A distinct domain organization of cystathionine β-synthase underlies cysteine and H2S biosynthesis in Pseudomonas aeruginosa and Klebsiella pneumoniae

Activation of the reverse transsulfuration pathway contributes to the survival and persistence of multidrug-resistant bacteria. The first step of this pathway, catalyzed by the enzyme cystathionine β-synthase (CBS), plays a crucial role in maintaining redox balance and cysteine homeostasis, making it an attractive therapeutic target against multidrug-resistant infections. In this study, we demonstrate that both Pseudomonas aeruginosa CBS and Klebsiella pneumoniae CBS exhibit high responsiveness to the allosteric activator S-adenosylmethionine (SAM) and preferentially utilize O-acetylserine (OAS) as a substrate over serine. The crystal structure of PaCBS and the AlphaFold-2 model of KpCBS reveal a novel domain organization characterized by an inverted arrangement of the regulatory Bateman module relative to the catalytic core. These findings provide a new structural and functional basis for the selective inhibition of CBS in multidrug-resistant pathogens. Conter et al. show that Pseudomonas aeruginosa and Klebsiella pneumoniae cystathionine βsynthases (CBS), key enzymes supporting redox balance and survival in these multidrug-resistant bacteria, are highly responsive to S-adenosylmethionine and preferentially utilize O-acetylserine. Their structural analyses revealed a novel CBS domain organization and provides a foundation for selective antimicrobial therapeutics development.