BACTERIAL EXPRESSION OF THE CATALYTIC DOMAIN OF 3-HYDROXY-3-METHYLGLUTARYL-COA REDUCTASE (ISOFORM HMGR1) FROM ARABIDOPSIS-THALIANA, AND ITSINACTIVATION BY PHOSPHORYLATION AT SER577 BY BRASSICA-OLERACEA 3-HYDROXY-3-METHYLGLUTARYL-COA REDUCTASE KINASE
S. Dale et al., BACTERIAL EXPRESSION OF THE CATALYTIC DOMAIN OF 3-HYDROXY-3-METHYLGLUTARYL-COA REDUCTASE (ISOFORM HMGR1) FROM ARABIDOPSIS-THALIANA, AND ITSINACTIVATION BY PHOSPHORYLATION AT SER577 BY BRASSICA-OLERACEA 3-HYDROXY-3-METHYLGLUTARYL-COA REDUCTASE KINASE, European journal of biochemistry, 233(2), 1995, pp. 506-513
The catalytic domain of 3-hydroxy-3-methylglutaryl-CoA reductase isofo
rm 1 (HMGR1cd) from Arabidopsis thaliana has been expressed in Escheri
chia coli in a catalytically active form and purified. The high effici
ency of the bacterial expression system together with the simplicity o
f the purification procedure used in this study resulted in the attain
ment of large quantities of pure enzyme (about 5 mg/l culture) with a
final specific activity of up to 17 U/mg. This specific activity is hi
gher than that reported to date for any 3-hydroxy-3-methylglutaryl-CoA
reductase (HMGR) purified from a plant source. HMGR1cd activity was c
ompletely blocked by the HMGR inhibitor mevinolin (IC50 = 12.5 nM). No
significant differences were observed between the K-m values of HMGR1
cd for NADPH (71 +/- 7 mu M) and (S)-3-hydroxy-3-methylglutaryl-CoA (8
.3 +/- 1.5 mu M) and those of pure HMGR preparations obtained from dif
ferent plant sources. The purified HMGR1cd was reversibly inactivated
by phosphorylation at a single site by Brassica oleracea HMGR kinase A
, which is functionally related to the mammalian AMP-activated protein
kinase. The site of phosphorylation is Ser577 in the complete sequenc
e of A. thaliana HMGR. The results in this paper represent the first e
vidence that a higher plant HMGR is regulated by direct phosphorylatio
n, at least in a cell-free system. Our results also reinforce the view
that the AMP-activated protein kinase/SNF1 family is an ancient and h
ighly conserved protein kinase system.