From knock-out phenotype to three-dimensional structure of a promising antibiotic target from Streptococcus pneumoniae

PloS One
Con DogovskiMatthew A Perugini

Abstract

Given the rise in drug-resistant Streptococcus pneumoniae, there is an urgent need to discover new antimicrobials targeting this pathogen and an equally urgent need to characterize new drug targets. A promising antibiotic target is dihydrodipicolinate synthase (DHDPS), which catalyzes the rate-limiting step in lysine biosynthesis. In this study, we firstly show by gene knock out studies that S. pneumoniae (sp) lacking the DHDPS gene is unable to grow unless supplemented with lysine-rich media. We subsequently set out to characterize the structure, function and stability of the enzyme drug target. Our studies show that sp-DHDPS is folded and active with a k(cat) = 22 s(-1), K(M)(PYR) = 2.55 ± 0.05 mM and K(M)(ASA) = 0.044 ± 0.003 mM. Thermal denaturation experiments demonstrate sp-DHDPS exhibits an apparent melting temperature (T(M)(app)) of 72 °C, which is significantly greater than Escherichia coli DHDPS (Ec-DHDPS) (T(M)(app) = 59 °C). Sedimentation studies show that sp-DHDPS exists in a dimer-tetramer equilibrium with a K(D)(4→2) = 1.7 nM, which is considerably tighter than its E. coli ortholog (K(D)(4→2) = 76 nM). To further characterize the structure of the enzyme and probe its enhanced stability, we solved the high resolut...Continue Reading

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Citations

Feb 3, 2016·Journal of the American Chemical Society·Yulia V SkovpenDavid R J Palmer
Mar 29, 2014·Proteins·Sarah C AtkinsonMatthew A Perugini
Nov 16, 2016·Scientific Reports·Janni B ChristensenMatthew A Perugini
Mar 12, 2016·PLoS Computational Biology·Shane E GordonMatthew A Perugini
Jan 17, 2020·FEBS Letters·Rachael E ImpeyTatiana P Soares da Costa
Feb 28, 2016·The Journal of Biological Chemistry·Martin G PeverelliMatthew A Perugini
Jul 28, 2017·Proteins·Tatiana P Soares da CostaMatthew A Perugini
Dec 6, 2017·Biophysics Reviews·Tatiana P Soares da CostaMatthew A Perugini
May 29, 2018·Structure·Sarah C AtkinsonMatthew A Perugini

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Methods Mentioned

BETA
gene knock
PCR
X-ray
circular dichroism
dynamic light scattering
SISA
PISA
gene
circular

Software Mentioned

SEDNTRP
SEDPHAT
SEDNTERP
ENZFITTER
SCALA
REFMAC5
COOT
HKL2000
PHENIX
ALV

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