W0408
AGT1: A Serine:Glyoxylate Aminotransferase from
Arabidopsis thaliana. Brian Hulsebus1, Aaron
Liepman2, Daniel Peisach1, J. Vijayalakshmi3,
Laura Olsen2, Mark Saper1,3, 1Dept. of
Biological Chemistry, 2Dept. of Molecular, Cellular, &
Developmental Biology, & 3Biophysics Res. Div., Univ. of
Michigan, Ann Arbor, MI 48109.
Photorespiration is a metabolite salvage pathway resulting in
the light-dependent consumption of O2 and evolution of CO2
in plants. This process significantly decreases the efficiency of
photosynthesis, but may also help prevent photoinhibition in intense sunlight.
Moreover, since photorespiration is temperature sensitive, slight changes in
temperature caused by global warming may have a great effect on photorespiration
rates and consequences for the planet's O2:CO2 ratio. Thus
efforts to understand the biochemistry and regulatory mechanisms involved in
photorespiration are of global importance.
Many of the photorespiratory reactions occur in peroxisomes,
where glycolate, a product of ribulose-1,5-bisphosphate oxidation is converted
to glyoxylate. To recycle glycolate back into a form capable of entering the
Calvin cycle requires a series of aminotransferases that convert glyoxylate to
glycine. Since glyoxylate in plants can be toxic to both plants and animals, its
metabolism is critical. A few of the essential glyoxylate aminotransferases from
the plant Arabidopsis thaliana have been purified and characterized. In
particular, Arabidopsis AGT1 is 24% identical to the human AGT1, an enzyme that,
when mutated, causes the human disease primary hyperoxaluria type I. Rather than
catalyzing the Ala:glyoxylate reaction, the Arabidopsis AGT1 is specific for
serine.
Two crystal structures of wild-type AGT1 (amino acids 3-401)
from Arabidopsis have been solved to 2.3 Å resolution. The first structure
with the cofactor PLP bound as an internal aldimine to K201 was solved by
multiple isomorphous replacement using 3 derivatives and refined to a
Rwork = 17.5% and Rfree = 20.7%. The second structure of
AGT1 was created by adding serine to crystals of the PLP bound AGT1, resulting
in the non-covalently bound PMP form. The structure of AGT1-PMP was solved by
molecular replacement, using AGT1-PLP as the initial model. The overall
structure is very similar to other aminotransferases but this is the first
structure of a glyoxylate aminotransferase. Interestingly, the catalytic dimer
makes nonpolar interactions in the crystal with another dimer related by
non-crystallographic symmetry. Both models contain formate ions, found in the
crystallization solution, and waters near the active site that may suggest areas
which could potentially contain substrate.
A single amino acid substitution (Pro to Leu) at residue 251
of AGT1 is responsible for the conditional lethal (air-sensitive) sat
mutant first described by Ogren and Somerville over 20 years ago. These mutants
photosynthesize at greatly reduced rates and accumulate serine and glycine when
grown under conditions promoting photorespiration. The crystal structures show
that P251 is in close proximity to the phosphate of PLP and PMP, and a P251L
mutant would likely require a significant perturbation in the local structure to
accommodate such a change.