The catalytic mechanism of Serine Hydroxymethyltransferase: a drug target against Malaria Henrique S. Fernandes1*, Maria J. Ramos2, Sérgio F. Sousa1, Nuno M.F.S.A. Cerqueira1 1
UCIBIO, BioSIM, Departamento de Biomedicina - Faculdade de Medicina da U. Porto 2 UCIBIO, GBC, Departamento de Química e Bioquímica - Faculdade de Ciências da U. Porto *
[email protected]
@Henrique_S_Fer
www.ucibio.pt +18.0
Introduction
+10.1
The last report of malaria from the WHO indicates an unpredictable increase of clinical cases where drug resistance has been observed. This demands an urgent need to develop new drugs targeting this parasite and search for new ways to inhibit it. (World Malaria Report 2016, World Health Organization, 2016) Serine HydroxyMethylTransferase (SHMT) has been pointed out as a promissory drug target to treat malaria. SHMT is a PLP-dependent enzyme(Fernandes HS, Chemistry, 2017) that catalyses the reversible transformation of ʟ-serine into glycine and tetrahydrofolate (THF) into 5,10-methylenetetrahydrofolate (5,10-methyleneTHF).(Stover P, J Biol Chem, 1990) In this work, we describe, for the first time, the complete catalytic mechanism of this enzyme.
+20. 0 +10. 0
+8.1
0.0
∆G kcal/mol
0.0
-10.0
-20.0
+8.1
GLU
GLU
51
51
-30.0
+8.8
THF GLU
51
EA
Anopheles mosquito
THF
(Fernandes HS, ACS Catal, 2018)
THF
-0.4
This knowledge can now be used to develop new transition-state analogues compounds as potent inhibitors of the enzyme.
GLU
51
P. falciparum sporozoites
+0.9 THF
Methodology GLU
The crystallographic structure of SHMT, deposited on Protein Data Bank with the code 1DFO(Scarsdale JN, J Mol Biol, 2000), was used to build the ONIOM QM/MM model to study the catalytic mechanism. One of the three available homodimers was selected to proceed the calculations. The enzyme was co-crystallised with the external aldimine (EA) with the product (glycine) bond to PLP and an analogue of the THF cofactor. The THF analogue was modulated to fit the natural THF configuration.
51
-8.7
THF
+0.9 GLU
51
-7.0
THF
The study of the catalytic mechanism followed the scheme: 5Å
THF GLU
51
THF EA
EA
...
Parameterization
Enzyme
-1.6 Serine Hydroxymethyltransferase
Complex Build all
THF
...
EA
Enzyme
side chain
H
scan
...
QM/MM Model Build
GLU
51
Minimizations
MM QM
-4.0
H 2O
SHMT
THF
TS opt
GLU
-5.3
51 GLU
51
5,10 methylene
Glycine
THF
opt
for each step R,TS,P
SP
IRC
-20.9
EA 5,10 methylene
R&P opt
THF
B3LYP/6-31G(d,p):ff99SB
-19.8
R,TS,P
freq
QI
-20.4
DLPNO-CCSD(T)/CBS:ff99SB
The input files were prepared using the molUP(Fernandes HS, J Comput Chem, 2018) extension for VMD(Humphrey W, J Mol Graph, 1996) as well as to analyse the results. The geometry optimisations were made using Gaussian09 (D version)(Frisch MJ, Gaussian, Inc.: Wallingford, 2009) and the single-point energy calculations (SP) were calculated using ORCA (4.0.1.2 version)(Frank N, Wiley Interdiscip Rev: Comput Mol Sci, 2018).
5,10 methylene
THF
-26.5
-11.4
5,10-methyleneTHF Purines Biosynthesis
Conclusion This enzyme catalyses the reversible conversion of ʟ-serine and THF into glycine and 5,10-methylene-THF, with the help of the PLP cofactor. The reaction requires eight sequential steps that can be divided into three main stages: the α-elimination, the cyclisation of the 5-hydroxymethyl-THF intermediate into 5,10-methylene-THF, and the protonation of the quinonoid intermediate (QI). The rate-limiting step is where the elimination of the hydroxymethyl group occurs, forming a formaldehyde intermediate that becomes immediately bonded to the THF cofactor. The rate-limiting step requires an energy boost of 18.0 kcal/mol. These results closely fit the experimental kinetic data that predicted an activation barrier of 15.7-16.2 kcal/mol (Florio R, FEBS J, 2009; Angelaccio S, J Mol Catal B: Enzym, 2014) . Together with the previous stages that are common to all PLP-dependent enzymes(Oliveira EF, J Am Chem Soc, 2011; Cerqueira NMFSA, J Chem Theory Comput, 2011), this work provides, for the first time and with an atomistic level of detail, a portrait of the entire mechanism catalysed by the SHMT. We believe that this knowledge provides valuable information for the rational molecular design of new drugs against malaria. This work was supported by the Applied Molecular Biosciences Unit - UCIBIO which is financed by national funds from FCT/MCTES (UID/Multi/04378/2013 and SFRH/BD/115396/2016) and co-financed by the ERDF under the PT2020 Partnership Agreement (POCI01-0145-FEDER-007728).
DNA and RNA synthesis
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