title: Discovery of novel allosteric effectors based on the predicted allosteric sites for Escherichia coli D-3-phosphoglycerate dehydrogenase.
abstract: D-3-phosphoglycerate dehydrogenase (PGDH) from Escherichia coli catalyzes the first critical step in serine biosynthesis, and can be allosterically inhibited by serine. In a previous study, we developed a computational method for allosteric site prediction using a coarse-grained two-state Gō Model and perturbation. Two potential allosteric sites were predicted for E. coli PGDH, one close to the active site and the nucleotide binding site (Site I) and the other near the regulatory domain (Site II). In the present study, we discovered allosteric inhibitors and activators based on site I, using a high-throughput virtual screen, and followed by using surface plasmon resonance (SPR) to eliminate false positives. Compounds 1 and 2 demonstrated a low-concentration activation and high-concentration inhibition phenomenon, with IC50 values of 34.8 and 58.0 µM in enzymatic bioassays, respectively, comparable to that of the endogenous allosteric effector, L-serine. For its activation activity, compound 2 exhibited an AC50 value of 34.7 nM. The novel allosteric site discovered in PGDH was L-serine- and substrate-independent. Enzyme kinetics studies showed that these compounds influenced Km, kcat, and kcat/Km. We have also performed structure-activity relationship studies to discover high potency allosteric effectors. Compound 2-2, an analog of compound 2, showed the best in vitro activity with an IC50 of 22.3 µM. Compounds targeting this site can be used as new chemical probes to study metabolic regulation in E. coli. Our study not only identified a novel allosteric site and effectors for PGDH, but also provided a general strategy for designing new regulators for metabolic enzymes.
authors.affiliation: BNLMS, State Key Laboratory for Structural Chemistry of Unstable and Stable Species, College of Chemistry and Molecular Engineering, Peking University, Beijing, China.
authors.forename: Qian
authors.initials: Q
authors.lastname: Wang
authors.affiliation: Center for Quantitative Biology, Peking University, Beijing, China.
authors.forename: Yifei
authors.initials: Y
authors.lastname: Qi
authors.affiliation: Center for Quantitative Biology, Peking University, Beijing, China.
authors.forename: Ning
authors.initials: N
authors.lastname: Yin
authors.affiliation: BNLMS, State Key Laboratory for Structural Chemistry of Unstable and Stable Species, College of Chemistry and Molecular Engineering, Peking University, Beijing, China; Center for Quantitative Biology, Peking University, Beijing, China.
authors.forename: Luhua
authors.initials: L
authors.lastname: Lai
mesh_terms: D000494:Allosteric Regulation / Q000187:drug effects; D000495:Allosteric Site; D001665:Binding Sites; D001667:Binding, Competitive / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D050543:Phosphoglycerate Dehydrogenase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D015203:Reproducibility of Results; D013329:Structure-Activity Relationship; D013379:Substrate Specificity / Q000187:drug effects
chemical_list: D004791:Enzyme Inhibitors; D050543:Phosphoglycerate Dehydrogenase
references.citation: J Biol Chem. 2004 Apr 2;279(14):13452-60
references.pmid: 14718528
references.citation: Arch Dis Child. 1996 Jun;74(6):542-5
references.pmid: 8758134
references.citation: Plant Physiol. 1968 Nov;43(11):1813-20
references.pmid: 5699148
references.citation: Pigment Cell Melanoma Res. 2011 Dec;24(6):1112-5
references.pmid: 21981974
references.citation: J Med Chem. 2006 Oct 19;49(21):6177-96
references.pmid: 17034125
references.citation: J Biol Chem. 1969 Jun 10;244(11):2816-23
references.pmid: 4306282
references.citation: J Biol Chem. 1968 May 10;243(9):2081-9
references.pmid: 4384871
references.citation: Biochemistry. 2008 Aug 12;47(32):8271-82
references.pmid: 18627175
references.citation: Adv Enzyme Regul. 1984;22:325-400
references.pmid: 6089514
references.citation: RNA. 2009 Jun;15(6):1219-30
references.pmid: 19369428
references.citation: J Biol Chem. 1977 Mar 10;252(5):1539-51
references.pmid: 14154
references.citation: Biochem J. 1997 Apr 15;323 ( Pt 2):365-70
references.pmid: 9163325
references.citation: Proc Natl Acad Sci U S A. 2004 Aug 24;101(34):12461-6
references.pmid: 15314233
references.citation: Biochim Biophys Acta. 1962 Jul 30;62:193-5
references.pmid: 13923751
references.citation: Eur J Biochem. 1975 Feb 21;51(2):415-27
references.pmid: 168067
references.citation: J Phys Chem B. 2006 Mar 16;110(10):5094-8
references.pmid: 16526752
references.citation: J Biol Chem. 2008 Oct 31;283(44):29706-14
references.pmid: 18776184
references.citation: J Biol Chem. 2005 Apr 15;280(15):14884-91
references.pmid: 15668250
references.citation: J Biol Chem. 1994 Mar 4;269(9):6784-9
references.pmid: 8120039
references.citation: J Comput Aided Mol Des. 2006 Oct-Nov;20(10-11):647-71
references.pmid: 17124629
references.citation: Nat Genet. 2011 Jul 31;43(9):869-74
references.pmid: 21804546
references.citation: J Biol Chem. 2003 May 16;278(20):18170-6
references.pmid: 12644455
references.citation: Nature. 2010 Feb 18;463(7283):899-905
references.pmid: 20164920
references.citation: FEBS Lett. 1986 Jul 28;203(2):139-43
references.pmid: 3089835
references.citation: J Comput Chem. 2010 Jan 30;31(2):455-61
references.pmid: 19499576
references.citation: J Bacteriol. 1996 Jan;178(1):232-9
references.pmid: 8550422
references.citation: J Biol Chem. 1963 Dec;238:3934-44
references.pmid: 14086727
references.citation: J Biol Chem. 1996 Sep 20;271(38):23235-8
references.pmid: 8798520
references.citation: Science. 2009 Nov 6;326(5954):853-8
references.pmid: 19892984
references.citation: Nat Struct Biol. 1995 Jan;2(1):69-76
references.pmid: 7719856
references.citation: J Chem Theory Comput. 2012 Aug 14;8(8):2962-71
references.pmid: 26592133
references.citation: Biochemistry. 1965 Jun;4(6):1076-85
references.pmid: 4378782
references.citation: Methods Enzymol. 1975;41:278-81
references.pmid: 236449
references.citation: J Biol Chem. 1966 Sep 10;241(17):4068-76
references.pmid: 5920812
references.citation: ACS Chem Biol. 2013 Jan 18;8(1):19-26
references.pmid: 23181429