doi:10.1016/j.jmb.2004.06.005
Copyright © 2004 Elsevier Ltd. All rights reserved.
Coenzyme Site-directed Mutants of Photosynthetic A4-GAPDH Show Selectively Reduced NADPH-dependent Catalysis, Similar to Regulatory AB-GAPDH Inhibited by Oxidized Thioredoxin
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Francesca Sparla1, †, Simona Fermani
, 
, 2, †, Giuseppe Falini2, Mirko Zaffagnini1, Alberto Ripamonti2, Piera Sabatino2, Paolo Pupillo1 and Paolo Trost1
1 Laboratorio di Fisiologia molecolare delle piante, Dipartimento di Biologia Evoluzionistica Sperimentale, via Irnerio 42, Università di Bologna, I-40126, Bologna, Italy
2 Dipartimento di Chimica “G. Ciamician”, via Selmi 2, Università di Bologna, I-40126, Bologna, Italy
Received 8 March 2004;
Revised 25 May 2004;
accepted 3 June 2004
Edited by I. Wilson
Available online 19 June 2004.
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.
Author Keywords: photosynthetic glyceraldehyde-3-phosphate dehydrogenase; NADP; light-regulation; site-specific mutants; enzyme structure
GAPDH, glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13); BPGA, 1,3-bisphosphoglycerate; CTE, C-terminal extension of subunit B of photosynthetic GAPDH; GapA, glyceraldehyde-3-phosphate dehydrogenase subunit A; GapB, glyceraldehyde-3-phosphate dehydrogenase subunit B; rmsd, root-mean-square deviation
Figure 1. (a) Ribbon model and nomenclature of the photosynthetic A4-GAPDH tetramer viewed along the molecular P axis. (b) The overall fold of a single monomer (R) of photosynthetic A4-GAPDH. The monomer is oriented as shown in the tetramer, the domains are represented in two different shades of blue and loop 31–36 is highlighted in orange. The images were produced using MOLSCRIPT[40.] and rendered using Raster3D. [41.]
Figure 2. Stereoview of the superimposition of the coenzyme-binding region between wild-type recombinant (dark grey) and native (light grey) A4-GAPDH structures, complexed with NADP. The image was produced using MOLSCRIPT[40.] and rendered using Raster3D. [41.]
Figure 3. Representation of the interactions between the NADP 2′-phosphate group and enzyme residues in: (a) the wild-type recombinant A4-GAPDH structure; (b) the native A4-GAPDH structure. The images were produced using MOLSCRIPT.[40.]
Figure 4. Stereoview of the superimposition of the coenzyme-binding region between wild-type recombinant A4-GAPDH (dark grey) and mutant T33A (light grey) structures, complexed with NADP. The image was produced using MOLSCRIPT[40.] and rendered using Raster3D. [41.]
Figure 5. Representation of the interactions between the NADP 2′-phosphate group and enzyme residues: (a) in the mutant T33A structure; (b) in the mutant S188A structure. The images were produced using MOLSCRIPT.[40.]
Figure 6. Superimposition of the tetramers (Cα trace) of wild-type recombinant A4-GAPDH (red) and mutant S188A (blue). Single superimposed subunits of the two structures, are shown in light colours. The image was produced using MOLSCRIPT[40.] and rendered using Raster3D. [41.]
Figure 7. Histograms showing the comparison between kinetic parameters reported in Table 3 determined by steady-state kinetic analysis, of wild-type recombinant A4-GAPDH and mutants T33A, S188A and T33A/S188A (left column) or native AB-GAPDH purified from spinach chloroplasts, either reduced or oxidized in the presence of thioredoxin (right-hand column). Only kinetic parameters relevant to the NADPH-dependent activity are shown. Histograms represent means, and error bars represent standard deviations. Statistically significant differences between wild-type and mutants are highlighted by one (*P<0.05) or two (**P<0.01) asterisks.
Table 1. Refinement and geometry statistics for wild-type recombinant A4-GAPDH and mutants T33A and S188A A4-GAPDH
Table 2. Conformational angles (deg.) for residues 31–36 for (1) wild-type recombinant and (2) native A4-GAPDHs complexed with NADP, (3) wild-type recombinant A4 complexed with NAD and (4) T33A and (5) S188A A4-GAPDH mutants complexed with NADP
The most important differences or similarities, discussed in the text, are highlighted in bold.
Table 3. Steady-state kinetic analysis of different GAPDH forms described in this work (wild-type recombinant A4-GAPDH, mutants T33A, S188A and T33A/S188A of the same enzyme, native AB-GAPDH purified from spinach chloroplasts, either reduced or oxidized in the presence of thioredoxin)
Kinetic experiments based on 70 single assays at varying concentrations of NADPH and 3-phosphoglycerate (see Materials and Methods) were repeated three to six times for each enzyme form. Data are presented as mean (±SD). nd, Not determined.
Table 4. Unit cell parameters and data collection statistics for wild-type recombinant A4-GAPDH and mutants T33A and S188A A4-GAPDH
Corresponding author
† F.S. and S.F. equally contributed to this work.
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