Structure and mechanism of the amphibolic enzyme D-ribulose-5-phosphate 3-epimerase from potato chloroplasts.
Ribulose-5-phosphate 3-epimerase (EC 5.1.3.1) catalyzes the interconversion of ribulose-5-phosphate and xylulose-5-phosphate in the Calvin cycle and in the oxidative pentose phosphate pathway. The enzyme from potato chloroplasts was expressed in Escherichia coli, isolated and crystallized. The crystal structure was elucidated by multiple isomorphous replacement and refined at 2.3 A resolution. The enzyme is a homohexamer with D3 symmetry. The subunit chain fold is a (beta alpha)8-barrel. A sequence comparison with homologous epimerases outlined the active center and indicated that all members of this family are likely to share the same catalytic mechanism. The substrate could be modeled by putting its phosphate onto the observed sulfate position and its epimerized C3 atom between two carboxylates that participate 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.
Kopp
J
J
Institut für Organische Chemie und Biochemie, Albertstr. 21, Freiburg im Breisgau, D-79104, Germany.
Kopriva
S
S
Süss
K H
KH
Schulz
G E
GE
D000595:Amino Acid Sequence; D001665:Binding Sites; D001704:Biopolymers / Q000737:chemistry; D002238:Carbohydrate Epimerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D002736:Chloroplasts / Q000201:enzymology*; D018360:Crystallography, X-Ray; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D011198:Solanum tuberosum / Q000201:enzymology*; D013379:Substrate Specificity
D001704:Biopolymers; D011994:Recombinant Proteins; D002238:Carbohydrate Epimerases; C022553:ribulosephosphate 3-epimerase