Cloning and Expression of the Unique Ca2+-ATPase from Flavobacterium odoratum

Abstract

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.

A Novel Calcium Uptake Transporter of Uncharacterized P-Type ATPase Family Supplies Calcium for Cell Surface Integrity in Mycobacterium smegmatis

“…Ca 2+ efflux requires transport against a stiff concentration gradient. Active transporters belonging to P-type ATPases of family 2 and antiporters have been identified from bacteria for their role in Ca 2+ efflux ( 10 , 18 – 21 ). Examples of molecular mechanisms of Ca 2+ uptake in bacteria are few and limited to Ca 2+ channels, viz ., a nonproteinaceous Ca 2+ -selective channel from Escherichia coli ( 22 ) and a recently identified pH-sensitive Ca 2+ channel from Bacillus subtilis ( 23 ).…”