Download the molecular mechanism of photosynthetic glyceraldehyde

THE MOLECULAR MECHANISM OF PHOTOSYNTHETIC GLYCERALDEHYDE-3PHOSPHATE DEHYDROGENASE REGULATION
Zaffagnini M, Sparla F, Pupillo P and Trost P
Department of Biology – Laboratory of Molecular Plant Physiology – University of Bologna
Photosynthetic GAPDH subunits (GapA and GapB) give rise in chloroplasts of higher plants to two
different isoforms with either A4 or AnBn stochiometry, the latter being more abundant and displaying
sophisticated regulatory properties. Photosynthetic GAPDH can use both NADPH and NADH as
electron donors, but only the NADPH-dependent activity is regulated. Thirty amino acids at the Cterminus of GapB (CTE, for C-terminal extension) constitute the only significant difference between
GAPDH subunits. Through mutagenesis approach it has been recently demonstrated that two
cysteine residues of the CTE (Cys-358 and Cys-349) are involved in the redox regulation of the
enzyme (Sparla et al., 2002). In the presence of oxidized thioredoxin f, these Cys are specifically
induced to form a disulfide bridge. Under these conditions the CTE assumes a hairpin structure and
the NADPH-dependent activity of GAPDH is depressed because kcat drops by a factor of 2.7 with no
major changes in affinity for the substrates (Km(BPGA), Km(NADPH)) .
In order to investigate the mechanism of GAPDH autoinhibition promoted by the CTE, a further
mutant was obtained in which the last, negatively charged residue of the CTE (Glu-362) was
exchanged into a neutral Gln (E362Q). The rationale of this experiment was to test whether the action
of the CTE was mediated by one of its abundant negative residues, since an exposed portion of
GAPDH (the so called S-loop), considered important for catalysis, was particularly reach in positive
arginines. Interestingly, the E362Q mutant proved to be almost insensitive to thioredoxin. The kcat ratio
between reduced and oxidized forms was 1.30.2 in the E362Q mutant, and 2.20.3 in recombinant wt
GapB, the latter showing a similar behaviour to native AnBn isoform. Effects of the mutation on
substrate affinity were minor if any. These results strongly support the participation of Glu362 to the
catalytic mechanism of GAPDH, when the CTE is in the oxidized state.
By comparying the crystal structures of A4-GAPDH complexed with either NADP or NAD (Fermani
et al., 2001; Falini et al., 2003) it was envisioned that only three residues are directly involved in
coenzyme specificity. Among these Ser188, belonging to the S-loop, has been demonstrated by site
specific mutagenesis to be essential for both coenzyme recognition and efficient NADPH-dependent
catalysis, suggesting that Ser188 might be a regulatory switch of the enzyme, possibly operated by
oxidized CTE. A kinetic and regulatory characterization of the S188A mutant of GapB is currently
under way.
Therefore the model we are going to test predicts that the formation of the disulfide bridge in the CTE
drives the C-terminal Glu-362 to approach the S-loop, thereby disturbing the maximal catalytic
efficiency which is induced by NADPH bound to the coenzyme site. According to this hypothesis
reduced GAPDH, similar to any GAPDH isoform which do not contain a functional CTE, reaches the
maximal catalytic efficiency because the CTE does not interact wih the S-loop.
Fermani S, Ripamonti A, Sabatino P, Zanotti G, Scagliarini S, Sparla F, Trost P, Pupillo P (2001) J Mol Biol, 314, 527-542
Sparla F, Pupillo P, Trost P (2002) J Biol Chem, 277, 44946-44952
Falini G, Fermani S, Ripamonti A, Sabatino P, Sparla F, Pupillo P and Trost P (2003) Biochemistry, 42(16): 4631-9