15th International Conference on Arabidopsis Research - TAIR

T07-065
Loss of the hydroxypyruvate reductase, AtHPR1,
does not lead to lethality under ambient CO2
conditions
Hartwig T.(1), Michl K.(1), Boldt R.(1), Kolukisaoglu Ü.(1), Bauwe H.(1)
1-University of Rostock, Dapartment of Life Sciences, Institute of Plant Physiology
The majority of carbon molecule losses by oxygenation of ribulose-1, 5bisphosphate
are rescued by the glycolate cycle. During the conversion of
2-phosphoglycolate to 3-phosphoglycerate the reduction of hydroxypyruvate
to glycerate is one of the last steps within this cyclic pathway. This reaction
is catalysed by hydroxypyruvate reductase (HPR) and in Arabidopsis thaliana
this enzyme is encoded by the single copy gene AtHPR1. We isolated a
mutant for this gene, hpr1-1, and it revealed to be viable under ambient air
conditions, unlike other mutants with defects in photorespiratory cycle. The
mutant shows an altered phenotype in normal air. In contrast these phenotypic
alterations disappear under elevated CO2. The loss of AtHPR1 leads to
an increase of photorespiratorial serine. Interestingly, we could only detect
low amounts of residuing HPR activity in the mutants, indicating that AtHPR1
confers the majority of HPR activity. Therefore, we conclude that the reduction
of hydroxypyruvate in the glycolate cycle is circumvented by another, yet
unknown, reaction.
15 th <strong>International</strong> <strong>Conference</strong> on Arabidopsis Research 2004 · Berlin
T07-066
Investigating the active sites of Arabidopsis thaliana
cytochrome P450 monooxygenases hydroxylating
aromatic rings
Sanjeewa Rupasinghe(1), Mary A. Schuler(1)
1-Department of Cell & Structural Biology, University of Illinois, Urbana, IL USA
Cytochrome P450 monooxygenases (P450s) are heme thiolate proteins that
catalyze difficult and extremely diverse chemistries. Despite the fact that they
share less than 13% sequence identity, they display common structural folds
that allow bacterial and mammalian P450 crystal structures to be used for
modeling of plant P450 sequences. To better understand the role of specific
amino acid residues in defining the ability of plant P450s to hydroxylate
aromatic substrates, four Arabidopsis P450s (CYP73A5 CYP84A1, CYP75B1,
CYP98A3) capable of hydroxylating aromatic rings with substituents at the
4-, 5- and 3-positions, respectively, have been homology modeled using
the MOE program. Analysis of the models by Ramachandran plot, PROSA
II and Profiles 3D as well as MD simulations have indicated that the P450
models are correctly folded and thermodynamically stable. Substrate docking
using SYBYL site ID, MOE dock and Insight II Affinity has identified residues
potentially contacting each substrate. Among these programs, MOE dock and
Insight II Affinity consistently positioned the aromatic ring of the substrate in
a similar orientation in all four proteins. Each aromatic ring was predicted to
be contacted by SRS6, the N-terminal of SRS5 and the C-terminus of SRS4
and each substrate tail was predicted to be contacted by SRS1, SRS2, the
N-terminal of SRS4 and the C-terminal of SRS5. Fifteen residues spanning
all SRS regions were selected for replacement mutagenesis in the active
site of CYP98A3 (p-coumaroylshikimic acid hydroxylase). Yeast expression
of mutants containing one or two replacements within the proposed SRS contact
regions followed by CO difference analysis indicated that most mutants
produced stable and correctly configured P450s with only two single mutants
(in SRS4) being completely unstable and two double mutants (in SRS1) being
partially unstable. Three geometric isomers of p-coumaroylshikimic acid were
chemically synthesized, purified by preparative HPLC and used for characterization
of these mutant proteins. Comparisons of hydroxylation rates have
indicated that mutations in SRS1, SRS2 and the N-terminus of SRS4 discriminate
between the isomers demonstrating that these regions are important
in recognizing the shikimic acid moiety of this substrate. Also, mutations in
SRS5 and SRS6 significantly impact catalytic activity without destabilizing the
catalytic site as is consistent with the substrate binding mode predicted for
this Arabidopsis P450.
T07 Metabolism (Primary, Secondary, Cross-Talk and Short Distance Metabolite Transport)