A QM/MM study of the catalytic mechanism of SAM methyltransferase RlmN from Escherichia coli

RlmN is a radical S‐adenosylmethionine (SAM) enzyme that catalyzes the C2 methylation of adenosine 2503 (A2503) in 23S rRNA and adenosine 37 (A37) in several Escherichia coli transfer RNAs (tRNA). The catalytic reaction of RlmN is distinctly different from that of typical SAM‐dependent methyltransferases that employs an S_N2 mechanism, but follows a ping‐pong mechanism which involves the intermediate methylation of a conserved cysteine residue. Recently, the x‐ray structure of a key intermediate in the RlmN reaction has been reported, allowing us to perform combined quantum mechanics and molecular mechanics (QM/MM) calculations to delineate the reaction details of RlmN at atomic level. Starting from the Cross‐Linked RlmN C118A?tRNA complex, the possible mechanisms for both the formation and the resolution of the cross‐linked species (IM2) have been illuminated. On the basis of our calculations, IM2 is formed by the attack of the C355‐based methylene radical on the sp²‐hybridized C2 of the adenosine ring, corresponding to energy barrier of 14.4 kcal/mol, and the resolution of IM2 is confirmed to follow a radical fragmentation mechanism. The cleavage of C′–S′ bond of mC355‐A37 cross‐link is in concert with the deprotonation of C2 by C118 residue, which is the rate‐limiting step with an energy barrier of 17.4 kcal/mol. Moreover, the cleavage of C′–S′ bond of IM2 can occur independently, that is, it does not require the loss of an electron of IM2 and the formation of disulfide bond between C355 and C118 as precondition. These findings would deepen the understanding of the catalysis of RlmN.     

S-adenosylmethionine synthase (BtSAMS) from Balsas teosinte confers resistance to peach aphids in Arabidopsis

The purpose of this study was to clone S-adenosylmethionine synthase (BtSAMS) from Balsas teosinte (Zea mays ssp. parviglumis) and investigate its function via heterologous expression in Arabidopsis plants. The BtSAMS coding sequence was cloned and its length is 1185 bp. The expression of BtSAMS in Z. mays ssp. parviglumis was a 12.2-fold increase after aphid infestation. Transgenic Arabidopsis plants heterologously expressing BtSAMS were generated and their morphological characters were observed. A prolonged vegetative growth period was displayed by BtSAMS-OX plants. The more and longer trichomes were observed on the upper surface of BtSAMS seedlings. The ethylene content in BtSAMS-OX plants was not significantly different compared with the untransformed controls. These BtSAMS-OX transgenic plants showed greater resistance to peach aphids compared with the untransformed control plants. Peach aphids appeared to prefer the untransformed controls in that aphids number reduced by more than 50% on BtSAMS plants compared to controls. When sprayed with the insecticide avi-imidacloprid, the number of peach aphids decreased on BtSAMS-OX plants and untransformed controls by 91.6% and 80.5%, respectively. Peach aphids also showed lower reproduction ability when forced to feed on the transgenic plants. The number of newly born nymphs on BtSAMS plants was less than 50% of that on the untransformed control. We therefore propose that BtSAMS may be a suitable candidate gene to confer resistance to peach aphids in plants.     

First principles model calculations of the biosynthetic pathway in selinadiene synthase

Terpenes comprise the largest class of natural products currently known. These ubiquitous molecules are synthesized by terpene synthases via complex carbocationic reactions, incorporating highly reactive intermediates. In the current study, we present a mechanistic investigation of the biosynthetic pathway for the formation of selina-4(15),7(11)-diene. We employ density functional theory to study a model carbocation system in the gas-phase, and delineate the energetic feasibility of a plausible reaction path. Our results suggests that during formation of selina-4(15),7(11)-diene, the substrate is likely folded in a conformation conducive to sequential cyclizations. We propose that a required proton transfer cannot occur intramolecularly in the gas-phase due to a high free energy barrier, and that enzyme assistance is essential for this step. Hybrid quantum mechanics-molecular mechanics docking studies suggest that enzyme intervention could be realized through electrostatic guidance.     

Comparison of the Structures of the Metal-thiolate Binding Site in Zn(II)-, Cd(II)-, and Hg(II)-Metallothioneins Using Molecular Modeling Techniques

Abstract

The first fully energy-minimized structures for a series of structurally related metal complexes of the important mammalian metal binding protein metallothionein are described. The structures were calculated based on structural information obtained from existing spectroscopic and crystallographic data, and minimized using molecular mechanics (MM2) techniques. A two domain structure, with stoichiometrics of M(II)₃?(S_cys)₉ and M(II)₄?(S_cys)₁₁ where M = zinc(II), cadmium(II), and mercury(II), was assembled and minimized. The resultant three-dimensional structure closely resembled that of rat liver Cd₅Zn₂?MT 1 obtained by analysis of x-ray diffraction data [A. H. Robbins, D. E. McRee, M. Williamson, S. A. Collett, N. H. Xuong, W. F. Furey, B. C. Wang and C. D. Stout, J. Mol. Biol. 221, 1269–1293 (1991)]. Minimized structures for Zn₇?MT, Cd₇?MT, and Hg₇?MT are reported. Deep crevices that expose the metal-thiolate clusters are seen in each structure. However, for the mercury-containing protein, much of the mercury-thiolate structure is visible and it is proposed that this provides access for extensive interaction between solvent water molecules and the mercury(II), resulting in the observed distortion away from tetrahedral geometry for Hg₇MT. Volume calculations are reported for the protein metallated with 7 Zn(II), Cd(II), or Hg(II). A series of structural changes calculated for the step-wise isomorphous replacement of Zn(II) by Cd(II) and Hg(II) in the Zn₄S₁₁ α domain are shown.     

Structural studies of the interaction of S-adenosylmethionine with the [4Fe-4S] clusters in biotin synthase and pyruvate formate-lyase activating enzyme

The diverse reactions catalyzed by the radical-SAM superfamily of enzymes are thought to proceed via a set of common mechanistic steps, key among which is the reductive cleavage of S-adenosyl-L-methionine (SAM) by a reduced [4Fe-4S] cluster to generate an intermediate deoxyadenosyl radical. A number of spectroscopic studies have provided evidence that SAM interacts directly with the [4Fe-4S] clusters in several of the radical-SAM enzymes; however, the molecular mechanism for the reductive cleavage has yet to be elucidated. Selenium X-ray absorption spectroscopy (Se-XAS) was used previously to provide evidence for a close interaction between the Se atom of selenomethionine (a cleavage product of Se-SAM) and an Fe atom of the [4Fe-4S] cluster of lysine-2,3-aminomutase (KAM). Here, we utilize the same approach to investigate the possibility of a similar interaction in pyruvate formate-lyase activating enzyme (PFL-AE) and biotin synthase (BioB), two additional members of the radical-SAM superfamily. The results show that the latter two enzymes do not exhibit the same Fe-Se interaction as was observed in KAM, indicating that the methionine product of reductive cleavage of SAM does not occupy a well-defined site close to the cluster in PFL-AE and BioB. These results are interpreted in terms of the differences among these enzymes in their use of SAM as either a cofactor or a substrate.     

The PLP-dependent biotin synthase from Escherichia coli: mechanistic studies

Biotin synthase (BioB), an iron-sulfur enzyme, catalyzes the last step of the biotin biosynthesis pathway. The reaction consists in the introduction of a sulfur atom into two non-activated C-H bonds of dethiobiotin. Substrate radical activation is initiated by the reductive cleavage of S-adenosylmethionine (AdoMet) into a 5'-deoxyadenosyl radical. The recently described pyridoxal 5'-phosphate-bound enzyme was used to show that only one molecule of AdoMet, and not two, is required for the formation of one molecule of biotin. Furthermore 5'-deoxyadenosine, a product of the reaction, strongly inhibited biotin formation, an observation that may explain why BioB is not able to make more than one turnover. However this enzyme inactivation is not irreversible.     

Loss of iron-sulfur clusters from biotin synthase as a result of catalysis promotes unfolding and degradation

Biotin synthase (BioB) is an S-adenosylmethionine radical enzyme that catalyzes addition of sulfur to dethiobiotin to form the biotin thiophane ring. In vitro, Escherichia coli BioB is active for only one turnover, during which the [2Fe-2S]²⁺ cluster is destroyed, one sulfide from the cluster is incorporated as the biotin thiophane sulfur, while Fe²⁺ ions and the remaining S²⁻ ion are released from the protein. The present work examines the fate of the protein following the loss of the FeS clusters. We examine the quaternary structure and thermal stability of active and inactive states of BioB, and find that loss of either the [4Fe-4S]²⁺ or [2Fe-2S]²⁺ clusters results in destabilization but not global unfolding of BioB. Using susceptibility to limited proteolysis as a guide, we find that specific regions of the protein appear to be transiently unfolded following loss of these clusters. We also examine the in vivo degradation of biotin synthase during growth in low-iron minimal media and find that BioB is degraded by an apparent ATP-dependent proteolysis mechanism that sequentially cleaves small fragments starting at the C-terminus. BioB appears to be resistant to degradation and capable of multiple turnovers only under high-iron conditions that favor repair of the FeS clusters, a process most likely mediated by the Isc or Suf iron-sulfur cluster assembly systems.     

The novel structure and chemistry of iron-sulfur clusters in the adenosylmethionine-dependent radical enzyme biotin synthase

Biotin synthase is an adenosylmethionine-dependent radical enzyme that catalyzes the substitution of sulfur for hydrogen at the saturated C6 and C9 positions in dethiobiotin. The structure of the biotin synthase monomer is an (alpha/beta)(8) barrel that contains one [4Fe-4S](2+) cluster and one [2Fe-2S](2+) cluster that encapsulate the substrates AdoMet and dethiobiotin. The air-sensitive [4Fe-4S](2+) cluster and the reductant-sensitive [2Fe-2S](2+) cluster have unique coordination environments that include close proximity to AdoMet and DTB, respectively. The relative positioning of these components, as well as several conserved protein residues, suggests at least two potential catalytic mechanisms that incorporate sulfur from either the [2Fe-2S](2+) cluster or a cysteine persulfide into the biotin thiophane ring. This review summarizes an accumulating consensus regarding the physical and spectroscopic properties of each FeS cluster, and discusses possible roles for the [4Fe-4S](2+) cluster in radical generation and the [2Fe-2S](2+) cluster in sulfur incorporation.     

Lysine enhances methionine content by modulating the expression of S-adenosylmethionine synthase

Lysine and methionine are two essential amino acids whose levels affect the nutritional quality of cereals and legume plants. Both amino acids are synthesized through the aspartate family biosynthesis pathway. Within this family, lysine and methionine are produced by two different branches, the lysine branch and the threonine-methionine branch, which compete for the same carbon/amino substrate. To elucidate the relationship between these biosynthetic branches, we crossed two lines of transgenic tobacco plants: one that overexpresses the feedback-insensitive bacterial enzyme dihydrodipicolinate synthase (DHPS) and contains a significantly higher level of lysine, and a second that overexpresses Arabidopsis cystathionine gamma-synthase (AtCGS), the first unique enzyme of methionine biosynthesis. Significantly higher levels of methionine and its metabolite, S-methylmethionine (SMM), accumulated in the newly produced plants compared with plants overexpressing AtCGS alone, while the level of lysine remained the same as in those overexpressing DHPS alone. The increased levels of methionine and SMM were correlated with increases in the mRNA and protein levels of AtCGS and a reduced mRNA level for the genes encoding S-adnosylmethionine (SAM) synthase, which converts methionine to SAM. Reduction in SAMS expression level leads most probably to the reduction of SAM found in plants that feed with lysine. As SAM is a negative regulator of CGS, this reduction leads to higher expression of CGS and consequently to an increased level of methionine. Elucidating the relationship between lysine and methionine synthesis may lead to new ways of producing transgenic crop plants containing increased methionine and lysine levels, thus improving their nutritional quality.     

The reaction mechanism of allene oxide synthase: Interplay of theoretical QM/MM calculations and experimental investigations

A combined theoretical and experimental study highlights the reaction mechanism of allene oxide synthase (AOS) and its possible link to hydroperoxide lyase (HPL) pathway. A previously published study (Lee et al., Nature 455 (2008) 363) has shown that the F137 residue is of central importance in differentiating between the AOS and HPL pathways after initial identical steps. In the experimental part of this study, we show that wild-type AOS from Arabidopsis or rice in fact produces both AOS and HPL products in a ratio of about 80:15, something that was found only in trace amounts before. Theoretical calculations successfully map the whole AOS pathway with 13(S)-hydroperoxy linolenic and linoleic acid as substrates. Subsequent calculations investigated the effects of in silico F137L mutation at the suggested diverging point of the two pathways. The results show that QM/MM calculations can reasonably reproduce three out of four experimentally available cases, and confirm that the pathways are energetically very close to each other, thus making a switch from one path to other plausible under different circumstances.     

Molecular modeling of cobalt(II) hyaluronate

Structural data for complexes of hyaluronic acid and 3d metals(II) of the fourth group of the periodic table are lacking. A combined QM/MM method was used to solve the structure of the first coordination sphere around the cobalt(II) ion. Some available experimental data were compared with the results obtained via computation and were found to be in good agreement. Our results open the way for using molecular modeling to solve the structure of other metal(II) hyaluronates.     

Suppression of the back proton-transfer from Asp85 to the retinal Schiff base in bacteriorhodopsin: a theoretical analysis of structural elements

The transfer of a proton from the retinal Schiff base to the nearby Asp85 protein group is an essential step in the directional proton-pumping by bacteriorhodopsin. To avoid the wasteful back reprotonation of the Schiff base from Asp85, the protein must ensure that, following Schiff base deprotonation, the energy barrier for back proton-transfer from Asp85 to the Schiff base is larger than that for proton-transfer from the Schiff base to Asp85. Here, three structural elements that may contribute to suppressing the back proton-transfer from Asp85 to the Schiff base are investigated: (i) retinal twisting; (ii) hydrogen-bonding distances in the active site; and (iii) the number and location of internal water molecules. The impact of the pattern of bond twisting on the retinal deprotonation energy is dissected by performing an extensive set of quantum-mechanical calculations. Structural rearrangements in the active site, such as changes of the Thr89:Asp85 distance and relocation of water molecules hydrogen-bonding to the Asp85 acceptor group, may participate in the mechanism which ensures that following the transfer of the Schiff base proton to Asp85 the protein proceeds with the subsequent photocycle steps, and not with back proton transfer from Asp85 to the Schiff base.     

S-Adenosylmethionine (SAM)-Accumulating Sake Yeast Suppresses Acute Alcohol-Induced Liver Injury in Mice

The suppressive effects on acute alcoholic liver injury of S-adenosylmethionine (SAM) and the sake yeast, Saccharomyces cerevisiae Kyokai No. 9, have been shown previously. To enhance the suppression of acute alcoholic liver injury by sake yeast, we prepared SAM-accumulating sake yeast (SAM yeast). Male C57BL/6 mice that had been fed on a diet containing 0.25% SAM yeast or sake yeast for two weeks received three doses of ethanol (5 g/kg BW). In the mice fed on the SAM yeast, the ethanol-induced increases in both triglyceride (TG) and alanine aminotransferase (ALT) were significantly repressed. In addition, the SAM yeast-fed mice did not show an ethanol-induced decrease in hepatic SAM level, suggesting that a disorder of methionine metabolism in the liver caused by ethanol was relieved by the SAM yeast. These results suggest that the SAM yeast had a stronger effect suppressing acute alcoholic liver injury in mice than the sake yeast.     

Structure and function of Mycobacterium smegmatis 7-keto-8-aminopelargonic acid (KAPA) synthase

The biotin biosynthesis pathway is an attractive target for development of novel drugs against mycobacterial pathogens, however there are as yet no suitable inhibitors that target this pathway in mycobacteria. 7-Keto-8-aminopelargonic acid synthase (KAPA synthase, BioF) is the enzyme which catalyzes the first committed step of the biotin synthesis pathway, but both its structure and function in mycobacteria remain unresolved. Here we present the crystal structure of Mycobacterium smegmatis BioF (MsBioF). The structure reveals an incomplete dimer, and the active site organization is similar to, but distinct from Escherichia coli 8-amino-7-oxononanoate synthase (EcAONS), the E. coli homologue of BioF. To investigate the influence of structural characteristics on the function of MsBioF, we deleted bioF in M. smegmatis and confirmed that BioF is required for growth in the absence of exogenous biotin. Based on structural and mutagenesis studies, we confirmed that pyridoxal 5′-phosphate (PLP) binding site residues His129, Lys235 and His200 are essential for MsBioF activity in vivo and residue Glu171 plays an important, but not essential role in MsBioF activity. The N-terminus (residues 1–37) is also essential for MsBioF activity in vivo. The structure and function of MsBioF reported here provides further insights for developing new anti-tuberculosis inhibitors aimed at the biotin synthesis pathway.     

Optimization of cutting schemes for the evaluation of molecular electrostatic potentials in proteins via Moving-Domain QM/MM

This work presents new developments of the moving-domain QM/MM (MoD-QM/MM) method for modeling protein electrostatic potentials. The underlying goal of the method is to map the electronic density of a specific protein configuration into a point-charge distribution. Important modifications of the general strategy of the MoD-QM/MM method involve new partitioning and fitting schemes and the incorporation of dynamic effects via a single-step free energy perturbation approach (FEP). Selection of moderately sized QM domains partitioned between and C (from C=O), with incorporation of delocalization of electrons over neighboring domains, results in a marked improvement of the calculated molecular electrostatic potential (MEP). More importantly, we show that the evaluation of the electrostatic potential can be carried out on a dynamic framework by evaluating the free energy difference between a non-polarized MEP and a polarized MEP. A simplified form of the potassium ion channel protein Gramicidin-A from Bacillus brevis is used as the model system for the calculation of MEP. Figure Schematic representation of the Moving Domain QM/MM method     

Assessment of metal-assisted nucleophile activation in the hepatitis delta virus ribozyme from molecular simulation and 3D-RISM

The hepatitis delta virus ribozyme is an efficient catalyst of RNA 2′-O-transphosphorylation and has emerged as a key experimental system for identifying and characterizing fundamental features of RNA catalysis. Recent structural and biochemical data have led to a proposed mechanistic model whereby an active site Mg²⁺ ion facilitates deprotonation of the O2′ nucleophile, and a protonated cytosine residue (C75) acts as an acid to donate a proton to the O5′ leaving group as noted in a previous study. This model assumes that the active site Mg²⁺ ion forms an inner-sphere coordination with the O2′ nucleophile and a nonbridging oxygen of the scissile phosphate. These contacts, however, are not fully resolved in the crystal structure, and biochemical data are not able to unambiguously exclude other mechanistic models. In order to explore the feasibility of this model, we exhaustively mapped the free energy surfaces with different active site ion occupancies via quantum mechanical/molecular mechanical (QM/MM) simulations. We further incorporate a three-dimensional reference interaction site model for the solvated ion atmosphere that allows these calculations to consider not only the rate associated with the chemical steps, but also the probability of observing the system in the presumed active state with the Mg²⁺ ion bound. The QM/MM results predict that a pathway involving metal-assisted nucleophile activation is feasible based on the rate-controlling transition state barrier departing from the presumed metal-bound active state. However, QM/MM results for a similar pathway in the absence of Mg²⁺ are not consistent with experimental data, suggesting that a structural model in which the crystallographically determined Mg²⁺ is simply replaced with Na⁺ is likely incorrect. It should be emphasized, however, that these results hinge upon the assumption of the validity of the presumed Mg²⁺-bound starting state, which has not yet been definitively verified experimentally, nor explored in depth computationally. Thus, further experimental and theoretical study is needed such that a consensus view of the catalytic mechanism emerges.