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Overall reaction for conversion of L-ornithine to L-proline as catalyzed by ornithine cyclodeaminase (OCD).
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Ornithine cyclodeaminase (OCD) is an NAD+-dependent deaminase that is found in bacterial species such as Pseudomonas putida. Importantly, it catalyzes the direct conversion of the amino acid L-ornithine to L-proline. Using molecular dynamics (MD) and a hybrid quantum mechanics/molecular mechanics (QM/MM) method in the ONIOM formalism, the catalytic...
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... Genes laterally transferred to protists seem to be mostly involved in the metabolism of amino acids and carbohydrates (39). Importantly, many prokaryotes express an ornithine cyclodeaminase (OCD), which catalyses the conversion of ornithine into Pro and ammonia (40). ...
Trypanosomatids are early-divergent eukaryotes that have adapted to parasitism. During their life cycles, these parasites switch between a mammalian and an invertebrate host, and the ability to adapt their metabolism to different nutritional sources is detrimental for their success. In the invertebrate host, these protists have access to high amounts of amino acids and efficiently utilise it for energy production. Proline is a particularly efficient energy source for trypanosomes. Glutamate is also efficiently used by Trypanosoma cruzi , but it needs to be converted into proline prior to its catabolism. By employing a series of genetic modifications and functional analysis, we show here that Leishmania parasites, the causative agents of leishmaniases, can utilise proline, glutamate and glutamine as energy sources, and although these parasites possess all the genes necessary for the biosynthesis of proline from glutamate, this pathway has, at best, limited function, with at least one of its components (pyrroline-5-carboxylate reductase) assuming divergent functions in different life cycle stages of the parasite. In fact, we show that the catabolism of glutamate is independent of proline biosynthesis and the former is most likely directly imported into the mitochondrion and catabolised to recover the cellular redox metabolism and increase mitochondrial membrane potential. Moreover, our data suggest a relevant role for glutamate dehydrogenase in nutritional stress response in Leishmania . These findings highlight relevant differences in amino acid metabolism between Trypanosoma and Leishmania and suggest a diversification in amino acid metabolic pathways within Trypanosomatidae.
... 40 This MD protocol has been successfully applied in other enzymatic studies. 41,42 Analyses. The trajectories of the 100 ns MD simulations were saved every picosecond. ...
Collagen IV networks are an essential component of basement membranes that are important for their structural integrity and thus that of an organism's tissues. Improper functioning of these networks has been associated with several diseases. Cross-links, such as sulfilimine bonds interconnecting NC1 domains, are critical for forming and mechanically stabilizing these collagen IV networks. More specifically, the sulfilimine cross-links form between methionine (Met93) and lysine/hydroxylsine (Lys211/Hyl211) residues of NC1 domains. Therefore, the dynamic nature of the sulfilimine bond in collagen IV is crucial for network formation. To understand the dynamic nature of a neutral and protonated sulfilimine bond in collagen IV, we performed molecular dynamics (MD) simulations on four sulfilimine cross-linked systems (i.e., Met93S-NLys211, Met93S-NHLys211 +, Met93S-NHyl211, and Met93S-NHHyl211 +) of collagen IV. The MD results showed that the neutral Met93S-NLys211 system has the smallest protein backbone and showed the cross-linked residues' RMSD value. The conformational change analyses showed that the conformations of the sulfilimine cross-linked residues take on a U-shape for the Met93S-NHyl211 and Met93S-HNHyl211 + systems, whereas the conformations of the sulfilimine cross-linked residues are more open for the Met93S-NLys211, and Met93S-NHLys211 + systems. Protonation is a crucial biochemical process to stabilize the protein structure or the biological cross-links. Furthermore, the protonation of the sulfilimine bond could potentially influence hydrogen bond interaction with near amino acid residues, and according to water distribution analyses, the sulfilimine bond can potentially exist in one or more protonation states.
... Although this barrier range is much higher than any of the following steps in the reaction, it is consistent with barriers for other enzyme mechanisms involving hydride transfer. 50 The energy barrier could additionally be inflated due to other contributing factors stemming from the methods used. For example, the level of theory used in this study, M06-2X, has been reported with errors of 4 -7 kJ mol -1 for barrier calculations involving hydrogen transfer. ...
A family of flavin/deazaflavin-dependent oxidoreductases (FDORs) from mycobacteria has been recently characterized and found to play a variety of catalytic roles, including the activation of prodrugs such as the candidate anti-tuberculosis drug, pretomanid (PA-824). However, our understanding of the catalytic mechanism used by these enzymes is relatively limited. To address this, in the present work we have used a combination of quantum mechanical and molecular dynamics calculations to study catalytic mechanism of the activation of pretomanid by the deazaflavin-dependent nitroreductase (Ddn) from Mycobacterium tuberculosis. The preferred pathway involves an initial hydride transfer step from the deprotonated cofactor (i.e. F420H–), with subsequent protonation, before undergoing a series of spontaneous intramolecular reactions to form the final reactive nitrogen species. The most likely proton source is a hydroxonium ion within the solvent accessible active site. Intriguingly, catalysis of the rate-determining hydride transfer step is aided by three tyrosine residues that form a hydrophobic barrier around the active site that, upon reaction, is then disrupted to allow increased water accessibility to facilitate the subsequent proton transfer step. The catalytic mechanism we propose is consistent with previous experimental observations of the Ddn enzyme, and will inform the design of improved pro-drugs in the future.
... 1,[12][13] In such cases they not only behave as electron carriers in oxidative phosphorylation, but also can act as substrates for ADP-ribosylation reactions as well as precursors of the calcium-mobilizing cyclic ADP-ribose. 14 More importantly, they are involved in proline metabolism with functions such as osmolytic control 15 and regulation of cellular stress responses. [16][17] NAD + /NADP + -dependent dehydrogenases are an important group of enzymes that play significant roles in metabolism [18][19][20] and as a result, are also thought to be important to several physiological disorders. ...
The NAD⁺-dependent enzyme, δ¹-pyrroline-5-carboxylate dehydrogenase (P5CDH), has an important role in proline and hydroxyproline catabolism for humans. Specifically, this aldehyde dehydrogenase is responsible for the oxidation of both l-glutamate-γ-semialdehyde (GSA) and 4-erythro-hydroxy-l-glutamate-γ-semialdehyde (4-OH-GSA) to their respective l-glutamate product forms. We have performed a detailed molecular dynamics (MD) study of both the reactant and product complex structures of P5CDH to gain insights into ligand binding (i.e., GSA, 4-OH-GSA, NAD⁺, GLU) in the active site. Moreover, our investigations were further extended to examine the structural impact of S352L, S352A, and E314A mutations on the deficiency in the P5CDH enzymatic activity. Our in silico mutation analysis indicated that the conserved Glu447 has significantly shifted in both the S352L and E314A mutants, causing NAD⁺ to be displaced from its predictive orientation in the binding site and hence forming a catalytically inactive enzyme. However in the case of S352A, the catalytic site including the oxyanion hole and Cys348 remain virtually unchanged, and the coenzyme maintains its binding position.
... The analysis of the trajectories has shown that, after the equilibration, these criteria are met in most of the structures, hence, the entropic cost for reaching a productive conformation should not be significant. To investigate the PES along the mechanism of the macrocyclization reaction, we performed QM/MM calculations, widely used in enzymatic studies [15][16][17][18] applying an ONIOM scheme, [19] as implemented in the Gaussian 09 software package. [20] The system, containing a total of 5200 atoms, was divided into a "QM layer" containing 91 atoms ( Figure 1) and an "MM layer," which were treated at DFT and classical MM levels, respectively. ...
Cyclic peptides are a class of compounds with high therapeutic potential, possessing bioactivities including antitumor and antiviral (including anti-HIV). Despite their desirability, efficient design and production of these compounds has not been achieved to date. The catalytic mechanism of patellamide macrocyclization by the PatG macrocyclase domain has been computationally investigated by using quantum mechanics/molecular mechanics methodology, specifically ONIOM(M06/6-311++G(2d,2p):ff94//B3LYP/6-31G(d):ff94). The mechanism proposed herein begins with a proton transfer from Ser783 to His 618 and from the latter to Asp548. Nucleophilic attack of Ser783 on the substrate leads to the formation of an acyl-enzyme covalent complex. The leaving group Ala-Tyr-Asp-Gly (AYDG) of the substrate is protonated by the substrate's N terminus, leading to the breakage of the P1-P1' bond. Finally, the substrate's N terminus attacks the P1 residue, decomposing the acyl-enzyme complex forming the macrocycle. The formation and decomposition of the acyl-enzyme complex have the highest activation free energies (21.1 kcal mol(-1) and 19.8 kcal mol(-1) respectively), typical of serine proteases. Understanding the mechanism behind the macrocyclization of patellamides will be important to the application of the enzymes in the pharmaceutical and biotechnological industries.
... MD simulations have been successfully used to evaluate the orientation of a substrate in the active site of the protein and to analyze the fluctuations of critical substrate-protein interactions [23]. MD simulations are sometimes utilized for predicting beneficial point mutations during re-design of active sites, both by in silico studying the dynamics of the catalytic residues of experimentally challenging systems [24][25][26], as well as predicting the role of substitutions within the active site cavity [27]. Regarding de novo designed enzymes, two main obstacles have been identified: low catalytic activities of the designed enzymes and a high percentage of design fails, where the variants might show no catalytic activity at all [2]. ...
The protein design toolbox has been greatly improved by the addition of enzyme computational simulations. Not only do they warrant a more ambitious and thorough exploration of sequence space, but a much higher number of variants and protein-ligand systems can be analyzed in silico compared to experimental engineering methods. Modern computational tools are being used to redesign and also for de novo generation of enzymes. These approaches are contingent on a deep understanding of the reaction mechanism and the enzyme’s three-dimensional structure coordinates, but the wealth of information produced by these analyses leads to greatly improved or even totally new types of catalysis.
... Schiff bases have been drawing considerable attention due to their potential application in many areas such as liquid crystals [10][11][12][13], organic dyes [14][15][16][17][18][19][20][21], catalysts [22][23][24], and intermediates in organic synthesis [25][26][27][28][29][30]. Moreover, many Schiff bases exhibit a broad range of biological activities [31][32][33][34][35]. For example, salicylideneaniline (1, Scheme 1) derivatives are effective against Mycobacterium tuberculosis H37Rv [36]. ...
A series of Schiff bases, salicylideneaniline derivatives 1-4, was synthesized under mild conditions and characterized by 1H NMR, HRMS, UV-Vis and fluorescence spectra, and single-crystal X-ray diffraction. In solid and aprotic solvents 1-4 exist mainly as E conformers that possess an intramolecular six-membered-ring hydrogen bond. A weak intramolecular C-H×××F hydrogen bond is also observed in fluoro-functionalized Schiff base 4, which generates another S(6) ring motif. The C-H×××F hydrogen bond further stabilizes its structure and leads it to form a planar configuration. Compounds 1-3 exhibit solely a long-wavelength proton-transfer tautomer emission, while dipole-functionalized Schiff base 4 shows remarkable dual emission originated from the excited-state intramolecular charge transfer (ESICT) and excited-state intramolecular proton transfer (ESIPT) states. Furthermore, the geometric structures, frontier molecular orbitals (MOs) and the potential energy curves for 1-4 in the ground and the first singlet excited state were fully rationalized by density functional theory (DFT) and time-dependent DFT calculations.
... This scheme is analogous to the oxidative deamination of Lamino acid to 2-oxo acids by AlaDH [8], and has been preliminarily proposed based on biochemical experiments [10]. However, the crystal structure and molecular dynamic analysis of OCD [2,11] indicate an alternative mechanism (scheme II) that no attack on the iminium by water occurs: intramolecular cyclic addition of C-5 nitrogen of the iminium to the C-2 carbon to form 2-aminoproline (step 4), loss of ammonia, and reduction of the resulting imine, Pyr2C (step 5). ...
... by which T3LHyp is converted to L-proline via Pyr2C (Fig. 1C). Interestingly, LhpI protein is a novel member of the OCD/CRYM superfamily, and catalyzes only the reduction of Pyr2C to L-proline (no OCD activity) [11]. Furthermore, there is no sequence similarity to DpkA protein as is known for the reductase of Pyr2C from bacteria [16], strongly suggesting their convergent evolution. ...
... Therefore, to obtain evolutional insight into Pyr2C reductase and OCD, we introduced several specific amino acid residues for OCD in TlLhpI by site-directed mutagenesis. Although Asp 228 of OCD (numbering from P. putida) plays an important role in all steps of catalysis [2,11], Asp 219 conserved in AfAlaDH is not involved in catalysis (steps 1 and 2) [8]. Therefore, it may be reasonable that the V244D (and A228K) mutant of TlLhpI has no significant effect on AlaDH activity (Tables 1 and 2, and Fig. 3C), because of the similar mechanism to AfAlaDH, as described above. ...
L-Ornithine cyclodeaminase (OCD) is involved in L-proline biosynthesis and catalyzes the unique deaminating cyclization of L-ornithine to L-proline via a Δ1-pyrroline-2-carboxyrate (Pyr2C) intermediate. Although this pathway functions in only a few bacteria, many archaea possess OCD-like genes (proteins), among which only AF1665 protein (gene) from Archaeoglobus fulgidus has been characterized as an NAD+-dependent L-alanine dehydrogenase (AfAlaDH). However, the physiological role of OCD-like proteins from archaea has been unclear. Recently, we revealed that Pyr2C reductase, involved in trans-3-hydroxy-L-proline (T3LHyp) metabolism of bacteria, belongs to the OCD protein superfamily and catalyzes only the reduction of Pyr2C to L-proline (no OCD activity) [FEBS Open Bio (2014) 4, 240-250]. In this study, based on bioinformatics analysis, we assumed that the OCD-like gene from Thermococcus litoralis DSM 5473 is related to T3LHyp and/or proline metabolism (TlLhpI). Interestingly, TlLhpI showed three different enzymatic activities: AlaDH; N-methyl-L-alanine dehydrogenase; Pyr2C reductase. Kinetic analysis suggested strongly that Pyr2C is the preferred substrate. In spite of their similar activity, TlLhpI had a poor phylogenetic relationship to the bacterial and mammalian reductases for Pyr2C and formed a close but distinct subfamily to AfAlaDH, indicating convergent evolution. Introduction of several specific amino acid residues for OCD and/or AfAlaDH by site-directed mutagenesis had marked effects on both AlaDH and Pyr2C reductase activities. The OCC_00387 gene, clustered with the TlLhpI gene on the genome, encoded T3LHyp dehydratase, homologous to the bacterial and mammalian enzymes. To our knowledge, this is the first report of T3LHyp metabolism from archaea.
... [13,17]). Homologous aspartate and glutamate residues, related to cyclization [24], are not found in LhpI (and other OCD/lcrystallin members), confirming only Pyr2C (and/or Pip2C) reductase activity. In OCD (from P. putida), Asp 161 forms hydrogen bonds with the 2 0 -and 3 0 -hydroxyl groups of the NADH ribose moiety [13,24] (Fig. 5B). ...
... Homologous aspartate and glutamate residues, related to cyclization [24], are not found in LhpI (and other OCD/lcrystallin members), confirming only Pyr2C (and/or Pip2C) reductase activity. In OCD (from P. putida), Asp 161 forms hydrogen bonds with the 2 0 -and 3 0 -hydroxyl groups of the NADH ribose moiety [13,24] (Fig. 5B). On the other hand, in l-crystallin (from human), the 2 0 -phosphate group of the NADPH ribose moiety interacts with side-chains of Asn 168 (equivalent to Asp 161 in OCD), Arg 169 and Thr 170 , in which the enzyme favors binding to NADP + (H) with negative charge [14]. ...
trans-4-Hydroxy-L-proline (T4LHyp) and trans-3-hydroxy-L-proline (T3LHyp) occur mainly in collagen. A few bacteria can convert T4LHyp to α-ketoglutarate, and we previously revealed a hypothetical pathway consisting of four enzymes at the molecular level (J Biol Chem (2007) 282, 6685-6695; J Biol Chem (2012) 287, 32674-32688). Here, we first found that Azospirillum brasilense has the ability to grow not only on T4LHyp but also T3LHyp as a sole carbon source. In A. brasilense cells, T3LHyp dehydratase and NAD(P)H-dependent Δ1-pyrroline-2-carboxylate (Pyr2C) reductase activities were induced by T3LHyp (and D-proline and D-lysine) but not T4LHyp, and no effect of T3LHyp was observed on the expression of T4LHyp metabolizing enzymes: a hypothetical pathway of T3LHyp→Pyr2C→L-proline was proposed. Bacterial T3LHyp dehydratase, encoded to LhpH gene, was homologous with the mammalian enzyme. On the other hand, Pyr2C reductase encoded to LhpI gene was a novel member of ornithine cyclodeaminase/μ-crystallin superfamily, differing from known bacterial protein. Furthermore, the LhpI enzymes of A. brasilense and another bacterium showed several different properties, including substrate and coenzyme specificities. T3LHyp was converted to proline by the purified LhpH and LhpI proteins. Furthermore, disruption of LhpI gene from A. brasilense led to loss of growth on T3LHyp, D-proline and D-lysine, indicating that this gene has dual metabolic functions as a reductase for Pyr2C and Δ1-piperidine-2-carboxylate in these pathways, and that the T3LHyp pathway is not linked to T4LHyp and L-proline metabolism.
Aminoacyl-tRNA synthetases (aaRSs) are essential enzymes that remarkably facilitate the aminoacylation process during translation. With a high fidelity, the mischarged tRNA is prevented through implementing pre- and post-transfer proofreading mechanisms. For instance, Lysine-tRNA synthetase charges the native substrate, lysine, to its cognate tRNA. In spite of the great structural similarity between lysine to the noncognate and toxic ornithine, with the side chain of lysine being only one methylene group longer, LysRS is able to achieve this discrimination with a high efficiency. In this work, the hybrid quantum mechanics/molecular mechanics (QM/MM) investigation was applied to probe the pre-transfer editing mechanism catalyzed by lysyl-tRNA synthetase to reject the noncognte aminoacyl, L-ornityl (Orn), compared to the cognate substrate, L-lysyl. Particularly, the self-cyclization pre-transfer editing mechanism was explored for the two substrates. The substrate-assisted self-cyclization editing of Orn-AMP, where its phosphate moiety acts as the catalytic base, is found to be the rate-determining step with an energy barrier of 101.2 kJ mol−1. Meanwhile, the corresponding rate-limiting pathway for the native Lys-AMP lies at 140.2 kJ mol−1. This observation clearly indicated the infeasibility of this catalytic scenario in the presence of the native substrate. Interestingly, a thermodynamically favorable cyclic product of −92.9 kJ mol−1 with respect to the aminoacyl reactant complex demonstrated evidence of a successful pre-transfer editing. This reaction resulted in the discharge of the on-cognate -ornithine derivative from LysU’s active site. These valuable mechanistic insights are valuable to enrich our knowledge of this extremely efficient and specific catalytic machinery of LysRS.
