A distinct gene expression mechanism for d ‐amino acid utilization in hyperthermophilic archaeon Pyrococcus horikoshii

Growing the hyperthermophilic archaeon Pyrococcus horikoshii OT‐3 in medium supplemented with d ‐ allo ‐Ile instead of l ‐Ile markedly upregulates the activity of broad substrate specificity amino acid racemase (BAR). In P. horikoshii genome, the BAR gene ( PH0138 ) forms a cluster with PH0137 , which encodes a putative transporter protein, and PH0140 that encodes a feast/famine regulatory protein (FFRP), involved in the transcriptional regulation of metabolic pathway genes. Here, we performed gene expression, protein–DNA interaction, and crystallographic analyses to elucidate the expression mechanism of the BAR cluster. Gene expression analysis revealed that d ‐ allo ‐Ile simultaneously induces PH0138 and PH0137 . Electrophoretic mobility shift assays demonstrated that the PH0140 protein binds to the PH0137 promoter in the presence of l ‐Ile, but this interaction is disrupted by d ‐ allo ‐Ile, identifying PH0140 as a d ‐amino acid‐responsive regulatory protein (DARP). The crystal structures of DARP bound to l ‐Ile and d ‐ allo ‐Ile were compared with those of another FFRP family member, P. horikoshii FL11. Our findings reveal a dimeric arrangement of l ‐Ile‐bound DARP resembling the DNA‐bound (open) form of FL11, whereas d ‐ allo ‐Ile‐bound DARP corresponds to the DNA‐unbound (closed) form. These conformational changes result from subtle alterations in hydrogen bonding around the coregulators. Furthermore, cultivation in d ‐ allo ‐Ile substitution medium only impacted the expression of PH0138 and PH0137 , and not the other genes, suggesting that DARP regulates a more limited gene set than FL11. Cumulatively, these results reveal a distinct mechanism by which an FFRP homolog controls d ‐amino acid utilization.