How Do Haloarchaea Synthesize Aromatic Amino Acids?

<div><p>Genomic analysis of <i>H. salinarum</i> indicated that the <i>de novo</i> pathway for aromatic amino acid (AroAA) biosynthesis does not follow the classical pathway but begins from non-classical precursors, as is the case for <i>M. jannaschii</i>. The first two steps in the pathway were predicted to be carried out by genes OE1472F and OE1475F, while the 3^rd step follows the canonical pathway involving gene OE1477R. The functions of these genes and their products were tested by biochemical and genetic methods. In this study, we provide evidence that supports the role of proteins OE1472F and OE1475F catalyzing consecutive enzymatic reactions leading to the production of 3-dehydroquinate (DHQ), after which AroAA production proceeds via the canonical pathway starting with the formation of DHS (dehydroshikimate), catalyzed by the product of ORF OE1477R. Nutritional requirements and AroAA uptake studies of the mutants gave results that were consistent with the proposed roles of these ORFs in AroAA biosynthesis. DNA microarray data indicated that the 13 genes of the canonical pathway appear to be utilised for AroAA biosynthesis in <i>H. salinarum</i>, as they are differentially expressed when cells are grown in medium lacking AroAA.</p></div

Protein sequence alignment of OE1472F from <i>H</i>. <i>salinarum</i> and MJ0400 from <i>M</i>. <i>jannaschii</i>.

<p>Conserved sequence motifs of archaea, described by Siebers et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107475#pone.0107475-Siebers1" target="_blank">[10]</a>, are marked with black boxes. Active site residues described by Morar et al. for <i>M</i>. <i>jannaschii</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107475#pone.0107475-Morar1" target="_blank">[9]</a> are marked in boldface letters and red asterisks. The catalytic lysine residue (Lys237) determined for the <i>E</i>. <i>coli</i> class IA aldolase (DhnA type aldolase), is marked with a black asterisk <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107475#pone.0107475-Thomson1" target="_blank">[44]</a>. Identical residues are colored yellow (43%), and similar residues are colored orange (61%). The alignment was done using ClustalW multiple sequence alignment program (<a href="http://us.expasy.org" target="_blank">http://us.expasy.org</a>). The secondary structure elements according to MJ0400 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107475#pone.0107475-Morar1" target="_blank">[9]</a> are shown at the top of the alignment. Lower panel: Superposition of MJ0400-F1,6-P complex (PDB:2QJG) with OE1472F. Colours indicate: Blue, MJ0400; red, OE1472F; green, F-1,6-P. The PDB file of OE1472F was generated by Phyre (<a href="http://us.expasy.org" target="_blank">http://us.expasy.org</a>) and 3D structure was superimposed and viewed by Pymol (<a href="http://www.pymol.org/" target="_blank">http://www.pymol.org/</a>).</p

Proposed pathways for the formation of methylglyoxal and DKFP.

<p>In <i>M</i>. <i>jannaschii</i>, reactions 1 and 3 were suggested to be catalyzed by the multifunctional glycolytic enzyme MJ1585. Reaction 2 was suggested to be catalyzed by triose phosphate isomerase EC 5.3.1.1 (ORFs MJ1528 and OE2500R of <i>M</i>. <i>jannaschii</i> and <i>H</i>. <i>salinarum</i>, respectively). Modified from White and Xu <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107475#pone.0107475-White2" target="_blank">[24]</a>.</p

Phe consumption during the growth of WT and mutants <i>Ins</i>OE1475F (left panel) ΔOE1477R (right panel).

<p>Culture media were supplemented with AroAAs, DHQ or shikimate, as indicated in the growth conditions column at left (see Methods for details). In each graph, the red circles represent Phe consumption by WT <i>H. salinarum</i> and blue circles represent consumption by the mutant strain. Simulation curves (dashed lines), are red for WT, and blue for the mutants. Error bars show the deviation of the average calculated from three biological repeats and two technical repeats. The uptake rates were calculated from the corresponding simulations.</p

Schematic representation of ORFs OE1472F, OE1475F and OE1477R in the <i>H. salinarum</i> R1 genome (accession number AM774415.1), and their relation to surrounding genes.

<p>CHY- conserved hypothetical protein. Arrows show the relative positions and orientations of ORFs (but are not drawn to scale). Coordinates 230688–237094 bp.</p

Aldolase and transaldolase activities reported in Archaea.

(1)<p> In the coupled assay, aldolase activity was determined using coupled assay, were the cleavage of F-1,6-P was coupled with glycerol-3-phosphate dehydrogenase (EC 1.1.1.8) and triose-phosphate isomerase (TIM, EC 5.3.1.1) of rabbit muscle. Enzymatic activities were measured by monitoring the increase in absorption of NADH at 366 nm (ε_50°C = 3.36 mm⁻¹cm⁻¹).</p>(2)<p> DHQ generated with recombinant proteins was determined by GC-MS.</p>(3)<p> Formation of DHAP was measured by Colorimetric assay (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107475#s4" target="_blank">materials and methods</a> for details).</p>(4)<p> <b>MJ0400</b> from <i>M</i>. <i>jannaschii</i> is homologous to OE1472F from <i>H</i>. <i>salinarum</i> and MMP0686 from <i>M</i>. <i>maripaludis</i>. Predicted transaldolase catalyzing the first reaction of the AroAA biosynthesis pathway in these organisms.</p>(5)<p> <b>MJ1585</b> from <i>M</i>. <i>jannaschii</i> is homologous to OE2019F from <i>H</i>. <i>salinarum</i> and MMP0293 from <i>M</i>. <i>maripaludis</i>. It is an aldolase.</p>(6)<p> <b>MJ1249</b> is homologous to OE1475F from <i>H</i>. <i>salinarum</i> and MMP0006 from <i>M</i>. <i>maripaludis</i>. It is believed to catalyze the second reaction in the AroAA biosynthesis, synthesizing DHQ.</p>(7)<p> <i>Thermoproteus tenax</i>, crenarchaeon.</p>(8)<p> <i>Pyrococcus furiosus</i>, euryarchaeon.</p>(9)<p> GenBank accession number.</p><p>Aldolase and transaldolase activities reported in Archaea.</p

Schematic representations of a single cross-over event between the chromosomal DNA of <i>H. salinarum</i> R1 and plasmid pMG501.

<p>P1,P2 and P3 and their associated arrows indicate the relative positions and orientations of the primers used for PCR. After in-frame integration of plasmid pMG501, PCR was expected to generate a product in transformed cells but not for the WT. <b>A</b>, Illustration of WT <i>H. salinarum</i> genotype. <b>B</b>, illustration of the mutant genotype after incorporation of plasmid pMG501. <b>C</b> and <b>D</b>, Agarose gel electrophoresis of PCR products using primers P1 and P2, and primers P3 and P2, respectively. Lane 1: MW markers (in bp), lane2: Chromosomal DNA of R1, lane 3: plasmid pMG501 (control), lanes 4–6- transformed colonies. <b>E</b>, Relative expression of OE1472F transcription in mutant <i>Stop</i>OE1472F (OE1471F::pMG501) grown in synthetic medium without AroAA to OD_600nm = 0.4 and 1.0. Results were obtained using RT-qPCR. See table S7 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107475#pone.0107475.s001" target="_blank">file S1</a> for details of the primers used.</p

Genomic contexts of ORFs involved AroAA biosynthesis in <i>H</i>. <i>salinarum</i>.

<p>Arrows show the relative positions and orientations of ORFs (but are not drawn to scale); those colored in red, green, blue or yellow represent ORFs assigned to the AroAA biosynthesis pathway while other, nearby ORFs, are uncoloured. <b>A</b> and <b>B</b>, ORFs assigned to convert precursors to DHQ. The first and second ORFs (OE1472F in red and OE1475F in green) are homologs of <i>M</i>. <i>jannaschii</i> ORFs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107475#pone.0107475-White1" target="_blank">[6]</a>. These form part of the non-canonical part of the pathway. ORF OE1477R (in blue) is the 3^rd ORF discussed in this paper. <b>C</b> and <b>D</b>, ORFs needed to convert DHQ to chorismate. <b>F</b> and <b>G</b>, the tryptophan branch. <b>H</b>, the tyrosine branch, <b>I</b>-the phenylalanine branch. J- the genomic context of ORF OE2019F, homolog to OE1472F. The numbers below the arrows represent the induction of AroAA-related genes in <i>H</i>. <i>salinarum</i> R1 cells grown in synthetic medium without AroAA relative to synthetic medium with AroAA. Induction values ≥2.0 are marked in red, and have p values of ≤10⁻³ unless indicated. * The two ORFs are involved in the conversion of chorismate to para-aminobenzoate, an intermediate of folate biosynthesis, <b>NS</b>, not significant; <b>ND</b>, not detected; <b>CHY</b>, conserved hypothetical protein; <b>HY</b>, hypothetical protein. ORF names and predicted functions are derived from the genome annotation at <a href="http://www.halolex.mpg.de" target="_blank">www.halolex.mpg.de</a>.</p

Proposed pathway for the biosynthesis of AroAA in <i>H</i>. <i>salinarum</i>, based on the pathway described for <i>M</i>. <i>jannaschii</i>.

<p><b>A</b>, The initial steps in the de novo pathway were proposed according to White <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107475#pone.0107475-White1" target="_blank">[6]</a>. Note that no transaldolase reaction with ASA+DKFP was detected in this study, whereas the detected aldolase activity of OE1472F suggests that in <i>H</i>. <i>salinarum</i> the precursor might be F-1,6-P rather than DKFP. For details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107475#pone-0107475-g010" target="_blank">Fig 10</a> and discussion. <b>B</b>, Downstream to DHQ, the canonical pathway is followed. Protein homologs found in <i>H</i>. <i>salinarum</i> are indicated above (or to the right of) the arrows, and the genes names are indicated below (or to the left). ASA-L-aspartate semialdehyde, DKFP-6-deoxy-5-ketofructose 1-phosphate, DHQ-dehydroquinate, DHS- dehydroshikimate.</p