Coenzyme site-directed mutants of photosynthetic A4-GAPDH show selectively reduced NADPH-dependent catalysis, similar to regulatory AB-GAPDH inhibited by oxidized thioredoxin

• Published in: Journal of molecular biology 2004-07, Vol.340 (5), p.1025-1037
• Main Authors: Sparla, Francesca, Fermani, Simona, Falini, Giuseppe, Zaffagnini, Mirko, Ripamonti, Alberto, Sabatino, Piera, Pupillo, Paolo, Trost, Paolo
• Format: Article
• Language: English
• Subjects: Binding Sites; Catalysis; Chloroplasts - enzymology; Chloroplasts - genetics; Crystallography, X-Ray; Escherichia coli; Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+) - antagonists & inhibitors; Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+) - chemistry; Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+) - genetics; Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+) - metabolism; Kinetics; Models, Molecular; Mutagenesis, Site-Directed - genetics; NADP - metabolism; Oxidation-Reduction; Photosynthesis; Protein Structure, Tertiary; Spinacia oleracea; Spinacia oleracea - enzymology; Spinacia oleracea - genetics; Thioredoxins - metabolism; Thioredoxins - pharmacology
• ISSN: 0022-2836
• DOI: 10.1016/j.jmb.2004.06.005

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.