Atypical LONELY GUY protein in Bordetella pertussis synthetizes a cytokinin-like compound negatively related to oxidative stress
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The Gram-negative bacterium Bordetella pertussis (Bp) is the causative agent of whooping cough. This infection is re-emerging and new features related to Bordetella pathogenesis and microbiology could be relevant to defeat it. BP1253 from Bp is a predicted exported protein erroneously classified as lysine decarboxylase, presenting sequence homology with some newly structurally characterized “LONELY GUY” proteins. These cytokinin-activating enzymes share with BP1253 the highly conserved motif PGGxGTxxE and the amino acid residues of the catalytic core. SEC and MALS analysis revealed a dimeric form as functional unit. Surface plasmon resonance studies showed that BP1253 selectively binds monophosphate nucleotides and dinucleotides such as AMP, GMP and NAD, NADP. Applying an enzymatic assay in the presence of nucleotides as substrates, we clearly showed that BP1253 is able to cleave the N-glycosidic linkage between the base and the ribose, leading to the formation of free bases. This phosphoribohydrolase activity, time- and dose- dependent, is the crucial reaction in producing active cytokinins, which are phytormones in plants. Moreover, the enzymatic assay with site-specific mutants confirmed the amino acids 120R and 143D as the catalytic residues and the positive charge in position 121 crucial for the reaction. Through LC-MS/MS we identified in growth medium of Tohama strain the 6-O-Methylguanine as physiological product of the BP1253, in agreement with the higher activity of the enzyme towards GMP. Although 6-O-Methylguanine resulted to be toxic for the same bacteria in oxidative stress conditions, BP1253 turned out to be also prevalently expressed in clinical isolates. Overall, the data presented show for the first time the presence of a cytokinin-activating enzyme in Bordetella pertussis.
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