Biochemical and genetic characterization of an early step in a novel pathway for the biosynthesis of aromatic amino acids and p‐aminobenzoic acid in the archaeon Methanococcus maripaludis

Abstract

SummaryMethanococcus maripaludis is a strictly anaerobic, methane-producing archaeon and facultative autotroph capable of biosynthesizing all the amino acids and vitamins required for growth. In this work, the novel 6-deoxy-5-ketofructose-1-phosphate (DKFP) pathway for the biosynthesis of aromatic amino acids (AroAAs) and p-aminobenzoic acid (PABA) was demonstrated in M. maripaludis. Moreover, PABA was shown to be derived from an early intermediate in AroAA biosynthesis and not from chorismate. Following metabolic labelling with [U-13 C]-acetate, the expected enrichments for phenylalanine and arylamine derived from PABA were observed. DKFP pathway activity was reduced following growth with aryl acids, an alternative source of the AroAAs. Lastly, a deletion mutant of aroAЈ, which encodes the first step in the DKFP pathway, required AroAAs and PABA for growth. Complementation of the mutants by an aroAЈ expression vector restored the wild-type phenotype. In contrast, a deletion of aroBЈ, which encodes the second step in the DKFP pathway, did not require AroAAs or PABA for growth. Presumably, methanococci contain an alternative activity for this step. These results identify the initial reactions of a new pathway for the biosynthesis of PABA in methanococci.

Carbohydrate Metabolism in Archaea: Current Insights into Unusual Enzymes and Pathways and Their Regulation

“…jannaschii homologs as well as analysis of the FBPA deletion strain (⌬aroA=; MMP0686) in Mco. maripaludis, which revealed FBPA activity similar to that of the wild-type strain (384). There is additional experimental evidence that the other FBPA homologs (MJ0400 and MMP0686) have a function in 6-deoxy-5-ketofructose 1-phosphate (DKFP) formation in Mca.…”

“…maripaludis, an E4P-independent pathway for the initial synthesis of 3-dehydrochinate (and further to chorismate) has been identified. The key intermediate of this pathway is 6-deoxy-5-ketofructose 1-phosphate (DKFP), differing from the canonical pathway, which starts with the condensation of E4P and PEP yielding 3-deoxy-arabino-heptulosanate 7-phosphate (382)(383)(384). This alternative pathway also appears to be present in other methanogens as well as in halophiles and might also be operative in other TKnegative organisms such as Arc.…”

“…There is additional experimental evidence that the other FBPA homologs (MJ0400 and MMP0686) have a function in 6-deoxy-5-ketofructose 1-phosphate (DKFP) formation in Mca. jannaschii, a key step in a novel pathway for the formation of aromatic amino acids (382,384). In the genome, two candidates for the noncanonical phosphoglycerate mutases were identified (MMP0112 and MMP1439); the Mca.…”

Characterization of Energy-Conserving Hydrogenase B in Methanococcus maripaludis

“…In the functional model of Porat et al (15), Ehb was proposed to generate the low potential electron donor to indolepyruvate oxidoreductase (Ior), which is required for one of the two pathways of aromatic amino acid biosynthesis in M. maripaludis (17,18). The deletion mutant ⌬iorA2 was unable to utilize the aryl acids phenylacetate, indoleacetate, and p-hydroxyphenylacetate to make the aromatic amino acids.…”

Cohesion Group Approach for Evolutionary Analysis of Aspartokinase, an Enzyme That Feeds a Branched Network of Many Biochemical Pathways

“…Some organisms lack the first two steps of the classic erythrose-4-phosphate (E4P) pathway for the synthesis of aromatic amino acids (75). Instead of utilizing E4P and phosphoenolpyruvate in the initial step, Laspartate semialdehyde and 6-deoxy-5-ketofructose 1-phosphate (DKFP) are combined to form 2-amino-3,7-dideoxy-Dthreo-hept-6-ulosonate via the action of an aldolase called AroAЈ.…”