Biology subjects

Christendat, D.

Publications and source records attributed to Christendat, D..

3 recordsLinked to original sources

bioRxiv2025

Evolutionary Adaptation of Prephenate Dehydrogenases: A regulatory ACT domain acquisition in ecological niche specialization

Bacteria prephenate dehydrogenase (PDH) participates in the metabolic pathway for tyrosine biosynthesis. PDHs within the Bacilaceae phylum contain an ACT domain which enables them to be allosterically regulated by tyrosine. The mechanism via which the ACT domain introduces allostery onto PDH enzymes remains elusive. Furthermore, the evolutionary and biological advantages of ACT domain mediated regulation of metabolic pathways are highly debated. Building on our previous study, in which we solved the crystal structure of a Bacillus antraces ACT-containing PDH and proposed a model for its allosteric regulation by tyrosine, we now present further structural, and functional analyses in support of this model. In this study, we generated truncated PDH protein constructs lacking the ACT domain, determined their crystal structure and evaluated the role of tyrosine in modulating their enzymatic activity. We determined that the truncated PDH remains catalytically active, however, it is no longer allosterically regulated by tyrosine. Comparative structural analysis between the truncated PDH and PDHs naturally lacking the ACT domain that are known to be competitively inhibited by tyrosine revealed only minor changes in a loop region in the prephenate binding site. Attempts to introduce amino acids identified from the competitively inhibited PDH into the truncated construct did not restore tyrosine sensitivity, even at high concentration. This indicates that additional main chain amino acids away from the substrate binding site also contribute competitive inhibition by tyrosine. Analysis of a highly represented phylogenetic tree revealed that ACT containing PDHs are predominantly distributed amongst Firmicute and Actinomycetota. Representative organisms from both groups colonize nutrient limited and extreme environments. This distribution suggests that acquisition of the ACT domain may confer an evolutionary advantage by enabling organisms to efficiently partition chorismate, the end product of the shikimate pathway, for the biosynthesis of tyrosine and other essential aromatic compounds.

biochemistry↗

bioRxiv2025

The interplay between glucose and aromatic compound regulation by two IclR-type transcription factors, LigR1 and LigR2, in Pseudomonas putida KT2440

The rhizosphere is a hotspot of microbial activity where plants release a diverse array of aromatic compounds, including shikimate pathway intermediates and monolignols. Pseudomonas putida KT2440, renowned for its metabolic versatility in this niche, uses largely uncharacterized regulatory and enzymatic strategies to utilize these compounds. We investigated two IclR-type transcriptional regulators, LigR1 and LigR2, that control expression of the uncharacterized lig1 and lig2 operons. We demonstrate that ligR1 deletion caused growth defects on glucose and 4-hydroxybenzoate accompanied by cell elongation and aggregation. Structural and functional analyses reveal that LigR1 and LigR2 activate the lig1 operon but repress the lig2 operon. LigR1 binding of 4-hydroxybenzoate induced repression by triggering tetramerization and increasing DNA-binding activity. In contrast, LigR2 responded to quinate, protocatechuate and 4-hydroxybenzoate to potently induce lig2 operon expression by relieving repression. While both operons cooperate in metabolizing these compounds, we propose the lig1 operon mediates influx through its major facilitator superfamily (MFS) transporter (PP_2604), whereas the lig2 operon catalyzes breakdown via a protocatechuate intermediate and the meta-cleavage pathway, supplying oxaloacetate to the TCA cycle. Importantly, we show that P. putida repurposes shikimate pathway intermediates for energy production. These findings challenge the canonical biosynthetic view of the shikimate pathway and redefine the metabolic flexibility of soil pseudomonads. We reveal a novel mechanism enabling P. putida to thrive in the chemically complex rhizosphere and open new avenues for exploring alternate roles of the shikimate pathway, emphasizing transcriptional regulators as tools to deconvolute complex metabolic landscapes. HighlightsO_LILigR1 and LigR2 transcriptionally regulate the lig1 and lig2 operons C_LIO_LILig1 operon is required for import of glucose and shikimate-derived compounds C_LIO_LILig2 operon metabolizes shikimate pathway compounds C_LIO_LIDysregulated LigR1/LigR2 expression impacts bacterial physiology C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=189 HEIGHT=200 SRC="FIGDIR/small/670189v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@9b461aorg.highwire.dtl.DTLVardef@175c73org.highwire.dtl.DTLVardef@1b6bd90org.highwire.dtl.DTLVardef@574e55_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2025

The coordinated regulatory roles of two LysR-Type Transcriptional Regulators balance chorismate and protocatechuate partition in Listeria organisms

Listeria monocytogenes is an economically deleterious foodborne pathogens that continually challenges the global food supply chain. Listeria species in general, synthesize protocatechuate from saprophytically-derived quinate and shikimate utilizing a novel class of bacterial dehydroshikimate dehydratase. Paradoxically, Listeria species are unable to metabolically utilize protocatechuate, as such, it was proposed that this compound is used as a currency to Listeria interactions with other microorganisms to improve their environmental proliferation. Therefore, an understanding of the regulatory mechanism for the metabolic pathway for protocatechuate biosynthesis is of great importance. Two LysR Type Transcriptional Regulators (LTTR), annotated QuiR and in this study QuiR2, are found upstream of genomic operons, qui1 and qui2, which transcribe genes for protocatechuate synthesis. QuiR, has been shown to activate the expression of genes from both operons with shikimate as a coinducer. However, the role of QuiR2, Lmo2233, is not clear. In this study, we conducted structural, biochemical and bioinformatics analyses of QuiR2 and demonstrated that it functions as a negative regulator of protocatechuate biosynthesis in Listeria species. Moreover, we determined that protocatechuate functions in modulating QuiR2 repressive properties through our mobility shift assay and LacZ reporter activity studies. Furthermore, phylogenetic analyses reveal that QuiR2 clusters closely but independently from QuiR thus supporting their distinct regulatory roles. We propose that QuiR2 prevents metabolic commitment of dehydroshikimate to protocatechuate when elevated and in limiting shikimate condition. In this study we revisited the biological role of the shikimate pathway in microbes and demonstrated that in addition to it producing chorismite for aromatic compound metabolism it is also important in allowing organisms to shuttle shikimate and quinate to produce protocatechuate which can be used as an energy source and more importantly in Listeria it is used to facilitate microbial interactions.

molecular biology↗