Inversion of the roles of the nucleophile and acid/base catalysts in the covalent binding of epoxyalkyl xyloside inhibitor to the catalytic glutamates of endo-1,4-β-xylanase (XYNII): a molecular dynamics study (original) (raw)
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NU. International Journal of Science, 2009
Xylanases hydrolyze the β-1,4-linked xylose backbone of xylans, the major hemicellulosic components of plant cell walls. They are especially useful in paper industry because they decrease the demand for chlorine-based chemicals in the wood pulp delignification process. In this study, 3D structure of Xyn10A, a family 10 xylanase from Bacillus firmus K-1 was generated by homology modeling using X-ray structure of xylanase from Bacillus sp. strain NG-27 as a template. The root mean square deviation (RMSD) of backbone atoms between the X-ray and homology modeled structures was 0.8 Å. Binding of xylopentaose (X 5) to the Xyn10A was investigated using molecular dynamics simulations via comparison the total energy, RMSD of Cα atom and root mean square fluctuation (RMSF) of free and complex forms of Xyn10A. In the reactive Xyn10A-X 5 conformation, in which the two catalytic sites, E149 and E255 are precisely positioned for the catalytic reaction, the-1 sugar moiety of the X 5 adopted a 1 C 4 chair conformation for the Xyn10A. According to the RMSF, 13 amino acid residues in active site of complex form showed more flexibility than those of free form. The result suggested that they may implicate in binding to X 5 at subsites-3 to +2 of substrate-binding site through hydrogen bonding and stacking interactions. Furthermore, the RMSF of X 5 revealed that the substrate binding site of Xyn10A was more specific to three middle xylose moieties than two terminal xylose moieties. The role of sugar moiety interaction with Xyn10A in catalytic mechanism is discussed.
Journal of Molecular Modeling, 2020
Lignin and phenolic compounds have been shown as the main recalcitrance for biomass decomposition, as they inhibit a number of lignocellulose-degrading enzymes. Understanding the inhibition mechanisms and energetic competitions with the native substrate is essential for the development of lignin resistive enzymes. In this study, atomistic detail of the size-dependent effects and binding modes of monomeric coniferyl alcohol, dimeric oligolignol, and tetrameric oligolignol made from coniferyl alcohols on the GH11 xylanase from Bacillus firmus strain K-1 was investigated by using molecular docking and atomistic molecular dynamics (MD) simulations. From the MD simulation results on the docked conformation of oligolignol binding within the "Cleft" and the "N-terminal," changes were observed both for protein conformations and positional binding of ligands, as binding with "Thumb" regions was found for all oligolignin models. Moreover, the uniquely stable "N-terminal" binding of the coniferyl alcohol monomer had no effect on the highly fluctuated Thumb region, showing no sign of inhibitory effect, and was in good agreement with recent studies. However, the inhibitory effect of oligolignols was size dependent, as the estimated binding energy of the tetrameric oligolignol became stronger than that of the xylohexaose substrate, and the important binding residues were identified for future protein engineering attempts to enhance the lignin resistivity of GH11.
Organic & Biomolecular Chemistry, 2009
Molecular dynamics simulations have been performed for non-covalent complexes of phenyl b-xylobioside with the retaining endo-b-1,4-xylanase from B. circulans (BCX) and its Tyr69Phe mutant using a hybrid QM/MM methodology. A trajectory initiated for the wild-type enzyme-substrate complex with the proximal xylose ring bound at the -1 subsite (adjacent to the scissile glycosidic bond) in the 4 C 1 chair conformation shows spontaneous transformation to the 2,5 B boat conformation, and potential of mean force calculations indicate that the boat is~30 kJ mol -1 lower in free energy than the chair. Analogous simulations for the mutant lacking one oxygen atom confirm the key role of Tyr69 in stabilizing the boat in preference to the 4 C 1 chair conformation, with a relative free energy difference of about 20 kJ mol -1 , by donating a hydrogen bond to the endocyclic oxygen of the proximal xylose ring. QM/MM MD simulations for phenyl b-xyloside in water, with and without a propionate/propionic acid pair to mimic the catalytic glutamate/glutamic acid pair of the enzyme, show the 4 C 1 chair to be stable, although a hydrogen bond between the OH group at C2 of xylose and the propionate moiety seems to provide some stabilization for the 2,5 B conformation. Fig. 1 Mechanism of retaining endo-1,4-b-xylanase: catalytic residues are Glu78 and Glu172. (Sugar-ring distortion not shown.) which computational modeling provides a powerful investigative tool. Many modelling studies have confirmed that substrate ring distortion is a common feature among glycosidases. 16-19 Molecular dynamics (MD) simulations have shown that the boat conformation at the -1 subsite is critical in the mechanism of family 18 chitinases, 16 and other studies have demonstrated that the -1 sugar moiety in cellulase Ce16A from Trichoderma reesi adopts a skew-boat conformation. 17 Similarly, modelling 460
Journal of Biological Chemistry, 2002
The first report of slow-tight inhibition of xylanase by a bifunctional inhibitor alkalo-thermophilic Bacillus inhibitor (ATBI), from an extremophilic Bacillus sp. is described. ATBI inhibits aspartic protease (Dash, C., and Rao, M. (2001) J. Biol. Chem., 276, 2487-2493) and xylanase (Xyl I) from a Thermomonospora sp. The steady-state kinetics revealed time-dependent competitive inhibition of Xyl I by ATBI, consistent with two-step inhibition mechanism. The inhibition followed a rapid equilibrium step to form a reversible enzyme-inhibitor complex (EI), which isomerizes to the second enzymeinhibitor complex (EI*), which dissociated at a very slow rate. The rate constants determined for the isomerization of EI to EI*, and the dissociation of EI* were 13 ؎ 1 ؋ 10 ؊6 s ؊1 and 5 ؎ 0.5 ؋ 10 ؊8 s ؊1 , respectively. The K i value for the formation of EI complex was 2.5 ؎ 0.5 M, whereas the overall inhibition constant K i * was 7 ؎ 1 nM. The conformational changes induced in Xyl I by ATBI were monitored by fluorescence spectroscopy and the rate constants derived were in agreement with the kinetic data. Thus, the conformational alterations were correlated to the isomerization of EI to EI*. ATBI binds to the active site of the enzyme and disturbs the native interaction between the histidine and lysine, as demonstrated by the abolished isoindole fluorescence of o-phthalaldehyde (OPTA)-labeled Xyl I. Our results revealed that the inactivation of Xyl I is due to the disruption of the hydrogen-bonding network between the essential histidine and other residues involved in catalysis and a model depicting the probable interaction between ATBI or OPTA with Xyl I has been proposed.
Biochemistry, 1996
The three-dimensional structures of endo-1,4-xylanase II (XYNII) from Trichoderma reesei complexed with 4,5-epoxypentyl-D-xyloside (X-O-C 5), 3,4-epoxybutyl-D-xyloside (X-O-C 4), and 2,3epoxypropyl-D-xyloside (X-O-C 3) were determined by X-ray crystallography. High-resolution measurement revealed clear electron densities for each ligand. Both X-O-C 5 and X-O-C 3 were found to form a covalent bond with the putative nucleophile Glu86. Unexpectedly, X-O-C 4 was found to bind to the putative acid/base catalyst Glu177. In all three complexes, clear conformational changes were found in XYNII compared to the native structure. These changes were largest in the X-O-C 3 complex structure. † Supported by the Academy of Finland and the Finnish Graduate School "Protein Structure and Function" (R.H.). ‡ Crystallographic coordinates have been deposited in the Brookhaven Protein Data Bank (reference 1RED, 1REE, 1REF).
Protein Science, 2004
A subject of great practical importance that has not received much attention is the question of the sensitivity of molecular dynamics simulations to the initial X-ray structure used to set up the calculation. We have found two cases in which seemingly similar structures lead to quite different results, and in this article we present a detailed analysis of these cases. The first case is acyl-CoA dehydrogenase, and the chief difference of the two structures is attributed to a slight shift in a backbone carbonyl that causes a key residue (the proton-abstracting base) to be in a bad conformation for reaction. The second case is xylose isomerase, and the chief difference of the two structures appears to be the ligand sphere of a Mg 2+ metal cofactor that plays an active role in catalysis.
Structure-function relationships of a catalytically efficient β-D-xylosidase
Applied Biochemistry and Biotechnology, 2007
β-D-Xylosidase from Selenomonas ruminantium is revealed as the best catalyst known (k cat , k cat /K m) for promoting hydrolysis of 1,4-β-D-xylooligosaccharides. 1 H nuclear magnetic resonance experiments indicate the family 43 glycoside hydrolase acts through an inversion mechanism on substrates 4-nitrophenylβ-D-xylopyranoside (4NPX) and 1,4-β-D-xylobiose (X2). Progress curves of 4-nitrophenyl-β-D-xylobioside, xylotetraose and xylohexaose reactions indicate that one residue from the nonreducing end of substrate is cleaved per catalytic cycle without processivity. Values of k cat and k cat /K m decrease for xylooligosaccharides longer than X2, illustrating the importance to catalysis of subsites-1 and +1 and the lack there of subsite +2. Homology models of the enzyme active site with docked substrates show that subsites beyond-1 are blocked by protein and subsites beyond +1 are not formed; they suggest that D14 and E186 serve catalysis as general base and general acid, respectively. Individual mutations, D14A and E186A, erode k cat and k cat /K m by <10 3 and to a similar extent for substrates 4NPX and 4-nitrophenyl-α-L-arabinofuranoside † The mention of firm names or trade products does not imply that they are endorsed or recommended by the US Department of Agriculture over other firms or similar products not mentioned.
Computational and Structural Biotechnology Journal, 2021
Glycoside hydrolases (GHs) are essential for plant biomass deconstruction. GH11 family consist of endob-1,4-xylanases which hydrolyze xylan, the second most abundant cell wall biopolymer after cellulose, into small bioavailable oligomers. Structural requirements for enzymatic mechanism of xylan hydrolysis is well described for GH11 members. However, over the last years, it has been discovered that some enzymes from GH11 family have a secondary binding sites (SBS), which modulate the enzymes activities, but mechanistic details of the molecular communication between the active site and SBS of the enzymes remain a conundrum. In the present work we structurally characterized GH11 xylanase from Paenibacillus xylanivorans A57 (PxXyn11B), a microorganism of agricultural importance, using protein crystallography and molecular dynamics simulations. The PxXyn11B structure was solved to 2.5 Å resolution and different substrates (xylo-oligosaccharides from X3 to X6), were modelled in its active and SBS sites. Molecular Dynamics (MD) simulations revealed an important role of SBS in the activity and conformational mobility of PxXyn11B, demonstrating that binding of the reaction products to the SBS of the enzyme stabilizes the N-terminal region and, consequently, the active site. Furthermore, MD simulations showed that the longer the ligand, the better is the stabilization within active site, and the positive subsites contribute less to the stabilization of the substrates than the negative ones. These findings provide rationale for the observed enzyme kinetics, shedding light on the conformational modulation of the GH11 enzymes via their SBS mediated by the positive molecular feedback loop which involve the products of the enzymatic reaction.