Substrate specificity screening of four plastid Haloacid
Dehydrogenase phosphatases in Arabidopsis thaliana
Hannah
+Institute
*
Raszka ,
Na
+
Sa ,
Dr. Sanja
+
Roje
of Biological Chemistry, Washington State University, Pullman WA 99163
*Department of Biology, Pacific Lutheran University, Tacoma WA 98447
HAD-like phosphatases which found in bacteria, archaea, and eukaryotes are predicted small molecule phosphatases. The HAD-like phosphatases
from E. coli, were previously investigated and shown to have broad or narrow substrate specificities to sugar phosphates, phosphorylated glycolytic
intermediates, nucleotide phosphates, and phosphorylated cofactors. However the function of plant HAD-like phosphatases is still uninvestigated.
This prompted an investigation into the substrate specificity of four Arabidopsis thaliana HAD phosphatases (At5g45170, At4g25840, At4g39970,
and At3g48420) located in the plastid.
Materials and Methods
At3g48420 At4g39970 At4g25840 At5g45170
Proteins At5g45170, At4g25840, At3g48420, At4g39970 were
overexpressed in E. coli clones with a polyhistidine tag. Crude protein
extracts were ran through Fast Protein Liquid Chromatography with a
HiTrap IMAC HP Column to isolate the target protein. After purification,
the concentration of the enzymes was determined by Bradford assay
against a BSA standard. The substrate specificity was tested by
spectrophotometrically measuring the formation of free-phosphate
using BIOMOL Green assay (Enzo Life Sciences, Plymouth Meeting, PA).
Substrates were phosphorylated cofactors (NADP, FMN, PLP), nucleoside
phosphates (ATP, GTP, CTP, UTP, ADP, AMP, XMP, IMP), phosphorylated
sugars (fructose-1,6-bisphosphate, glucose-1-phosphate, glucose-6phosphate) and other phosphorylated molecules (TSPP, PG2 and PG3).
Specific activity for each substrate was calculated (Table 1).
At4g39970
At3g48420
A
A
B
B
At4g25840
B
A
A
0.160
1.27
0.351
1.03
FMN
0.400
nd
nd
7.17
NADP
1.74
0.413
nd
1.13
ATP
16.3
nd
nd
0.815
GTP
nd
nd
nd
nd
CTP
2.32
0.413
0.398
0.777
UTP
7.57
2.58
nd
14.9
ADP
5.73
nd
1.90
2.80
AMP
nd
nd
0.0566
0.153
XMP
0.768
0.748
0.776
3.53
IMP
nd
0.268
0.147
0.0510
TSPP
30.4
0.603
nd
2.57
PG2
3.40
0.4.75
0.776
8.38
PG3
Fructose-1,6bisphosphate
Glucose-1phosphate
Glucose-6phosphate
nd
0.167
nd
0.573
0.118
0.100
0.0611
nd
nd
0.145
0.140
nd
0.394
0.000223
nd
0.484
Table 1: Specific activity (nmol/mg/min) of each enzyme substrate
pair
At5g45170
B
Fig 1: SDS-PAGE gel protein
gel purification.
At4g39970 A: Crude extract,
B: Purified protein 34.6 kDa.
At3g48420 A: Crude extract,
B: Purified protein 34.2 kDa.
At4g25840 A: Purified protein
33.1 kDa, B: Crude extract.
At5g45170 A: Purified
protein 40.7 kDa, B: Crude
extract
PLP
Results and Discussion
All four Arabidopsis thaliana plastid HAD phosphatases show a high degree of variability in
the substrates they work on. In general, the enzymes tend to have the lowest specific activity
for the phosphorylated sugars while they have higher activity for the neucleosides and the
phosphorylated organic acids. However, there is a great deal of variability of specific activity
within each substrate group. For future investigation, it is predicted that these four
phosphatases might play a role in the riboflavin biosynthetic pathway. This is to be
investigated next.
This work was supported by the National Science Foundation to Dr. Sanja
Roje under grant number NSF MCB-1052492