base linkage with Lys258.nAdditionally, loss of this anchor causes a 10u0001 greater tilt of deazaPLP toward the substrate in the external aldimine. An importantnmechanistic difference between the L-aspartate 3ndeazaPLP and R-methyl-L-aspartate 3nPLP external aldimines is a hydrogen bondnbetween Gly38 and Lys258 in the former, positioning the catalytic base above and approximately equidistant between CR and C40n.nIn contrast, in the R-methyl-L-aspartate 3nPLP external aldimine, the ε-amino group of Lys258 is rotated ∼70u0001 to form a hydrogennbond to Tyr70 because of the steric bulk of the methyl group.