Purification and characterization of an aldehyde oxidase fromPseudomonassp. KY 4690

Abstract

An aldehyde oxidase, which oxidizes various aliphatic and aromatic aldehydes using O 2 as an electron acceptor, was purified from the cell-free extracts of Pseudomonas sp. KY 4690, a soil isolate, to an electrophoretically homogeneous state. The purified enzyme had a molecular mass of 132 kDa and consisted of three non-identical subunits with molecular masses of 88, 39, and 18 kDa. The absorption spectrum of the purified enzyme showed characteristics of an enzyme belonging to the xanthine oxidase family. The enzyme contained 0.89 mol of flavin adenine dinucleotide, 1.0 mol of molybdenum, 3.6 mol of acid-labile sulfur, and 0.90 mol of 5P-CMP per mol of enzyme protein, on the basis of its molecular mass of 145 kDa. Molecular oxygen served as the sole electron acceptor. These results suggest that aldehyde oxidase from Pseudomonas sp. KY 4690 is a new member of the xanthine oxidase family and might contain 1 mol of molybdenum^molybdpterin^cytosine dinucleotide, 1 mol of flavin adenine dinucleotide, and 2 mol of [2Fe^2S] clusters per mol of enzyme protein. The enzyme showed high reaction rates toward various aliphatic and aromatic aldehydes and high thermostability.

Purification and Some Properties of An Aldehyde Oxidase from Streptomyces Rimosus ATCC10970

“…These results suggested that the enzyme consisted of three non-identical subunits, as do ALODs from Pseudomonas sp. KY4690 and P. stutzeri IFO12695 (Uchida et al 2003(Uchida et al , 2005. Although the MM of the medium subunit of S. rimosus ATCC10970 ALOD was similar to those of the Pseudomonas ALODs, the MMs of the other subunits of the former ALOD were different from those of the Pseudomonas ALODs.…”

“…After both electrophoreses, the sample was stained for protein with 0.25% Coomassie brilliant blue R-250. After native-PAGE, stain for enzyme activity was also done, as described previously (Uchida et al 2003). Standard proteins for SDS-PAGE used were rabbit muscle phosphorylase (MM, 97.4 kDa), bovine serum albumin (MM, 66.2 kDa), chicken egg albumin (MW, 45.0 kDa), bovine carbonic anhydrase (MW, 31.0 kDa), soybean trypsin inhibitor (MW, 21.5 kDa), and lysozyme (MW, 14.4 kDa) (BioRad Laboratories).…”

“…The N-terminal amino acid sequences of the large and medium subunits of Pseudomonas sp. KY4690 ALOD were VVHLQKAVPRVDGPL and MNRFGYAKP-NAVPDA, respectively (Uchida et al 2003).…”

“…KY4690 and Pseudomonas stutzeri IFO12695, xanthine dehydrogenase from Veillonella atypica, and CO dehy- drogenase from Pseudomonas carboxidovorans (Gremer & Meyer 1996;Ha¨nzelmann et al 1999;Santiago et al 1999;Uchida et al 2003Uchida et al , 2005. The four enzymes consisted of three non-identical subunits, had molybdenum-molybdpterin cytosine dinucleotide, FAD, and [2Fe-2S] clusters, and belonged to the xanthine oxidase family.…”

“…We recently reported the molecular and catalytic properties of ALODs purified to electrophoretically homogeneous states from Pseudomonas sp. KY4690 and P. stutzeri IFO12695 (Uchida et al 2003(Uchida et al , 2005. The Pseudomonas ALODs were hetero-trimeric proteins; the MMs of the subunits of the former enzyme were 18, 39, and 88 kDa and those of the latter enzyme were 18, 38, and 83 kDa.…”