TY - JOUR
T1 - Structure and inhibition of a quorum sensing target from Streptococcus pneumoniae
AU - Singh, Vipender
AU - Shi, Wuxian
AU - Almo, Steven C.
AU - Evans, Gary B.
AU - Furneaux, Richard H.
AU - Tyler, Peter C.
AU - Painter, Gavin F.
AU - Lenz, Dirk H.
AU - Mee, Simon
AU - Zheng, Renjian
AU - Schramm, Vern L.
PY - 2006/10/31
Y1 - 2006/10/31
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=33751083094&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=33751083094&partnerID=8YFLogxK
U2 - 10.1021/bi061184i
DO - 10.1021/bi061184i
M3 - Article
C2 - 17059210
AN - SCOPUS:33751083094
SN - 0006-2960
VL - 45
SP - 12929
EP - 12941
JO - Biochemistry
JF - Biochemistry
IS - 43
ER -