title: Coenzyme site-directed mutants of photosynthetic A4-GAPDH show selectively reduced NADPH-dependent catalysis, similar to regulatory AB-GAPDH inhibited by oxidized thioredoxin.
abstract: Chloroplast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of higher plants uses both NADP(H) and NAD(H) as coenzyme and consists of one (GapA) or two types of subunits (GapA, GapB). AB-GAPDH is regulated in vivo through the action of thioredoxin and metabolites, showing higher kinetic preference for NADPH in the light than in darkness due to a specific effect on kcat(NADPH). Previous crystallographic studies on spinach chloroplast A4-GAPDH complexed with NADP or NAD showed that residues Thr33 and Ser188 are involved in NADP over NAD selectivity by interacting with the 2'-phosphate group of NADP. This suggested a possible involvement of these residues in the regulatory mechanism. Mutants of recombinant spinach GapA (A4-GAPDH) with Thr33 or Ser188 replaced by Ala (T33A, S188A and double mutant T33A/S188A) were produced, expressed in Escherichia coli, and compared to wild-type recombinant A4-GAPDH, in terms of crystal structures and kinetic properties. Affinity for NADPH was decreased significantly in all mutants, and kcat(NADPH) was lowered in mutants carrying the substitution of Ser188. NADH-dependent activity was unaffected. The decrease of kcat/Km of the NADPH-dependent reaction in Ser188 mutants resembles the behaviour of AB-GAPDH inhibited by oxidized thioredoxin, as confirmed by steady-state kinetic analysis of native enzyme. A significant expansion of size of the A4-tetramer was observed in the S188A mutant compared to wild-type A4. We conclude that in the absence of interactions between Ser188 and the 2'-phosphate group of NADP, the enzyme structure relaxes to a less compact conformation, which negatively affects the complex catalytic cycle of GADPH. A model based on this concept might be developed to explain the in vivo light-regulation of the GAPDH.
authors.affiliation: Laboratorio di Fisiologia molecolare delle piante, Dipartimento di Biologia Evoluzionistica Sperimentale, via Irnerio 42, Università di Bologna, I-40126 Bologna, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Sparla
authors.forename: Simona
authors.initials: S
authors.lastname: Fermani
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Falini
authors.forename: Mirko
authors.initials: M
authors.lastname: Zaffagnini
authors.forename: Alberto
authors.initials: A
authors.lastname: Ripamonti
authors.forename: Piera
authors.initials: P
authors.lastname: Sabatino
authors.forename: Paolo
authors.initials: P
authors.lastname: Pupillo
authors.forename: Paolo
authors.initials: P
authors.lastname: Trost
mesh_terms: D001665:Binding Sites; D002384:Catalysis; D002736:Chloroplasts / Q000201:enzymology / Q000235:genetics; D018360:Crystallography, X-Ray; D024601:Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+) / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed / Q000235:genetics*; D009249:NADP / Q000378:metabolism*; D010084:Oxidation-Reduction; D010788:Photosynthesis; D017434:Protein Structure, Tertiary; D018724:Spinacia oleracea / Q000201:enzymology / Q000235:genetics; D013879:Thioredoxins / Q000378:metabolism* / Q000494:pharmacology*
chemical_list: D013879:Thioredoxins; D009249:NADP; D024601:Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+)