Abstract
The gene encoding the flavin-containing monoamine oxidase (MAO-N) of the filamentous fungus Aspergillus niger was cloned. MAO-N is the first nonvertebrate monoamine oxidase described to date. Three partial cDNA clones, isolated from an expression library, were used to identify and clone the structural gene (maoN) from an A. niger genomic DNA library. The maoN gene was sequenced, and analysis revealed an open reading frame that codes for a protein of 495 amino acids with a calculated molecular mass of 55.6 kDa. Sequencing of an internal proteolytic fragment of the purified enzyme confirmed the derived amino acid sequence. Analysis of the deduced amino acid sequence indicates that MAO-N is structurally related to the human monoamine oxidases MAO-A and MAO-B. In particular, the regions known to be involved in the binding of the FAD cofactor show a high degree of homology; however, the conserved cysteine residue to which the flavin cofactor is covalently bound in the mammalian forms is absent in the fungal enzyme. MAO-N has the C-terminal tripeptide Ala-Arg-Leu, which corresponds to the consensus targeting sequence found in many peroxisomal enzymes. The full-length cDNA for MAO-N was expressed in Escherichia coli from the T7 promoter of the expression vector pET3a, yielding a soluble and fully active enzyme form.
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References
Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K (1989) Current protocols in molecular biology, John Wiley and Sons, New York
Bach AWJ, Lan NC, Johnson DL, Abell CW, Bembenek ME, Kwan S-W, Seeburg PH, Shih JC (1988) cDNA cloning of human liver monoamine oxidase A and B: molecular basis of differences in enzymatic properties. Proc Nail Acad Sci USA 85:4934–4938
Blaschko H (1974) The natural history of amine oxidases. Rev Physiol Biochem Pharmacol 70:83–418
Didion T, Roggenkamp R (1992) Targeting signal of the peroxisomal catalase in the methylotrophic yeast Hansenula polymorpha. FEBS Lett 303:113–116
Garrard LJ, Goodman JM (1989) Two genes encode the major membrane-associated protein of methanol-induced peroxisome's from Candida boidinii. J Biol Chem 264:13929–13937
Gottowik J, Cesura AM, Malherbe P, Lang G, Da Prada M (1993) Characterization of wild-type and mutant forms of human monoamine oxidase A and B expressed in a mammalian cell line. FEBS Letters 317:152–156
Gurr SJ, Unkles SE, Kinghorn JR (1987) The structure and organization of nuclear genes of filamentous fungi. In: Kinghorn JR (ed) Gene structure in eukaryotic microbes. IRL Press, Oxfrord, UK, pp 93–139
Hsu Y-PP, Weyler W, Chen S, Sims KB, Rinehart WB, Utterback MC, Powell JF, Breakefield XO (1988) Structural features of human monoamine oxidase A elucidated from cDNA and peptide sequences. J Neurochem 51:1321–1324
Hsu Y-PP, Powell JF, Sims KB, Breakefield XO (1989) Molecular genetics of the monoamine oxidases. J Neurochem 53:12–18
Isobe K, Tani Y, Yamada H (1980) Crystallization and characterization of polyamine oxidase from Aspergillus terreus. Agric Biol Chem 44:2749–2751
Ito A, Kuwahara T, Inadome S, Sagara Y (1988) Molecular cloning of a cDNA for rat liver monoamine oxidase B. Biochem Biophys Res Commun 157:970–976
Johnston JP (1968) Some observations upon a new inhibitor of monoamine oxidase in brain tissue. Biochem Pharmacol 17:1285–1297
Kearney E, Salach JI, Walker WH, Seng RL, Keaney W, Zeszotek E, Singer TP (1971) The covalently-bound flavin of hepatic monoamine oxidase: isolation and sequence of a flavin peptide and evidence for binding at the 8α position. Eur J Biochem 24:312–327
Knoll J, Magyar K (1972) Some puzzling pharmacological effects of monoamine oxidase inhibitors. Adv Biochem Psychopharmacol 5:393–408
Lerch K, Longoni C, Jordi E (1982) Primary structure of tyrosinase from Neurospora crassa: purification and amino acid sequence of the cyanogen bromide fragments. J Biol Chem 257:6408–6413
Marzluf GA (1993) Regulation of sulfur and nitrogen metabolism in filamentous fungi. Annu Rev Microbiol 47:31–55
Mitoma J-Y, Ito A (1992) Mitochondrial targeting signal of rat liver monoamine oxidase B is located at its carboxy terminus. Japan J Biochem 111:20–24
Miyazawa S, Osumi T, Hashitomo T, Ohno K, Miura S, Fujiki Y (1989) Peroxisome targeting signal of rat liver acyl-coenzyme A oxidase resides at the carboxy terminus. Mol Cell Biol 9:83–91
Nagy J, Salach JI (1981) Identity of the active site flavin fragments from the human A-form of monoamine oxidase and the bovine B-form. Arch Biochem Biophys 208:388–394
Powell JF, Hsu Y-PP, Weyler W, Chen S, Salach JI, Andrikopoulos K, Mallet J, Breakefield XO (1988) The primary structure of bovine monoamine oxidase type A: comparison with peptide sequences of bovine monoamine oxidase type B and other flavoenzymes. Biochem J 259:407–413
Powell JF (1991) Molecular biological studies of monoamine oxidase: structure and function. Biochem Soc Transact 19:199–201
Roggenkamp R (1992) Targeting signals for protein import into peroxisomes. Cell Biochem Funct 10:193–199
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual (2nd edn). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
Saura Marti J, Kettler R, Da Prada M, Richards JG (1990) Molecular neuroanatomy of MAO-A and MAO-B. J Neural Transm 32:49–53
Schilling B, Lerch K (1995) Amine oxidase from Aspergillus niger: identification of a novel flavin dependent enzyme. Biochim. Biophys. Acta, in press
Steffan JS, McAllister-Henn L (1992) Isolation and characterization of the yeast gene encoding the MDH3 isozyme of malate dehydrogenase. J Biol Chem 267:24708–24715
Stevens JN, Metzenberg RL (1982) Preparation of Neurospora DNA: some improvements. Neurospora Newslett 29:27–28
Spina MB, Cohen G (1988) Exposure of striatal synaptosomes to l-dopa increases levels of oxidized glutathione. J Pharmacol Exp Ther 247:502–507
Subramani S (1992) Targeting of proteins into the peroxisomal matrix. J Membrane Biol 125:99–106
Subramani S (1993) Protein import into peroxisomes and biogenesis of the organelle. Annu Rev Cell Biol 9:445–478
Tabor CW, Tabor H, Rosenthal SM (1954) Purification of amine oxidase from beef plasma. J Biol Chem 208:645–661
Van Dijken JP, Veenhuis M (1980) Cytochemical localization of glucose oxidase in peroxisomes of Aspergillus niger. Eur J Appl Microbiol 9:275–283
Vogel HJ (1956) A convenient growth medium for Neurospora. Microbial Genet Bull 13:42–43
Wierenga RK, Terpstra P, Hot WGJ (1986) Prediction of the occurrence of the ADP-binding βαβ-fold in proteins, using an amino acid sequence fingerprint. J Mol Biol 187:101–107
Wu H-F, Chen K, Shih JC (1993) Site-directed mutagenesis of monoamine oxidase A and B: role of cysteines. Mol Pharmacol 43:888–893
Yamada H, Adachi O, Ogata K (1965) Amine oxidases of microorganisms: purification and crystallization of amine oxidase of Aspergillus niger. Agric Biol Chem 29:649–654
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Communicated by C. A. M. J. J. van den Hondel
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Schilling, B., Lerch, K. Cloning, sequencing and heterologous expression of the monoamine oxidase gene from Aspergillus niger . Molec. Gen. Genet. 247, 430–438 (1995). https://doi.org/10.1007/BF00293144
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DOI: https://doi.org/10.1007/BF00293144