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Characterization of a novel β-alanine biosynthetic pathway consisting of promiscuous metabolic enzymes.

ABSTRACT: Bacteria adapt to utilize the nutrients available in their environment through a sophisticated metabolic system composed of highly specialized enzymes. Although these enzymes can metabolize molecules other than those for which they evolved, their efficiency toward promiscuous substrates is considered too low to be of physiological relevance. Herein, we investigated the possibility that these promiscuous enzymes are actually efficient enough at metabolizing secondary substrates to modify the phenotype of the cell. For example, in the bacterium Acinetobacter baylyi ADP1 (ADP1), panD (coding for l-aspartate decarboxylase) encodes the only protein known to catalyze the synthesis of β-alanine, an obligate intermediate in CoA synthesis. However, we show that the ADP1 ΔpanD mutant could also form this molecule through an unknown metabolic pathway arising from promiscuous enzymes and grow as efficiently as the wildtype strain. Using metabolomic analyses, we identified 1,3-diaminopropane and 3-aminopropanal as intermediates in this novel pathway. We also conducted activity screening and enzyme kinetics to elucidate candidate enzymes involved in this pathway, including 2,4-diaminobutyrate aminotransferase (Dat) and 2,4-diaminobutyrate decarboxylase (Ddc) and validated this pathway in vivo by analyzing the phenotype of mutant bacterial strains. Finally, we experimentally demonstrate that this novel metabolic route is not restricted to ADP1. We propose that the occurrence of conserved genes in hundreds of genomes across many phyla suggests that this previously undescribed pathway is widespread in prokaryotes.

SUBMITTER: Perchat N

PROVIDER: S-EPMC9213253 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Folates are tripartite molecules comprising pterin, para-aminobenzoate (PABA), and glutamate moieties, which are essential cofactors involved in DNA and amino acid synthesis. The obligately intracellular Chlamydia species have lost several biosynthetic pathways for essential nutrients which they can obtain from their host but have retained the capacity to synthesize folate. In most bacteria, synthesis of the pterin moiety of folate requires the FolEQBK enzymes, while synthesis of the PABA moiety is carried out by the PabABC enzymes. Bioinformatic analyses reveal that while members of Chlamydia are missing the genes for FolE (GTP cyclohydrolase) and FolQ, which catalyze the initial steps in de novo synthesis of the pterin moiety, they have genes for the rest of the pterin pathway. We screened a chlamydial genomic library in deletion mutants of Escherichia coli to identify the "missing genes" and identified a novel enzyme, TrpFCtL2, which has broad substrate specificity. TrpFCtL2, in combination with GTP cyclohydrolase II (RibA), the first enzyme of riboflavin synthesis, provides a bypass of the first two canonical steps in folate synthesis catalyzed by FolE and FolQ. Notably, TrpFCtL2 retains the phosphoribosyl anthranilate isomerase activity of the original annotation. Additionally, we independently confirmed the recent discovery of a novel enzyme, CT610, which uses an unknown precursor to synthesize PABA and complements E. coli mutants with deletions of pabA, pabB, or pabC. Thus, Chlamydia species have evolved a variant folate synthesis pathway that employs a patchwork of promiscuous and adaptable enzymes recruited from other biosynthetic pathways. Importance: Collectively, the involvement of TrpFCtL2 and CT610 in the tetrahydrofolate pathway completes our understanding of folate biosynthesis in Chlamydia. Moreover, the novel roles for TrpFCtL2 and CT610 in the tetrahydrofolate pathway are sophisticated examples of how enzyme evolution plays a vital role in the adaptation of obligately intracellular organisms to host-specific niches. Enzymes like TrpFCtL2 which possess an enzyme fold common to many other enzymes are highly versatile and possess the capacity to evolve to catalyze related reactions in two different metabolic pathways. The continued identification of unique enzymes such as these in bacterial pathogens is important for development of antimicrobial compounds, as drugs that inhibit such enzymes would likely not have any targets in the host or the host's normal microbial flora.

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Characterization of a novel β-alanine biosynthetic pathway consisting of promiscuous metabolic enzymes.

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