Highly Conserved Small Subunit Residues Influence Rubisco Large Subunit Catalysis*

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American Society for Biochemistry and Molecular Biology

RESUMO

The chloroplast enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalyzes the rate-limiting step of photosynthetic CO2 fixation. With a deeper understanding of its structure-function relationships and competitive inhibition by O2, it may be possible to engineer an increase in agricultural productivity and renewable energy. The chloroplast-encoded large subunits form the active site, but the nuclear-encoded small subunits can also influence catalytic efficiency and CO2/O2 specificity. To further define the role of the small subunit in Rubisco function, the 10 most conserved residues in all small subunits were substituted with alanine by transformation of a Chlamydomonas reinhardtii mutant that lacks the small subunit gene family. All the mutant strains were able to grow photosynthetically, indicating that none of the residues is essential for function. Three of the substitutions have little or no effect (S16A, P19A, and E92A), one primarily affects holoenzyme stability (L18A), and the remainder affect catalysis with or without some level of associated structural instability (Y32A, E43A, W73A, L78A, P79A, and F81A). Y32A and E43A cause decreases in CO2/O2 specificity. Based on the x-ray crystal structure of Chlamydomonas Rubisco, all but one (Glu-92) of the conserved residues are in contact with large subunits and cluster near the amino- or carboxyl-terminal ends of large subunit α-helix 8, which is a structural element of the α/β-barrel active site. Small subunit residues Glu-43 and Trp-73 identify a possible structural connection between active site α-helix 8 and the highly variable small subunit loop between β-strands A and B, which can also influence Rubisco CO2/O2 specificity.

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