Cloning and Functional Expression of a Mammalian Gene for a Peroxisomal Sarcosine Oxidase

Abstract

Sarcosine oxidation in mammals occurs via a mitochondrial dehydrogenase closely linked to the electron transport chain. An additional H 2 O 2 -producing sarcosine oxidase has now been purified from rabbit kidney. A corresponding cDNA was cloned from rabbit liver and the gene designated sox. This rabbit sox gene encodes a protein of 390 amino acids and a molecular mass of 44 kDa identical to the molecular mass estimated for the purified enzyme. Sequence analysis revealed an N-terminal ADP-␤␣␤-binding fold, a motif highly conserved in tightly bound flavoproteins, and a C-terminal peroxisomal targeting signal 1. Sarcosine oxidase from rabbit liver exhibits high sequence homology (25-28% identity) to monomeric bacterial sarcosine oxidases. Both purified sarcosine oxidase and a recombinant fusion protein synthesized in Escherichia coli contain a covalently bound flavin, metabolize sarcosine, L-pipecolic acid, and L-proline, and cross-react with antibodies raised against L-pipecolic acid oxidase from monkey liver. Subcellular fractionation demonstrated that sarcosine oxidase is a peroxisomal enzyme in rabbit kidney. Transfection of human fibroblast cell lines and CV-1 cells (monkey kidney epithelial cells) with the sox cDNA resulted in a peroxisomal localization of sarcosine oxidase and revealed that the import into the peroxisomes is mediated by the peroxisomal targeting signal 1 pathway.In mammals a variety of H 2 O 2 -producing oxidases including D-amino-acid oxidase, D-aspartate oxidase, L-hydroxy-acid oxidase, acyl-CoA oxidase, and L-pipecolic acid oxidase are compartmentalized in peroxisomes. The H 2 O 2 generated from these reactions is then converted to H 2 O and O 2 by the peroxisomal matrix enzyme catalase (1). Several disorders have been described in which there is a defect in peroxisomal assembly that results in a partial or total absence of peroxisomal functions (for a review see Ref.2). Patients with these peroxisomal disorders such as Zellweger syndrome, neonatal adrenoleukodystrophy, infantile Refsum disease, and hyperpipecolatemia all have elevated levels of L-pipecolic acid, an imino acid, which in human and monkey liver is oxidized by a peroxisomal L-pipecolic acid oxidase (3). Indeed, L-pipecolic acid oxidase activity was not detected in liver samples from patients with Zellweger syndrome (4). Primates dehydrogenate L-pipecolic acid to ␦-piperideine-6-carboxylate which is spontaneously converted to ␣-aminoadipic acid ␥-semialdehyde.The subcellular localization of this pathway seems to differ in other mammalian species. In rabbits, guinea pigs, dogs, and sheep L-pipecolic acid oxidation is primarily mitochondrial (5). However, during our studies examining the subcellular distribution of L-pipecolic acid oxidation in rabbits, a considerable amount of L-pipecolic acid oxidation was detected in the peroxisomes, in addition to the previously reported mitochondrial activity (6). Interestingly, this peroxisomal enzyme showed a high specific activity for sarcosine and also oxidized L-pipecolic acid and L-p...

Arginine metabolism: nitric oxide and beyond

“…Proline can also be converted into P5C by -pipecolic acid oxidase and sarcosine oxidase [49], but it is not known whether these enzymes play any significant role in endogenous arginine synthesis. The differential expression of the various enzymes has resulted in highly compartmentalized metabolic functions of different organs.…”

Covalent attachment of flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) to enzymes: The current state of affairs

“…The gene for SOX from Streptomyces sp. IU3210-8SY was cloned and overexpressed in Streptomyces lividuns (Suzuki et al, 1992), and genes for SOX from another Bacillus species and an Arthrobactor species have also been cloned and sequenced (see Reuber et al, 1997). …”

“…This year, the gene for a monomeric rabbit liver peroxisomal SOX was cloned and functionally expressed in E. coli (Reuber et al, 1997). This liver enzyme is probably the same as L-pipecolic acid oxidase, an enzyme thought to be involved in Zellweger syndrome, and possibly other human maladies.…”

“…Comparison of the amino acid sequences of these monomeric oxidases with that of the FMN-binding protein of the heterotetrameric SOX (see below) is of no aid in this regard, because there are no sequence homologies. On the other hand, His 49 of rabbit SOX aligns with the FAD-binding His 84 of dimethylglycine dehydrogenase (Reuber et al, 1997). …”

Identification of a Stable Flavin-thiolate Adduct in Heterotetrameric Sarcosine Oxidase

“…This family is mainly composed of considerably smaller, monomeric proteins (~44 kDa) that contain covalently bound FAD as the only prosthetic group and exhibit modest sequence homology (~20% identity) with the β subunit of TSOX. Members of this family include monomeric sarcosine oxidase (MSOX), N-methyltryptophan oxidase (MTOX), pipecolate oxidase and nikD [11][12][13][14][15][16][17][18] . Crystal structures have been determined for MSOX 11, 14 , a monofunctional enzyme that exhibits sarcosine oxidase but not 5,10-CH 2 -H 4 folate synthase activity 5 .…”