Structure and Inhibition of a Quorum Sensing Target from Streptococcus pneumoniae

• Published in: Biochemistry (Easton) Vol. 45; no. 43; pp. 12929 - 12941
• Main Authors: Singh, Vipender, Shi, Wuxian, Almo, Steven C, Evans, Gary B, Furneaux, Richard H, Tyler, Peter C, Painter, Gavin F, Lenz, Dirk H, Mee, Simon, Zheng, Renjian, Schramm, Vern L
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 31-10-2006
• Subjects: ADENINES; AFFINITY; Amino Acid Sequence; ANTIBIOTICS; BACTERIA; Binding Sites; BIOCHEMISTRY; Catalysis - drug effects; CRYSTAL STRUCTURE; Crystallography, X-Ray - methods; DIMERS; EFFICIENCY; ELECTROSTATICS; Enzyme Activation - drug effects; ENZYMES; ESCHERICHIA COLI; Escherichia coli - enzymology; Escherichia coli - genetics; GENES; GROWTH; HYDROLASES; INHIBITION; ISOTOPE EFFECTS; KINETICS; MAMMALS; MATERIALS SCIENCE; Models, Molecular; Molecular Sequence Data; N-Glycosyl Hydrolases - chemistry; N-Glycosyl Hydrolases - genetics; N-Glycosyl Hydrolases - metabolism; national synchrotron light source; Protein Binding - drug effects; Protein Structure, Secondary; Protein Structure, Tertiary; Ribitol - analogs & derivatives; Ribitol - pharmacology; Sequence Analysis, Protein; Signal Transduction - drug effects; STREPTOCOCCUS; Streptococcus pneumoniae; Streptococcus pneumoniae - enzymology; Streptococcus pneumoniae - genetics; Streptococcus pneumoniae - metabolism; Structural Homology, Protein; Substrate Specificity; SUBSTRATES
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi061184i

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 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-TolT-ImmA), or ethyl (EtT-ImmA) groups increases the affinity to give K i 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 K i value of 24 nM, and replacing the 5‘-methyl group with p-Cl-phenyl (p-Cl-PhT-DADMe-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 103−104-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 decreased 845-fold relative to that of E. coli MTAN.