Structure and mechanism of the amphibolic enzyme D-ribulose-5-phosphate 3-epimerase from potato chloroplasts

Ribulose-5-phosphate S-epimerase (EC 5.1.3.1) catalyzes the interconversion of ribulose-5-phosphate and xylulose-5-phosphate in the Calvin cycle and i n the oxidative pentose phosphate pathway. The enzyme from potato chloropla sts was expressed in Escherichia coli, isolated and crystallized. The cryst al structure was elucidated by multiple isomorphous replacement and refined at 2.3 Angstrom resolution. The enzyme is a homohexamer with D-3 symmetry. The subunit chain fold is a (beta alpha)(8)-barrel. A sequence comparison with homologous epimerases outlined the active center and indicated that al l members of this family are likely to share the same catalytic mechanism. The substrate could be modeled by putting its phosphate onto the observed s ulfate position and its epimerized C3 atom between two carboxylates that pa rticipate in an extensive hydrogen bonding system. A mutation confirmed the crucial role of one of these carboxylates. The geometry together with the conservation pattern suggests that the negative charge of the putative cis- ene-diolate intermediate is stabilized by the transient induced dipoles of a methionine sulfur "cushion", which is proton-free and therefore prevents isomerization instead of epimerization. (C) 1999 Academic Press.