Bacterial expression of the catalytic domain of 3-hydroxy-3-methylglutaryl-CoA reductase (isoform HMGR1) from Arabidopsis thaliana, and its inactivation by phosphorylation at Ser577 by Brassica oleracea 3-hydroxy-3-methylglutaryl-CoA reductase kinase.
The catalytic domain of 3-hydroxy-3-methylglutaryl-CoA reductase isoform 1 (HMGR1cd) from Arabidopsis thaliana has been expressed in Escherichia coli in a catalytically active form and purified. The high efficiency of the bacterial expression system together with the simplicity of the purification procedure used in this study resulted in the attainment 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 higher than that reported to date for any 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) purified from a plant source. HMGR1cd activity was completely blocked by the HMGR inhibitor mevinolin (IC50 = 12.5 nM). No significant differences were observed between the Km values of HMGR1cd for NADPH (71 +/- 7 microM) and (S)-3-hydroxy-3-methylglutaryl-CoA (8.3 +/- 1.5 microM) and those of pure HMGR preparations obtained from different 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 sequence of A. thaliana HMGR1. The results in this paper represent the first evidence that a higher plant HMGR is regulated by direct phosphorylation, 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 highly conserved protein kinase system.
Dale
S
S
Biochemistry Department, The University, Dundee, UK.
Arró
M
M
Becerra
B
B
Morrice
N G
NG
Boronat
A
A
Hardie
D G
DG
Ferrer
A
A
D055372:AMP-Activated Protein Kinases; D000595:Amino Acid Sequence; D001483:Base Sequence; D001937:Brassica / Q000201:enzymology; D004926:Escherichia coli / Q000235:genetics; D006903:Hydroxymethylglutaryl CoA Reductases / Q000737:chemistry / Q000378:metabolism*; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000502:physiology*; D010766:Phosphorylation; D011494:Protein Kinases / Q000502:physiology*; D017346:Protein Serine-Threonine Kinases; D011994:Recombinant Proteins / Q000378:metabolism; D012694:Serine / Q000378:metabolism
D007527:Isoenzymes; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D012694:Serine; D006903:Hydroxymethylglutaryl CoA Reductases; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D055372:AMP-Activated Protein Kinases