Cloning of the cDNAs Coding for Two Novel Molybdo-flavoproteins Showing High Similarity with Aldehyde Oxidase and Xanthine Oxidoreductase

Abstract

The cDNAs coding for two novel mouse molybdo-flavoproteins, AOH1 and AOH2 (aldehyde oxidase homolog 1 and 2), were isolated. The AOH1 and AOH2 cDNAs code for polypeptides of 1336 amino acids. The two proteins have similar primary structure and show striking amino acid identity with aldehyde oxidase and xanthine oxidoreductase, two other molybdo-flavoenzymes. AOH1 and AOH2 contain consensus sequences for a molybdopterin-binding site and two distinct 2Fe-2S redox centers. In its native conformation, AOH1 has a molecular weight consistent with a homotetrameric structure. Transfection of the AOH1 and AOH2 cDNAs results in the production of proteins with phenanthridine but not hypoxanthine oxidizing activity. Furthermore, the AOH1 protein has benzaldehyde oxidizing activity with electrophoretic characteristics identical to those of a previously identified aldehyde oxidase isoenzyme (Holmes, R. S. (1979) Biochem. Genet. 17, 517-528). The AOH1 transcript is expressed in the hepatocytes of the adult and fetal liver and in spermatogonia. In liver, the AOH1 protein is synthesized in a gender-specific fashion. The expression of AOH2 is limited to keratinized epithelia and the basal layer of the epidermis and hair folliculi. The selective cell and tissue distribution of AOH1 and AOH2 mRNAs is consistent with the localization of the respective protein products.

Regulation and Biochemistry of Mouse Molybdo-flavoenzymes

“…Recently we demonstrated that the molybdo-flavoenzyme family is larger than previously anticipated (12,13). In fact, we identified two novel proteins characterized by high structural similarities with AOX1 and XOR (12,13).…”

“…The polyclonal anti-XOR antibody was raised against the synthetic peptide, NH 2 -NTMKTQSFIAKH-COOH, according to an established methodology (12). The anti-AOH1, anti-AOX1, anti-AOH2, and anti-XOR rabbit antisera were used for Western blot analyses, which were carried out with a chemiluminescence-based protocol as described previously (12,13,18,19). SDS-PAGE was performed according to standard techniques (20).…”

“…In fact, we identified two novel proteins characterized by high structural similarities with AOX1 and XOR (12,13). Given the closer relationship to AOX1, we named the two proteins aldehyde oxidase homologues 1 and 2 (AOH1 and AOH2).…”

“…Given the closer relationship to AOX1, we named the two proteins aldehyde oxidase homologues 1 and 2 (AOH1 and AOH2). AOH1 and AOH2 are encoded by two distinct genes that map at a very short distance from the AOX1 locus on mouse chromosome 1 (1,13). The tissue distribution of AOH1 is superimposable to that of AOX1, and the two enzymes are predominantly synthesized in the hepatic, lung, and testicular tissue (12).…”

“…This has focused our attention on the development of efficient methods for the purification of the enzymes from their native sources (7,13,14). Comparison of the enzymatic properties of AOX1 and AOH1 is particularly relevant and difficult.…”