Hao-Bo Guo

Paradigm for industrial strain improvement identifies sodium acetate tolerance loci in Zymomonas mobilis and Saccharomyces cerevisiae

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+) 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+ and Ac- ions exerts a synergistic inhibitory effect for strain ZM4. A structural model for the NhaA sodium-proton 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.

Time-Dependent Density Functional Theory Assessment of UV Absorption of Benzoic Acid Derivatives

Benzoic acid (BA) derivatives of environmental relevance exhibit various photophysical and photochemical characteristics. Here, time-dependent density functional theory (TDDFT) is used to calculate photoexcitations of eight selected BAs and the results are compared with UV spectra determined experimentally. High-level gas-phase EOM-CCSD calculations and experimental aqueous-phase spectra were used as the references for the gas-phase and aqueous-phase TDDFT results, respectively. A cluster-continuum model was used in the aqueous-phase calculations. Among the 15 exchange-correlation (XC) functionals assessed, five functionals, including the meta-GGA hybrid M06-2X, double hybrid B2PLYPD, and range-separated functionals CAM-B3LYP, ωB97XD, and LC-ωPBE, were found to be in excellent agreement with the EOM-CCSD gas-phase calculations. These functionals furnished excitation energies consistent with the pH dependence of the experimental spectra with a standard deviation (STDEV) of ∼0.20 eV. A molecular orbital analysis revealed a πσ* feature of the low-lying transitions of the BAs. The CAM-B3LYP functional showed the best overall performance and therefore shows promise for TDDFT calculations of processes involving photoexcitations of benzoic acid derivatives.

Structure and conformational dynamics of the metalloregulator MerR upon binding of Hg(II).

The bacterial metalloregulator MerR is the index case of an eponymous family of regulatory proteins, which controls the transcription of a set of genes (the mer operon) conferring mercury resistance in many bacteria. Homodimeric MerR represses transcription in the absence of mercury and activates transcription upon Hg(II) binding. Here, the average structures of the apo and Hg(II)-bound forms of MerR in aqueous solution are examined using small-angle X-ray scattering, indicating an extended conformation of the metal-bound protein and revealing the existence of a novel compact conformation in the absence of Hg(II). Molecular dynamics (MD) simulations are performed to characterize the conformational dynamics of the Hg(II)-bound form. In both small-angle X-ray scattering and MD, the average torsional angle between DNA-binding domains is approximately 65 degrees. Furthermore, in MD, interdomain motions on a timescale of approximately 10 ns involving large-amplitude (approximately 20 A) domain opening-and-closing, coupled to approximately 40 degrees variations of interdomain torsional angle, are revealed. This correlated domain motion may propagate allosteric changes from the metal-binding site to the DNA-binding site while maintaining DNA contacts required to initiate DNA underwinding.

Cluster-Continuum Calculations of Hydration Free Energies of Anions and Group 12 Divalent Cations.

Understanding aqueous phase processes involving group 12 metal cations is relevant to both environmental and biological sciences. Here, quantum chemical methods and polarizable continuum models are used to compute the hydration free energies of a series of divalent group 12 metal cations (Zn(2+), Cd(2+), and Hg(2+)) together with Cu(2+) and the anions OH(-), SH(-), Cl(-), and F(-). A cluster-continuum method is employed, in which gas-phase clusters of the ion and explicit solvent molecules are immersed in a dielectric continuum. Two approaches to define the size of the solute-water cluster are compared, in which the number of explicit waters used is either held constant or determined variationally as that of the most favorable hydration free energy. Results obtained with various polarizable continuum models are also presented. Each leg of the relevant thermodynamic cycle is analyzed in detail to determine how different terms contribute to the observed mean signed error (MSE) and the standard deviation of the error (STDEV) between theory and experiment. The use of a constant number of water molecules for each set of ions is found to lead to predicted relative trends that benefit from error cancellation. Overall, the best results are obtained with MP2 and the Solvent Model D polarizable continuum model (SMD), with eight explicit water molecules for anions and 10 for the metal cations, yielding a STDEV of 2.3 kcal mol(-1) and MSE of 0.9 kcal mol(-1) between theoretical and experimental hydration free energies, which range from -72.4 kcal mol(-1) for SH(-) to -505.9 kcal mol(-1) for Cu(2+). Using B3PW91 with DFT-D3 dispersion corrections (B3PW91-D) and SMD yields a STDEV of 3.3 kcal mol(-1) and MSE of 1.6 kcal mol(-1), to which adding MP2 corrections from smaller divalent metal cation water molecule clusters yields very good agreement with the full MP2 results. Using B3PW91-D and SMD, with two explicit water molecules for anions and six for divalent metal cations, also yields reasonable agreement with experimental values, due in part to fortuitous error cancellation associated with the metal cations. Overall, the results indicate that the careful application of quantum chemical cluster-continuum methods provides valuable insight into aqueous ionic processes that depend on both local and long-range electrostatic interactions with the solvent.

X-ray Structure of a Hg2+ Complex of Mercuric Reductase (MerA) and Quantum Mechanical/Molecular Mechanical Study of Hg2+ Transfer between the C-Terminal and Buried Catalytic Site Cysteine Pairs

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.

Energy Triplets for Writing Epigenetic Marks: Insights from QM/MM Free‐Energy Simulations of Protein Lysine Methyltransferases

The nucleosome is the fundamental building block of eukaryotic chromatin, within which histone proteins play an important role in packaging of DNA. 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. One important modification is histone lysine methylation catalyzed by protein lysine methyltransferases (PKMTs). The biological consequences of histone lysine methylation (e.g., gene activation and repression) depend on the methylation states of the lysine residue (mono-, dior tri-methylated; see Figure 1). 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. 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 [a] Dr. Q. Xu, Y.-z. Chu, Dr. H.-B. Guo, Prof. J. C. Smith, Prof. H. Guo Department of Biochemistry and Cellular and Molecular Biology University of Tennessee Knoxville, TN 37996 (USA) Fax: (+1) 865-974-6306 E-mail : hguo1@utk.edu [b] Prof. J. C. Smith, Prof. H. Guo UT/ORNL Center for Molecular Biophysics Oak Ridge National Laboratory Oak Ridge TN 37831 (USA) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.200902297 and contains a comparison of the results of the B3LYP/6-31G** and corrected SCCDFTB calculations for the methyl transfer in a model system, a detailed description of the methods, and an estimate of the free energy for formation of the reactive conformations.. Figure 1. A) Mono-, dior tri-methylation of Lys. B) The reaction coordinate: R= r ACHTUNGTRENNUNG(CM···Sd) r ACHTUNGTRENNUNG(CM···Nz). The parameters for monitoring the orientation of AdoMet and lysine/methyl lysine are r ACHTUNGTRENNUNG(CM···Nz) and q. q is defined as the angle between the two vectors r1 (the direction of the lone pair of electrons) and r2 (the direction of CM Sd bond pointing from CM to Sd).

Chapter 5:Molecular Simulation in the Energy Biosciences

Molecular simulation can be used to understand key physical processes in the energy biosciences. Both molecular mechanical (MM) and quantum mechanical (QM) simulation techniques provide atomic-detailed insight into mechanisms at the core of research in bioenergy and bioremediation. The present article describes molecular simulation in the energy biosciences in two sections: Methods and Applications. In the Methods section, we provide a synopsis of current progress in developing simulation techniques that make efficient use of large-scale supercomputers. This is done with two examples: scalable implicit solvation models and scaling molecular dynamics (MD) to O(100k) cores. In the Applications section, we discuss modeling and simulation of cellulosic biomass, an effort aimed at shedding light on biomass recalcitrance to hydrolysis (a bottleneck in biofuel production) and simulations describing the fate of mercury in contaminated biogeochemical systems. We outline research aimed at understanding the dynamics and function of the proteins and enzymes that confer mercury resistance to bacteria.