Biochimica et Biophysica Acta (BBA) - General Subjects
Regular paperIdentification and characterization of a l-tyrosine decarboxylase in Methanocaldococcus jannaschii
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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2021, Journal of Biological ChemistryCitation Excerpt :The discovery of MYFRTyramine prompted us to investigate the genetic and biosynthetic basis for the incorporation of tyrosine or tyramine into MYFR. In the biosynthesis of MFR, tyramine is generated from tyrosine by MfnA, a pyridoxal phosphate-dependent l-tyrosine decarboxylase first identified in the methanogenic archaeon Methanocaldococcus jannaschii (16). To determine whether a similar enzyme is present in bacteria, we used BLAST (23) to search for homologs of MfnA in all strains analyzed above (Table S3).
Crystallographic Snapshots of the Dunathan and Quinonoid Intermediates provide Insights into the Reaction Mechanism of Group II Decarboxylases
2020, Journal of Molecular BiologyCitation Excerpt :More so, the current study will help better understand the structural basis of the evolution of PLP-dependent enzymes that display such exquisite reaction specificity and substrate selectivity. pSpeedET vector containing MJ0050 gene that encodes for MjDC (Uniprot Id Q60358) was purchased from DNASU (JCSG 390948; PSI: Biology Materials Repository Clone ID: MjCD00289364, http://psimr.asu.edu/).17,43 Oligonucleotides not listed in our previous study and used for mutagenesis in the current study are listed in Table S3.18
Structural insights into the mechanism of internal aldimine formation and catalytic loop dynamics in an archaeal Group II decarboxylase
2019, Journal of Structural BiologyCitation Excerpt :The apparent molecular weight of the eluting species was determined by comparing their elution volume to that of a set of molecular weight standards under the same experimental conditions (Fig. S1). Decarboxylase assay was performed as described by previously with some modifications (Kezmarsky et al., 2005; Phan et al., 1983). Briefly, 10 µg of enzyme was incubated for 5 min with 0.5 mM L-tyrosine and 20 μM PLP in 50 mM HEPES, pH 8.0, at 80 °C (final volume 250 μl).
Engineering a bacterial platform for total biosynthesis of caffeic acid derived phenethyl esters and amides
2017, Metabolic EngineeringCitation Excerpt :Notably, E. coli also consumed phenylpyruvate, 4-hydroxyphenylpyruvate and 3, 4-hydroxyphenylpyruvate to produce corresponding aromatic amino acids (data not shown). To generate aromatic amines including PA, tyramine (TA) and dopamine (DA), aromatic amino acid decarboxylases (AADCs) of different bacterial origins were introduced and screened (Kezmarsky et al., 2005; Koyanagi et al., 2012; Zhang and Ni, 2014). The phenylalanine decarboxylase (LbPDC) from Lactobacillus brevis, tyrosine decarboxylase (MjTDC) from Methanocaldococcus jannaschii and L-Dopa decarboxylase (PpDDC) from Pseudomonas putida were over-expressed on pCS27 plasmids and tested by feeding aromatic amino acids including phenylalanine, tyrosine and L-Dopa (500 mg/L).
The one-carbon carrier methylofuran from methylobacterium extorquens AM1 contains a large number of α- and γ-Linked glutamic acid residues
2016, Journal of Biological ChemistryCitation Excerpt :Considering the two methylene groups in the tyramine-like moiety, the position next to the amine group was considered more likely to be modified, as the attachment of a carboxylic acid group at that position would result in a tyrosine residue instead of a tyramine. Furthermore, in the biosynthesis of the methanogenic MFR, the tyramine residue is formed by the decarboxylation of tyrosine (23). In methylofuran, a tyrosine residue instead of a tyramine could, therefore, easily be explained by a modified biosynthesis where the decarboxylation step is missing.
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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.