Structure of Soybean β-Cyanoalanine Synthase and the Molecular Basis for Cyanide Detoxification in Plants<sup>[W]</sup>
Abstract
Plants produce cyanide (CN) during ethylene biosynthesis in the mitochondria and require β-cyanoalanine synthase (CAS) for CN detoxification. Recent studies show that CAS is a member of the β-substituted alanine synthase (BSAS) family, which also includes the Cys biosynthesis enzyme O-acetylserine sulfhydrylase (OASS), but how the BSAS evolved distinct metabolic functions is not understood. Here we show that soybean (Glycine max) CAS and OASS form α-aminoacrylate reaction intermediates from Cys and O-acetylserine, respectively. To understand the molecular evolution of CAS and OASS in the BSAS enzyme family, the crystal structures of Gm-CAS and the Gm-CAS K95A mutant with a linked pyridoxal phosphate (PLP)-Cys molecule in the active site were determined. These structures establish a common fold for the plant BSAS family and reveal a substrate-induced conformational change that encloses the active site for catalysis. Comparison of CAS and OASS identified residues that covary in the PLP binding site. The Gm-OASS T81M, S181M, and T185S mutants altered the ratio of OASS:CAS activity but did not convert substrate preference to that of a CAS. Generation of a triple mutant Gm-OASS successfully switched reaction chemistry to that of a CAS. This study provides new molecular insight into the evolution of diverse enzyme functions across the BSAS family in plants.
All values are expressed as a mean ± se (n = 3).
All values are expressed as a mean ± se (n = 3).
Acknowledgments
This article was funded by a grant from the U.S. Department of Agriculture (NRI-2005-02518) to J.M.J. M.J. received an American Society of Plant Biologists Summer Undergraduate Research Fellowship. Portions of this research were carried out at the Argonne National Laboratory Structural Biology Center of the Advanced Photon Source, a national user facility operated by the University of Chicago for the Department of Energy Office of Biological and Environmental Research (DE-AC02-06CH11357).
AUTHOR CONTRIBUTIONS
H.Y., M.J., and J.M.J. designed the research; H.Y., M.J., and J.M.J. performed research; H.Y., M.J., and J.M.J. analyzed data; and H.Y. and J.M.J. wrote the article.
Notes
Glossary
CAS | β-cyanoalanine synthase |
BSAS | β-substituted alanine synthase |
OASS | O-acetylserine sulfhydrylase |
PLP | pyridoxal phosphate |
Amax | photosynthetic capacity |
TSGN | to be defined |
DES | desulfhydrase |
SSCS | S-sulfocysteine synthase |
PDB | Protein Data Bank |
Glossary
CAS | β-cyanoalanine synthase |
BSAS | β-substituted alanine synthase |
OASS | O-acetylserine sulfhydrylase |
PLP | pyridoxal phosphate |
Amax | photosynthetic capacity |
TSGN | to be defined |
DES | desulfhydrase |
SSCS | S-sulfocysteine synthase |
PDB | Protein Data Bank |