Structure and Inhibition of a Quorum Sensing Target from Streptococcus pneumoniae

Abstract

Streptococcus pneumoniae 5′-methylthioadenosine/S-adenosylhomocysteine hydrolase (MTAN) catalyzes the hydrolytic deadenylation of its substrates to form adenine and 5-methylthioribose or S-ribosylhomocysteine (SRH). MTAN is not found in mammals but is involved in bacterial quorum sensing. MTAN gene disruption affects growth and pathogenicity of bacteria, making it a target for antibiotic design. Kinetic isotope effects and computational studies have established a dissociative SN1 transition state for E. coli MTAN and transition state analogues resembling the transition state are powerful inhibitors of the enzyme (Singh, V., Lee, J. L., Núñez, S., Howell, P. L. and Schramm, V. L. (2005) Biochemistry 44, 11647-11659). The MTAN from S. pneumoniae has 40% sequence identity to E. coli MTAN, but exhibits remarkably distinct kinetic and inhibitory properties. 5′-Methylthio-Immucillin-A (MT-ImmA) is a transition state analogue resembling an early SN1 transition state. It is a weak inhibitor of S. pneumoniae MTAN with a K i of 1.0 μM. The X-ray structure of S. pneumoniae MTAN with MT-ImmA indicates a dimer with the methylthio group in a flexible hydrophobic pocket. Replacing the methyl group with phenyl (PhT-ImmA), tolyl (p-TolTImmA) or ethyl (EtT-ImmA) groups increases the affinity to give K i values of 335 nM, 60 nM and 40 nM, respectively. DADMe-Immucillins are geometric and electrostatic mimics of a fullydissociated transition state and bind more tightly than Immucillins. MT-DADMe-Immucillin-A inhibits with a K i value of 24 nM and replacing the 5′-methyl group with p-Cl-phenyl (p-Cl-PhTDADMe-ImmA) gave a K i * value of 0.36 nM. The inhibitory potential of DADMe-Immucillins relative to the Immucillins supports a fully dissociated transition state structure for S. pneumoniae MTAN. Comparison of active site contacts in the X-ray crystal structures of E. coli and S. pneumoniae MTAN with MT-ImmA would predict equal binding, yet most analogues bind 10 3 to 10 4 fold more tightly to the E. coli enzyme. Catalytic site efficiency is primarily responsible for this difference since k cat /K m for S. pneumoniae MTAN is <10 -2 that of E. coli MTAN. Keywords5′-methylthioadenosine; 5′-methylthioadenosine nucleosidase; quorum sensing; nucleoside hydrolase; transition state; transition state analogue inhibitors; polyamines; MTAN structure; catalytic efficiency *Corresponding author: telephone (718) 430-2813, Fax (718) Email vern@aecom.yu.edu. 3 Immucillin-H is (1S)-1-(9-deazahypoxanthin-9-yl)-1,4-dideoxy-1,4-imino-D-ribitol and has been shown to have a pK a > 10 at N7 (47). MT-ImmA is chemically similar in the 9-deazaadenine ring and is expected to have similar pK a . NIH Public Access NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript5′-Methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN 1 ) is a bacterial enzyme encoded by the pfs gene and catalyzes hydrolytic depurination of 5′-methylthioadenosine (MTA) to form 5-methylthioribose (MTR) and S-adenosylhomocysteine (SAH) to S-ribosylh...

The Monomeric, Tetrameric, and Fibrillar Organization of Fib: The Dynamic Building Block of the Bacterial Linear Motor of Spiroplasma melliferum BC3

“…Autophosphorylation has been known to occur in a 57-kDa protein in Spiroplasma. 39 The best match is with the 3bsf phosphorylase nucleosidase from Arabidopsis thaliana 42 Incidentally, Fib of Spiroplasma melliferum BC3, using antibodies against serine, threonine, and tyrosine, appears to be phosphorylated (S.T., unpublished results).…”

Exploiting Quorum Sensing To Confuse Bacterial Pathogens

“…Studies utilizing kinetic isotope effects (KIEs) and crystal structures of MTANs in complex with known transition state analogues have allowed for transition state modeling of MTANs from E. coli, S. pneumoniae, Neisseria meningitidis, S. aureus, and V. cholerae and the subsequent design of analogue inhibitors (120)(121)(122)(149)(150)(151)(152)(153). Thus far, it appears that most MTANs share a fully dissociated S N 1 transition state characterized by a ribooxacarbenium ion (121,122,149,152), with the exception of MTANs from (149).…”

“…Thus far, it appears that most MTANs share a fully dissociated S N 1 transition state characterized by a ribooxacarbenium ion (121,122,149,152), with the exception of MTANs from (149). The majority of the transition state analogue inhibitor characterization has been performed using purified MTANs in vitro.…”

Quorum Sensing in Gram-Negative Bacteria: Small-Molecule Modulation of AHL and AI-2 Quorum Sensing Pathways

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