Exploring alternative catalytic mechanisms of the Cas9 HNH domain

Kaldis

2022

PLoS Comput Biol

Self Cite

Methylenetetrahydrofolate dehydrogenase/cyclohydrolase (MTHFD2) is a new drug target that is expressed in cancer cells but not in normal adult cells, which provides an Achilles heel to selectively kill cancer cells. Despite the availability of crystal structures of MTHFD2 in the inhibitor- and cofactor-bound forms, key information is missing due to technical limitations, including (a) the location of absolutely required Mg2+ ion, and (b) the substrate-bound form of MTHFD2. Using computational modeling and simulations, we propose that two magnesium ions are present at the active site whereby (i) Arg233, Asp225, and two water molecules coordinate MgA2+, while MgA2+ together with Arg233 stabilize the inorganic phosphate (Pi); (ii) Asp168 and three water molecules coordinate MgB2+, and MgB2+ further stabilizes Pi by forming a hydrogen bond with two oxygens of Pi; (iii) Arg201 directly coordinates the Pi; and (iv) through three water-mediated interactions, Asp168 contributes to the positioning and stabilization of MgA2+, MgB2+ and Pi. Our computational study at the empirical valence bond level allowed us also to elucidate the detailed reaction mechanisms. We found that the dehydrogenase activity features a proton-coupled electron transfer with charge redistribution connected to the reorganization of the surrounding water molecules which further facilitates the subsequent cyclohydrolase activity. The cyclohydrolase activity then drives the hydration of the imidazoline ring and the ring opening in a concerted way. Furthermore, we have uncovered that two key residues, Ser197/Arg233, are important factors in determining the cofactor (NADP+/NAD+) preference of the dehydrogenase activity. Our work sheds new light on the structural and kinetic framework of MTHFD2, which will be helpful to design small molecule inhibitors that can be used for cancer treatment.

Section: Discussionmentioning

confidence: 99%

“…The EVB atoms are given in each section below. Detailed EVB simulation procedures are described in our previous work [ 18 – 20 ] and S2 Text . Our system consists of 10,032 atoms including 177 molaris-generated water molecules.…”

Section: Methodsmentioning

confidence: 99%

The catalytic mechanism of the mitochondrial methylenetetrahydrofolate dehydrogenase/cyclohydrolase (MTHFD2)

Kaldis

2022

PLoS Comput Biol

Self Cite

“…Hence, this has been a useful approach for this study 30 . In addition, EVB has been a reliable method to study the energetics of various enzymatic reactions 31‐33 . Thus, in our EVB formalism we compare the barriers between the reference reaction and that of the reaction in protein.…”

Section: Discussionmentioning

confidence: 99%

Histidine protonation states are key in the LigI catalytic reaction mechanism

Mondal

et al. 2021

Proteins

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Lignin is one of the world's most abundant organic polymers, and 2‐pyrone‐4,6‐dicarboxylate lactonase (LigI) catalyzes the hydrolysis of 2‐pyrone‐4,6‐dicarboxylate (PDC) in the degradation of lignin. The pH has profound effects on enzyme catalysis and therefore we studied this in the context of LigI. We found that changes of the pH mostly affects surface residues, while the residues at the active site are more subject to changes of the surrounding microenvironment. In accordance with this, a high pH facilitates the deprotonation of the substrate. Detailed free energy calculations by the empirical valence bond (EVB) approach revealed that the overall hydrolysis reaction is more likely when the three active site histidines (His31, His33 and His180) are protonated at the ɛ site, however, protonation at the δ site may be favored during specific steps of the reaction. Our studies have uncovered the determinant role of the protonation state of the active site residues His31, His33 and His180 in the hydrolysis of PDC.

“…Detailed EVB simulation procedures are described in our previous work. [17][18][19] All the EVB calculations were done using the Enzymix module within the MOLARIS-XG package. 16,20 The Water Flooding (WF) Approach…”

Section: Evb Simulationsmentioning

confidence: 99%

Catalytic mechanism of the mitochondrial methylenetetrahydrofolate dehydrogenase/cyclohydrolase (MTHFD2)

Kaldis

2021

Preprint

Self Cite

Methylenetetrahydrofolate dehydrogenase/cyclohydrolase (MTHFD2) is a new drug target that is expressed in cancer cells but not in normal adult cells, which provides an Achilles heel to selectively kill cancer cells. Despite the availability of crystal structures of MTHFD2 in the inhibitor- and cofactor-bound forms, key information is missing due to technical limitations, including (a) the location of absolutely required Mg2+ ion, and (b) the substrate-bound form of MTHFD2. Using homology modeling and simulation studies, we propose that two magnesium ions are present at the active site whereby (i) Arg233, Asp225, and two water molecules coordinate MgA, while MgA together with Arg233 stabilize the inorganic phosphate (Pi); (ii) Asp168 and three water molecules coordinate MgB, and MgB further stabilizes Pi by forming a hydrogen bond with two oxygens of Pi; (iii) Arg201 directly coordinates the Pi; and (iv) through three water-mediated interactions, Asp168 contributes to the positioning and stabilization of MgA, MgB and Pi. Our computational study at the empirical valence bond level allowed us to elucidate the detailed reaction mechanisms. We found that the dehydrogenase activity features a proton-coupled electron transfer with charge redistribution coupled to the reorganization of the surrounding water molecules which further facilitates the subsequent cyclohydrolase activity. The cyclohydrolase activity then drives the hydration of the imidazoline ring and the ring opening in a concerted way. Furthermore, we have uncovered that two key residues Ser197/Arg233 are key factors in determining the cofactor (NADP+/NAD+) preference of the dehydrogenase activity. Our work sheds new light on the structural and kinetic framework of MTHFD2, which will be helpful to design small molecule inhibitors that can be used for cancer therapy.