Inhibiting the Actions of Essential Biomolecule Phosphopantetheine.

Coenzyme A;Phosphopantothenoylcysteine synthetase;Saturation Mutagenesis;Lipid A;Acyltransferases;Biological Chemistry;Pharmacy and Pharmacology;Chemistry;Health Sciences;Science;Medicinal Chemistry

Heslip, Kyle AnthonySoellner, Matthew Bryan ;

University of Michigan

关键词: Coenzyme A;    Phosphopantothenoylcysteine synthetase;    Saturation Mutagenesis;    Lipid A;    Acyltransferases;    Biological Chemistry;    Pharmacy and Pharmacology;    Chemistry;    Health Sciences;    Science;    Medicinal Chemistry;   

Others  :  https://deepblue.lib.umich.edu/bitstream/handle/2027.42/111574/heslipky_1.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

Phosphopantetheine is an essential biomolecule required for life. The enzyme phosphopantothenoylcysteine synthetase (PPCS) incorporates the reactive thiol moiety in the Coenzyme A (CoA) biosynthetic pathway. Two types of PPCS exist in bacteria: bifunctional fusion protein Type 1 and monofunctional Type 3. Previously developed cytidylate mimics were determined to have stronger binding affinity for Type 3 Streptococcus pneumoniae PPCS compared to Type 1. To explore the structure activity relationship of Type 3 PPCS, completely conserved residues of bacterial PPCS were probed via saturation mutagenesis in a developed knockout system. Mutagenesis revealed that conserved S. pneumoniae PPCS residues K123 and D93 were stringent, as only K123N/R/M and D93E/N were viable mutations. D93 was determined to be responsible for the association of divalent cations and CTP to the active site, as D93E and D93N exhibited a 1.5-2.7 fold (151-267 μM apparent Km) less affinity for CTP and D93N resulted in a 2-fold drop in MgCl2 affinity. K123 was identified as catalytically significant to the first half CTP utilizing reaction as the K123M and K123R mutations had 1.4-8.8 fold (142-872 μM apparent Km) loss in the binding affinity for CTP.Additionally, phosphopantetheine is the essential prosthetic group of acyl carrier protein (ACP), utilized by gram negative specific acyltransferases (LpxA and LpxD) to construct lipopolysaccharide (LPS). Peptide RJPXD33 (TNLYMLPKWDIP) was discovered to bind and inhibit both LpxA and LpxD (binding constants of 22 μM and 6.5 μM respectively), offering a probe for the development of a dual targeting small molecule inhibitor. The molecular binding mechanism by which RJPXD33 interacts with LpxD was elucidated via fluorescence polarization based binding assays of truncated and alanine-mutated peptide. RJPXD33 P7 was identified to be important to binding LpxD as mutation of the residue to alanine resulted in a binding constant >50 μM, while similar tests in LpxA had a limited effect on binding (9.4 ± 0.6 μM). Residue K8 appeared inconsequential, as mutation to alanine had little effect on binding to LpxA or LpxD (5.1 μM and 3.1 μM binding constants, respectively), but essential to inhibition. Photo-labile RJPXD33 probes were implemented for mapping LpxD-RJPXD33 protein-peptide interactions.