Summary Crassulacean acid metabolism (CAM) is a specialized mode of photosynthesis that features nocturnal CO2 uptake, facilitates increased water‐use efficiency (WUE), and enables CAM plants to inhabit water‐limited environments such as semi‐arid deserts or seasonally dry forests. Human population growth and global climate change now present challenges for agricultural production systems to increase food, feed, forage, fiber, and fuel production. One approach to meet these challenges is to increase reliance on CAM crops, such as Agave and Opuntia, for biomass production on semi‐arid, abandoned, marginal, or degraded agricultural lands. Major research efforts are now underway to assess the productivity of CAM crop species and to harness the WUE of CAM by engineering this pathway into existing food, feed, and bioenergy crops. An improved understanding of CAM has potential for high returns on research investment. To exploit the potential of CAM crops and CAM bioengineering, it will be necessary to elucidate the evolution, genomic features, and regulatory mechanisms of CAM. Field trials and predictive models will be required to assess the productivity of CAM crops, while new synthetic biology approaches need to be developed for CAM engineering. Infrastructure will be needed for CAM model systems, field trials, mutant collections, and data management.
The application of systems biology tools holds promise for rational industrial microbial strain development. Here, we characterize a Zymomonas mobilis mutant (AcR) demonstrating sodium acetate tolerance that has potential importance in biofuel development. The genome changes associated with AcR are determined using microarray comparative genome sequencing (CGS) and 454-pyrosequencing. Sanger sequencing analysis is employed to validate genomic differences and to investigate CGS and 454-pyrosequencing limitations. Transcriptomics, genetic data and growth studies indicate that over-expression of the sodium-proton antiporter gene nhaA confers the elevated AcR sodium acetate tolerance phenotype. nhaA over-expression mostly confers enhanced sodium (Na þ ) tolerance and not acetate (Ac − ) tolerance, unless both ions are present in sufficient quantities. NaAc is more inhibitory than potassium and ammonium acetate for Z. mobilis and the combination of elevated Na þ and Ac − ions exerts a synergistic inhibitory effect for strain ZM4. A structural model for the NhaA sodiumproton antiporter is constructed to provide mechanistic insights. We demonstrate that Saccharomyces cerevisiae sodium-proton antiporter genes also contribute to sodium acetate, potassium acetate, and ammonium acetate tolerances. The present combination of classical and systems biology tools is a paradigm for accelerated industrial strain improvement and combines benefits of few a priori assumptions with detailed, rapid, mechanistic studies.ethanol | inhibitor | microarray | sequencing | systems biology
Methylation of certain lysine residues in the N-terminal tails of core histone proteins in nucleosome is of fundamental importance in the regulation of chromatin structure and gene expression. Such histone modification is catalyzed by protein lysine methyltransferases (PKMTs). PKMTs contain a conserved SET domain in almost all of the cases and may transfer one to three methyl groups from S-adenosyl-L-methionine (AdoMet) to the -amino group of the target lysine residue. Here, quantum mechanical/molecular mechanical molecular dynamics and free-energy simulations are performed on human PKMT SET7/9 and its mutants to understand two outstanding questions for the reaction catalyzed by PKMTs: the mechanism for deprotonation of positively charged methyl lysine (lysine) and origin of product specificity. The results of the simulations suggest that Tyr-335 (an absolute conserved residue in PKMTs) may play the role as the general base for the deprotonation after dissociation of AdoHcy (S-adenosyl-L-homocysteine) and before binding of AdoMet. It is shown that conformational changes could bring Y335 to the target methyl lysine (lysine) for proton abstraction. This mechanism provides an explanation why methyl transfers could be catalyzed by PKMTs processively. The freeenergy profiles for methyl transfers are reported and analyzed for wild type and certain mutants (Y305F and Y335F) and the activesite interactions that are of importance for the enzyme's function are discussed. The results of the simulations provide important insights into the catalytic process and lead to a better understanding of experimental observations concerning the origin of product specificity for PKMTs.enzyme catalysis ͉ quantum mechanical/molecular mechanical molecular dynamics simulations ͉ potential of mean force
Long-lived perennial plants, with distinctive habits of inter-annual growth, defense, and physiology, are of great economic and ecological importance. However, some biological mechanisms resulting from genome duplication and functional divergence of genes in these systems remain poorly studied. Here, we discovered an association between a poplar ( 5-enolpyruvylshikimate 3-phosphate synthase gene () and lignin biosynthesis. Functional characterization of PtrEPSP revealed that this isoform possesses a helix-turn-helix motif in the N terminus and can function as a transcriptional repressor that regulates expression of genes in the phenylpropanoid pathway in addition to performing its canonical biosynthesis function in the shikimate pathway. We demonstrated that this isoform can localize in the nucleus and specifically binds to the promoter and represses the expression of a -like transcriptional regulator, which itself specifically binds to the promoter and represses the expression of (known as in), a master regulator of the phenylpropanoid pathway and lignin biosynthesis. Analyses of overexpression and RNAi lines targeting PtrEPSP confirmed the predicted changes in expression patterns. These results demonstrate that PtrEPSP in its regulatory form and PtrhAT form a transcriptional hierarchy regulating phenylpropanoid pathway and lignin biosynthesis in.
Mercury (Hg) is a major global pollutant arising from both natural and anthropogenic sources. Defining the factors that determine the relative affinities of different ligands for the mercuric ion, Hg 2+ , is critical to understanding its speciation, transformation, and bioaccumulation in the environment. Here, we use quantum chemistry to dissect the relative binding free energies for a series of inorganic anion complexes of Hg 2+ . Comparison of Hg 2+ −ligand interactions in the gaseous and aqueous phases shows that differences in interactions with a few, local water molecules led to a clear periodic trend within the chalcogenide and halide groups and resulted in the well-known experimentally observed preference of Hg 2+ for soft ligands such as thiols. Our approach establishes a basis for understanding Hg speciation in the biosphere.
The nucleosome is the fundamental building block of eukaryotic chromatin, within which histone proteins play an important role in packaging of DNA.[1] The tails of histone proteins are subject to different post-translational covalent modifications, and these modifications correspond to an important epigenetic mechanism to lead to distinct downstream events in the regulation of chromatin structure and gene expression.[2] One important modification is histone lysine methylation catalyzed by protein lysine methyltransferases (PKMTs). [3][4][5][6] The biological consequences of histone lysine methylation (e.g., gene activation and repression) depend on the methylation states of the lysine residue (mono-, di-or tri-methylated; see Figure 1). [7,8] Therefore, it is of fundamental importance to understand why different PKMTs have their unique ability to direct specific degrees of lysine methylation which is called product specificity. Such knowledge may have important implications for developing strategies in the manipulation of the signaling properties.In this Communication, the free-energy profiles are obtained from quantum mechanical/molecular mechanical (QM/MM) free-energy simulations for the first, second and third methyl transfers in DIM-5 (a trimethylase) as well as in some of its mutants with different product specificity. The free-energy profile for the third methyl transfer in SET7/9 (a mono-methylase) is also obtained and compared with the data published earlier. [9] It is found that in each case the three free-energy barriers are well correlated with experimentally observed product specificity. The results of the simulations suggest that the relative efficiencies of the chemical steps involving the three methyl transfers in PKMTs from Sadenosyl-l-methionine (AdoMet) to the e-amino group of the target lysine may determine, at least in some cases, how the epigenetic marks of lysine methylation are written. Two different energy triplets are proposed as important parameters for the prediction of product specificity.The free-energy profiles for the first, second and third methyl transfers are plotted in Figure 2 A for DIM-5 as a function of the reaction coordinate. It is of interest to note that the free-energy barriers are rather similar. Thus, if the first methyl transfer from AdoMet to the target lysine can
Mercuric reductase, MerA, is a key enzyme in bacterial mercury resistance. This homodimeric enzyme captures and reduces toxic Hg2+ to Hg0, which is relatively unreactive and can exit the cell passively. Prior to reduction, the Hg2+ is transferred from a pair of cysteines (C558′ and C559′ using Tn501 numbering) at the C-terminus of one monomer to another pair of cysteines (C136 and C141) in the catalytic site of the other monomer. Here, we present the X-ray structure of the C-terminal Hg2+ complex of the C136A/C141A double mutant of the Tn501 MerA catalytic core and explore the molecular mechanism of this Hg transfer with quantum mechanical/molecular mechanical (QM/MM) calculations. The transfer is found to be nearly thermoneutral and to pass through a stable tricoordinated intermediate that is marginally less stable than the two end states. For the overall process, Hg2+ is always paired with at least two thiolates and thus is present at both the C-terminal and catalytic binding sites as a neutral complex. Prior to Hg2+ transfer, C141 is negatively charged. As Hg2+ is transferred into the catalytic site, a proton is transferred from C136 to C559′ while C558′ becomes negatively charged, resulting in the net transfer of a negative charge over a distance of ∼7.5 Å. Thus, the transport of this soft divalent cation is made energetically feasible by pairing a competition between multiple Cys thiols and/or thiolates for Hg2+ with a competition between the Hg2+ and protons for the thiolates.
The QM/MM MD and free energy simulations show that serine-carboxyl peptidases (sedolisins) may stabilize the tetrahedral intermediates and tetrahedral adducts primarily through a general acid-base mechanism involving Asp (Asp164 for kumamolisin-As) rather than the oxyanion-hole interactions as in the cases of serine proteases.
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