Methylthioadenosine Deaminase in an Alternative Quorum Sensing Pathway in Pseudomonas aeruginosa

摘要

Pseudomonas aeruginosa possesses an unusual pathway for 5′-methylthioadenosine (MTA) metabolism involving deamination to 5′-methylthioinosine (MTI) followed by N-ribosyl phosphorolysis to hypoxanthine and 5-methylthio-α-D-ribose 1-phosphate. The specific MTI phosphorylase of P. aeruginosa has been reported (Guan, R., Ho, M. C., Almo, S. C. and Schramm, V. L. (2011) Biochemistry 50, 1247–1254) and here we characterize MTA deaminase from P. aeruginosa (PaMTADA). Genomic analysis indicated the PA3170 locus to be a candidate for MTA deaminase (MTADA). Protein encoded by PA3170 was expressed and shown to deaminate MTA with 40-fold greater catalytic efficiency for MTA than for adenosine. The kcat/Km value of 1.6 × 10⁷ M⁻¹s⁻¹ for MTA is the highest catalytic efficiency known for an MTA deaminase. 5′-Methylthiocoformycin (MTCF) is a 4.8 pM transition state analogue for PaMTADA but causes no significant inhibition of human adenosine deaminase or MTA phosphorylase. MTCF is permeable to P. aeruginosa and exhibits an IC50 of 3 nM on cellular PaMTADA activity. PaMTADA is the only activity in P. aeruginosa extracts to act on MTA. MTA and 5-methylthio-α-D-ribose are involved in quorum sensing pathways, thus PaMTADA is a potential target for quorum sensing. The crystal structure of PaMTADA in complex with MTCF shows the transition state mimic 8-R-hydroxyl group in contact with a catalytic site Zn²⁺, the 5′-methylthio group in a hydrophobic pocket and the transition state mimic of the diazepine ring in contact with a catalytic site Glu.