Baoping Ling

Molecular dynamics simulations of the coenzyme induced conformational changes of Mycobacterium tuberculosis L-alanine dehydrogenase

Mycobacterium tuberculosis L-alanine dehydrogenase (L-MtAlaDH) catalyzes the NADH-dependent reversible oxidative deamination of L-alanine to pyruvate and ammonia. L-MtAlaDH has been proposed to be a potential target in the treatment of tuberculosis. Based on the crystal structures of this enzyme, molecular dynamics simulations were performed to investigate the conformational changes of L-MtAlaDH induced by coenzyme NADH. The results show that the presence of NADH in the binding domain restricts the motions and conformational distributions of L-MtAlaDH. There are two loops (residues 94-99 and 238-251) playing important roles for the binding of NADH, while another loop (residues 267-293) is responsible for the binding of substrate. The opening/closing and twisting motions of two domains are closely related to the conformational changes of L-MtAlaDH induced by NADH.

Molecular dynamics simulations of mutated Mycobacterium tuberculosis l-alanine dehydrogenase to illuminate the role of key residues

L-Alanine dehydrogenase from Mycobacterium tuberculosis (L-MtAlaDH) catalyzes the NADH-dependent interconversion of l-alanine and pyruvate, and it is considered to be a potential target for the treatment of tuberculosis. The experiment has verified that amino acid replacement of the conserved active-site residues which have strong stability and no great changes in biological evolutionary process, such as His96 and Asp270, could lead to inactive mutants [Ågren et al., J. Mol. Biol. 377 (2008) 1161-1173]. However, the role of these conserved residues in catalytic reaction still remains unclear. Based on the crystal structures, a series of mutant structures were constructed to investigate the role of the conserved residues in enzymatic reaction by using molecular dynamics simulations. The results show that whatever the conserved residues were mutated, the protein can still convert its conformation from open state to closed state as long as NADH is present in active site. Asp270 maintains the stability of nicotinamide ring and ribose of NADH through hydrogen bond interactions, and His96 is helpful to convert the protein conformation by interactions with Gln271, whereas, they would lead to the structural rearrangement in active site and lose the catalytic activity when they were mutated. Additionally, we deduce that Met301 plays a major role in catalytic reaction due to fixing the nicotinamide ring of NADH to prevent its rotation, and we propose that Met301 would be mutated to the hydrophobic residue with large steric hindrance in side chain to test the activity of the protein in future experiment.

2-[4-(Diethyl­amino)phen­yl]-1-ethyl­imidazo[4,5-f][1,10]­phenanthroline

In the title compound, C33H25N5, the central benzene ring is not coplanar with any other rings. The molxadecules are linked through an interxadmolecular C—H⋯N hydrogen bond.

Theoretical study of the hydrolysis mechanism of 2-pyrone-4,6-dicarboxylate (PDC) catalyzed by LigI

2-Pyrone-4,6-dicarboxylate lactonase (LigI) is the first identified enzyme from amidohydrolase superfamily that does not require a divalent metal ion for catalytic activity. It catalyzes the reversible hydrolysis of 2-pyrone-4,6-dicarboxylate (PDC) to 4-oxalomesaconate (OMA) and 4-carboxy-2-hydroxymuconate (CHM) in the degradation of lignin. In this paper, a combined quantum mechanics and molecule mechanics (QM/MM) approach was employed to study the reaction mechanism of LigI from Sphingomonas paucimobilis. According to the results of our calculations, the whole catalytic reaction contains three elementary steps, including the nucleophilic attack, the cleavage of CO of lactone (substrate) and the intramolecular proton transfer. The intermediate has two intramolecular proton transfer pathways, due to which, two final hydrolysis products can be obtained. The energy profile indicates that 4-carboxy-2-hydroxymuconate (CHM) is the main hydrolysis product, therefore, the isomerization between 4-carboxy-2-hydroxymuconate (CHM) and 4-oxalomesaconate (OMA) is suggested to occur in solvent. During the catalytic reaction, residue Asp248 acts as a general base to activate the hydrolytic water molecule. Although His31, His33 and His180 do not directly participate in the chemical process, they play assistant roles by forming electrostatic interactions with the substrate and its involved species in activating the carbonyl group of the substrate and stabilizing the intermediates and transition states.

Mechanistic Unveiling into C=C Double-Bond Rotation and Origins of Regioselectivity and Products E/Z Selectivity of Pd-Catalyzed Olefinic C-H Functionalization of (E)-N-Methoxy Cinnamamide

Density functional theory (DFT) calculations have been performed to study the Pd-catalyzed C-H functionalization of (E)-N-methoxy cinnamamide (E1), which selectively provides the α-C-H activation products (EP as minor product and its C═C rotation isomer ZP as major product). Three crucial issues are solved: (i) The detailed mechanism leading to ZP is one issue. The computational analyses of the mechanisms proposed in previously experimental and theoretical literature do not seem to be consistent with the experimental findings due to the high barriers involved. Alternatively, we present a novel oxidation/reduction-promoted mechanism featuring the Pd(0) → Pd(II) → Pd(0) transformation. The newly proposed mechanism involves the initial coordination of the active catalyst PdL2 (L = t-BuCN) with the C═C bond in EP, followed by the oxidative cyclization/reductive decyclization-assisted C═C double-bond rotation processes resulting in ZP and regeneration of PdL2. (ii) The origin of the product E/Z selectivity is the second issue. On the basis of the calculated results, it is found that, at the initial stage of the reaction, EP is certainly completely generated, while no ZP formation occurred. Once E1 is used up, EP immediately acts as the partner of the new catalytic cycle and sluggishly evolves into ZP. A small amount of generated ZP would reversibly transform to EP due to the higher barrier involved. (iii) The intrinsic reasons for the regioselectivity are the third issue. The calculated results indicate that the regioselectivity for α-C-H activation is mainly attributed to the stronger electrostatic attraction between the α-C and the metal center.

Identification of the active site of human mitochondrial malonyl-coenzyme a decarboxylase: A combined computational study.

Malonyl‐CoA decarboxylase (MCD) can control the level of malonyl‐CoA in cell through the decarboxylation of malonyl‐CoA to acetyl‐CoA, and plays an essential role in regulating fatty acid metabolism, thus it is a potential target for drug discovery. However, the interactions of MCD with CoA derivatives are not well understood owing to unavailable crystal structure with a complete occupancy in the active site. To identify the active site of MCD, molecular docking and molecular dynamics simulations were performed to explore the interactions of human mitochondrial MCD (HmMCD) and CoA derivatives. The findings reveal that the active site of HmMCD indeed resides in the prominent groove which resembles that of CurA. However, the binding modes are slightly different from the one observed in CurA due to the occupancy of the side chain of Lys183 from the N‐terminal helical domain instead of the adenine ring of CoA. The residues 300u2009−u2009305 play an essential role in maintaining the stability of complex mainly through hydrogen bond interactions with the pyrophosphate moiety of acetyl‐CoA. Principle component analysis elucidates the conformational distribution and dominant concerted motions of HmMCD. MM_PBSA calculations present the crucial residues and the major driving force responsible for the binding of acetyl‐CoA. These results provide useful information for understanding the interactions of HmMCD with CoA derivatives. Proteins 2016; 84:792–802.

Theoretical investigations on the interactions of glucokinase regulatory protein with fructose phosphates

Glucokinase (GK) plays a critical role in maintaining glucose homeostasis in the human liver and pancreas. In the liver, the activity of GK is modulated by the glucokinase regulatory protein (GKRP) which functions as a competitive inhibitor of glucose to bind to GK. Moreover, the inhibitory intensity of GKRP-GK is suppressed by fructose 1-phosphate (F1P), and reinforced by fructose 6-phosphate (F6P). Here, we employed a series of computational techniques to explore the interactions of fructose phosphates with GKRP. Calculation results reveal that F1P and F6P can bind to the same active site of GKRP with different binding modes, and electrostatic interaction provides a major driving force for the ligand binding. The presence of fructose phosphate severely influences the motions of protein and the conformational space, and the structural change of sugar phosphate influences its interactions with GKRP, leading to a large conformational rearrangement of loop2 in the SIS2 domain. In particular, the binding of F6P to GKRP facilitates the protruding loop2 contacting with GK to form the stable GK-GKRP complex. The conserved residues 179-184 of GKRP play a major role in the binding of phosphate group and maintaining the stability of GKRP. These results may provide deep insight into the regulatory mechanism of GKRP to the activity of GK.

Identification of a potential proton donor to the linking oxygen atom in a three-metal ion assisted catalysis pathway catalyzed by Fructose-1, 6-bisphosphatase

In this paper, the dephosphorylation mechanism of FBP to F6P catalyzed by the Fructose-1, 6-bisphosphatase (St-Fbp) from Sulfolobus tokodaii was studied using quantum mechanical/molecular mechanical (QM/MM) approach. Based on the experimental results, total five possible catalytic mechanisms (path1-path4) were designed. The most possible dephosphorylation reaction follows a two-step mechanism (path2): a dephosphorylation process (with D12 being an base of W6 and residue K133 being the proton donor of the linking FBP:O4) and a proton exchange process (between K133 and the water W1). Furthermore, the three-step of path4 is also possible: a dephosphorylation process (with D54 being the base of W6 and residue K133 being the proton donor of the linking FBP:O4) and two proton exchange processes (first between residues D54 and D12 then between K133 and the water W1). The relative low energy of this pathway suggests that D54 might also be a base except D12. Our calculations indicate that K133 is the preferred proton donor during the breaking of the phosphate bond O4-P1, with the W1 being an alternative proton donor to access to a more stable product. Findings here give a new insight into the understanding of catalytic mechanism of FBPase.

Molecular dynamics simulations of isoleucine-release pathway in GAF domain of N-CodY from Bacillus Subtilis.

The GAF domain located in the N-terminal motifs of CodY (N-CodY) is responsible for increasing the affinity of CodY to its target sites on DNA by its interaction with the branched chain amino acids (BCAAs) involving isoleucine, leucine and valine. The study of the interaction of GAF domain with isoleucine gains much attention in recent years, but the mechanism of isoleucine release still remains unclear. In this paper, a conventional molecular dynamics (MD) and force probe molecular dynamics (FPMD) simulations have been performed with the aim to understand how the isoleucine ligand escapes from the GAF domain of N-CodY from Bacillus subtilis. The MD results reveal that the ligand release is a gradual process, which is accompanied by the movement of the loop between β3 and β4. During the periods of ligand escaping from the bottom to the top of binding pocket, isoleucine forms hydrogen bonds one after another with series of residues, such as ARG61, THR96, PHE98, VAL100, GLU101 and ASN102, under the mediation of hydrophobic contacts. The FPMD results show that the easiest way to pull ligand out of the cavity is along x direction (i.e. the direction is opposite to MET62).