Multiple isoforms of choline kinase from Caenorhabditis elegans: cloning, expression, purification, and characterization.
Choline kinase is the first enzymatic step in the CDP-choline pathway for phosphatidylcholine biosynthesis. The genome of the nematode, Caenorhabditis elegans, contains seven genes that appear likely to encode choline and/or ethanolamine kinases. We cloned five and expressed four of these genes, and purified or partially purified three of the encoded enzymes. All expressed proteins had choline kinase activity; those that most closely resemble the mammalian choline kinases were the most active. CKA-2, a very active form, was purified to near homogeneity. The K(m) values for CKA-2 were 1.6 and 2.4 mM for choline and ATP, respectively, and k(cat) was 74 s(-1). CKA-2 was predominantly a homodimer as assessed by glycerol gradient sedimentation and dynamic light scattering. CKB-2, which was less similar to mammalian choline kinases, had K(m) values for choline and ATP of 13 and 0.7 mM, and k(cat) was 3.8 s(-1). Both of these highly purified enzymes required magnesium, had very alkaline pH optima, and were much more active with choline as substrate than with ethanolamine. These results provide a foundation for future studies on the structure and function of choline kinases, as well as studies on the genetic analysis of the function of the multiple isoforms in this organism.
Gee
Patricia
P
Department of Biological Chemistry, 4417 Medical Science I, University of Michigan Medical Center, 1301 Catherine Road, Ann Arbor 48109-0606, USA.
Kent
Claudia
C
D000595:Amino Acid Sequence; D000818:Animals; D017173:Caenorhabditis elegans / Q000201:enzymology* / Q000235:genetics; D002794:Choline / Q000378:metabolism; D002797:Choline Kinase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D018388:Codon, Terminator; D007527:Isoenzymes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008563:Membrane Lipids / Q000378:metabolism; D008969:Molecular Sequence Data; D010713:Phosphatidylcholines / Q000378:metabolism; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment
D018388:Codon, Terminator; D007527:Isoenzymes; D008563:Membrane Lipids; D010713:Phosphatidylcholines; D011994:Recombinant Proteins; D002797:Choline Kinase; D008274:Magnesium; D002794:Choline