Cloning and expression of the unique Ca2+-ATPase from Flavobacterium odoratum.
The 60-kDa Ca2+-ATPase from Flavobacterium odoratum is kinetically and mechanistically similar to other P-type ATPases, suggesting its use as a model system for structure-function studies of ion transport. A portion of the gene was amplified by polymerase chain reaction of genomic DNA with degenerate oligonucleotide primers, one based on the N-terminal amino acid sequence of the purified protein and the other based on a consensus sequence for the phosphorylation site of P-type ATPases. This gene fragment was used to screen a lambda library of F. odoratum 29979 DNA. Clone "C" is 3.3 kilobases in length and contains one complete and part of a second open reading frame, the first of which encodes a 58-kDa protein containing the exact N-terminal amino acid sequence of the purified protein. We have named this gene cda, for calcium-dependent ATPase. Escherichia coli, transformed with clone C, demonstrates high levels of calcium-dependent and vanadate-sensitive ATP hydrolysis activity, forms a 60-kDa phosphointermediate, and cross-reacts with antibodies to the purified Ca2+-ATPase. The gene has almost no sequence homology to even the highly conserved regions characteristic of P-type ATPases but does possess significant homology to a protein with alkaline phosphatase activity (PhoD) from Zymomonas mobilis. The putative phosphorylation site is a Walker A (P-loop) ATP binding sequence and is modified relative to P-type ATPases, suggesting that the F. odoratum Ca2+-ATPase may represent an ancestral link between the F- and the P-type ATPases or perhaps a new class of ATPases.
Peiffer
W E
WE
Department of Medicine, Medical University of South Carolina, Charleston, 29425-2221, USA.
Desrosiers
M G
MG
Menick
D R
DR
D000469:Alkaline Phosphatase / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D000252:Calcium-Transporting ATPases / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D017931:DNA Primers; D005417:Flavobacterium / Q000201:enzymology* / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D015698:Genomic Library; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D017355:Zymomonas / Q000201:enzymology
D017931:DNA Primers; D011994:Recombinant Proteins; D000469:Alkaline Phosphatase; D000252:Calcium-Transporting ATPases