Chloroplast NADP-malate dehydrogenase: Structural basis of light- dependent regulation of activity by thiol oxidation and reduction

Background: NADP-dependent malate dehydrogenase (EC 1.1.1.82) is a light-activated chloroplast enzyme that functions in the C₄ pathway of photosynthesis. The light regulation is believed to be mediated in vivo by thioredoxin-catalyzed reduction and re-oxidation of cystine residues. The rates of reversible activation and inactivation of the enzyme are strongly influenced by the coenzyme substrates that seem to ultimately determine the steady-state extent of activation in vivo. Results: The X-ray structure of the inactive, oxidized enzyme was determined at 2.8 Å resolution. The core structure is homologous to NAD-dependent malate dehydrogenases. Two surface- exposed and thioredoxin-accessible disulfide bonds are present, one in the N- terminal extension and the other in the C-terminal extension. The C-terminal peptide of the inactive, oxidized enzyme is constrained by its disulfide bond to fold into the active site over NADP⁺, hydrogen bonding to the catalytic His225 as well as obstructing access of the C₄ acid substrate. Two loops flanking the active site, termed the Arg₂ and Trp loops, that contain the C₄ acid substrate binding residues are prevented from closing by the C- terminal extension. Conclusions: The structure explains the role of the C- terminal extension in inhibiting activity. The negative C terminus will interact more strongly with the positively charged nicotinamide of NADP⁺ than NADPH, explaining why the coenzyme-binding affinities of the enzyme differ so markedly from those of all other homologous α-hydroxy acid dehydrogenases. NADP⁺ may also slow dissociation of the C terminus upon reduction, providing a mechanism for the inhibition of activation by NADP⁺ but not NADPH.