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Identification and characterization of a l-tyrosine decarboxylase in Methanocaldococcus jannaschii

https://doi.org/10.1016/j.bbagen.2004.12.003Get rights and content

Abstract

Methanofuran is the first coenzyme in the methanogenic pathway used by the archaeon Methanocaldococcus jannaschii, as well as other methanogens, to reduce CO2 to methane. The details of the pathway for the biosynthesis of methanofuran and the responsible genes have yet to be established. A clear structural element in all known methanofurans is tyramine, likely produced by the decarboxylation of l-tyrosine. We show here that the mfnA gene at M. jannaschii locus MJ0050 encodes a thermostable pyridoxal phosphate-dependent l-tyrosine decarboxylase that specifically produces tyramine. Homologs of this gene are widely distributed among euryarchaea but are not specifically related to known bacterial or plant tyrosine decarboxylases.

Introduction

Methanofuran [1] is the first coenzyme in the pathway used by methanogens to reduce CO2 to methane [2]. The methyltrophic bacteria also appear to use methanofuran as a coenzyme in formaldehyde oxidation [3], [4]. In the first step of CO2 reduction in methanogenesis, the benzylic amino group of methanofuran reacts with CO2 to form a carbamate, which is then reduced to N-formylmethanofuran [5]. The formate of the resulting N-formylmethanofuran is then transferred to tetrahydromethanopterin and subsequently reduced to methane [6]. Of all the methanogenic coenzymes, the biosynthesis of methanofuran has received the least attention [7], [8]. Although methanogens produce various forms of methanofuran that differ in their side chains, all contain a conserved core structure with tyramine as a dominant element [9]. We proposed that this tyramine moiety is produced by the decarboxylation of l-tyrosine (Fig. 1) [7]. A search of the available genome sequences of methanogens failed to identify any gene annotated as a tyrosine decarboxylase but did identify a gene encoding a putative glutamate decarboxylase in Methanocaldococcus jannaschii (locus MJ0050). Considering that the known glutamate decarboxylases [10] and l-tyrosine decarboxylases are all members of same group II of pyridoxal 5′-phosphate (PLP)-dependent decarboxylases [11] and that there is no apparent reason for methanogens to produce a glutamate decarboxylase, we considered that the M. jannaschii enzyme could catalyze the decarboxylation of l-tyrosine, producing tyramine for methanofuran biosynthesis.

To establish the functional role of the MJ0050 gene, its protein product was heterologously expressed, purified and tested for decarboxylase activity against a wide range of amino acids. Unlike aromatic decarboxylases that catalyze the decarboxylation of many analogous amino acids [12] this enzyme is quite specific for l-tyrosine, which is consistent with its proposed role of supplying tyramine for methanofuran biosynthesis. As the first protein proposed to function specifically in methanofuran biosynthesis, this enzyme is designated the MfnA enzyme and the respective gene, mfnA.

Section snippets

Chemicals

The amino acids l-alanine, l-aspartic acid, 3,4-dihydroxy-l-phenylalanine, l-glutamic acid, l-homotyrosine, l-4-hydroxyphenylglycine, l-lysine, l-ornithine, O-phospho-l-threonine, l-phenylalanine, l-phenylglycine, l-serine, l-threonine, l-tryptophan, l-m-tyrosine, l-p-tyrosine (l-tyrosine) and d-tyrosine; the amines tryptamine, hydroxylamine and O-methylhydroxylamine; and the pH buffers 2-(N-cyclohexylamino)ethanesulfonic acid (CHES) and

Purification of the MfnA protein

The final purified MfnA protein was yellow and showed absorbance maxima at 277, 335 nm and 419 nm (Fig. 4) expected for a PLP containing protein [20], [21] and the same as observed in the aromatic amino acid decarboxylases [12]. The protein formed a single band when analyzed by SDS-PAGE with an apparent mass of 45 kDa, consistent with the predicted mass of 48 kDa based on nucleotide sequence. The MfnA protein eluted from the analytical gel filtration column with an apparent mass of 90 kDa,

Discussion

Glutamate decarboxylase has been isolated from a wide range of biological materials from bacteria to brains [28], where it catalyzes the formation of 4-aminobutyrate from l-glutamate. The family of group II PLP-dependent amino acid decarboxylases, which includes previously characterized glutamate and tyrosine decarboxylases has a sole representative in the genome of M. jannaschii. This MJ0050 gene was previously annotated as a l-glutamate decarboxylase. However, the requirement for such a

Acknowledgements

This work was supported by U. S. National Science Foundation Grant MCB 0231319 to R. H. W. We thank Kim Harich for assistance with the GC–MS analyses.

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    1

    Current address: Department of Chemistry and Biochemistry and The Institute for Cellular and Molecular Biology, The University of Texas at Austin, 1 University Station A5300, Austin, TX 78712-0165, USA.

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