Cloning and expression of the unique Ca2+-ATPase from Flavobacterium odoratum

J Biol Chem. 1996 Mar 1;271(9):5095-100. doi: 10.1074/jbc.271.9.5095.


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.

Publication types

  • Comparative Study
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Alkaline Phosphatase / chemistry
  • Alkaline Phosphatase / metabolism
  • Amino Acid Sequence
  • Base Sequence
  • Calcium-Transporting ATPases / biosynthesis*
  • Calcium-Transporting ATPases / genetics
  • Calcium-Transporting ATPases / metabolism
  • Cloning, Molecular
  • DNA Primers
  • Flavobacterium / enzymology*
  • Flavobacterium / genetics
  • Gene Expression
  • Genes, Bacterial
  • Genomic Library
  • Kinetics
  • Molecular Sequence Data
  • Molecular Weight
  • Polymerase Chain Reaction
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Sequence Homology, Amino Acid
  • Zymomonas / enzymology


  • DNA Primers
  • Recombinant Proteins
  • Alkaline Phosphatase
  • Calcium-Transporting ATPases

Associated data

  • GENBANK/L42816