Structure and inhibition of a quorum sensing target from Streptococcus pneumoniae

Vipender Singh, Wuxian Shi, Steven C. Almo, Gary B. Evans, Richard H. Furneaux, Peter C. Tyler, Gavin F. Painter, Dirk H. Lenz, Simon Mee, Renjian Zheng, Vern L. Schramm

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51 Scopus citations

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 the 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 Escherichia 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 sequence of MTAN from S. pneumoniae is 40% identical to that of E. coli MTAN, but S. pneumoniae MTAN 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 Ki 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-TolT-ImmA), or ethyl (EtT-ImmA) groups increases the affinity to give Ki values of 335, 60, and 40 nM, respectively. DADMe-Immucillins are geometric and electrostatic mimics of a fully dissociated transition state and bind more tightly than Immucillins. MT-DADMe-Immucillin-A inhibits with a Ki value of 24 nM, and replacing the 5′-methyl group with p-Cl-phenyl (p-Cl-PhT-DADMe-ImmA) gave a Ki* 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 3-104-fold more tightly to the E. coli enzyme. Catalytic site efficiency is primarily responsible for this difference since k cat/Km for S. pneumoniae MTAN is decreased 845-fold relative to that of E. coli MTAN.

Original languageEnglish (US)
Pages (from-to)12929-12941
Number of pages13
JournalBiochemistry
Volume45
Issue number43
DOIs
StatePublished - Oct 31 2006

ASJC Scopus subject areas

  • Biochemistry

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    Singh, V., Shi, W., Almo, S. C., Evans, G. B., Furneaux, R. H., Tyler, P. C., Painter, G. F., Lenz, D. H., Mee, S., Zheng, R., & Schramm, V. L. (2006). Structure and inhibition of a quorum sensing target from Streptococcus pneumoniae. Biochemistry, 45(43), 12929-12941. https://doi.org/10.1021/bi061184i