TITLE:
Molecular cloning and characterization of an ECTO-NOX3 (ENOX3) of Saccharomyces cerevisiae
AUTHORS:
Sara Dick, Connie Phung, Dorothy M. Morré, D. James Morré
KEYWORDS:
Age-Related NADH Oxidase (arNOX) ECTO-NOX3; TM9 Superfamily of Transmembrane Proteins; Molecular Cloning; Saccharomyces cerevisiae
JOURNAL NAME:
Advances in Biological Chemistry,
Vol.3 No.5,
October
30,
2013
ABSTRACT:
Exfoliated ECTO-NOX3 (ENOX3) proteins, are
members of the human TM9 superfamily of transmembrane proteins that generate
superoxide, are present in blood and other body fluids, and increase activity
with age beginning about age 30, hence age-related NOX (arNOX or ENOX3). A
yeast deletion library was screened based on NADH fluorescence using a 384 well
plate assay to identify a yeast isolate lacking a previously identified cell
surface oxidase exhibiting an oscillatory pattern with a period length of 26
min and capable of generating superoxide. The cDNA was cloned from a yeast over
expression library using NADH as an impermeant substrate with analysis by Fast
Fourier Transform and decomposition fits. The objective was to identify and
sequence an ENOX homologue in Saccharomyces cerevisiae with a 26 min
rather than a 24 or 25 min period length. The finding identified YER113C as the
yeast ENOX3 protein with a 26 min period and capable of generating superoxide.
The encoded protein was expressed in bacteria and characterized. Gel slices of
expressed proteins revealed a protein of ca. 81,545 kDa with properties
paralleling those of human ar-NOX (periodic NADH oxidation, protein disulfide thiol
interchange, inhibited by mammalian arNOX inhibitors and superoxide production
inhibited by superoxide dismutase). The YER113C sequence exhibited a 44%
similarity and a 26% identity with the mammalian ENOX3 SF4 (arNOX SF4) of the
TM9 superfamily of transmembrane proteins1.
The YER113C deletion mutant lacked arNOX activity.