Multiple time scale backbone dynamics of homologous thermophilic and mesophilic ribonuclease HI enzymes

Backbone conformational fluctuations on multiple time scales in a cysteine-free Thermus thermophilus ribonuclease HI mutant (ttRNH(*)) are quantified using (15)N nuclear magnetic spin relaxation. Laboratory-frame relaxation data acquired at 310 K and at static magnetic field strengths of 11.7, 14.1 and 18.8 T are analysed using reduced spectral density mapping and model-free approaches. Chemical exchange line broadening is characterized using Hahn-echo transverse and multiple quantum relaxation data acquired over a temperature range of 290-320 K and at a static magnetic field strength of 14.1 T. Results for ttRNH(*) are compared to previously published data for a mesophilic homologue, Escherichia coli ribonuclease HI (ecRNH). Intramolecular conformational fluctuations on the picosecond-to-nanosecond time scale generally are similar for ttRNH(*) and ecRNH. beta-Strands 3 and 5 and the glycine-rich region are more rigid while the substrate-binding handle region and C-terminal tail are more flexible in ttRNH(*) than in ecRNH. Rigidity in the two beta-strands and the glycine-rich region, located along the periphery of the central beta-sheet, may be associated with the increased thermodynamic stability of the thermophilic enzyme. Chemical exchange line broadening, reflecting microsecond-to-millisecond time scale conformational changes, is more pronounced in ttRNH(*) than in ecRNH, particularly for residues in the handle and surrounding the catalytic site. The temperature dependence of chemical exchange show an increase of approximately 15 kJ/mol in the apparent activation energies for ttRNH(*) residues in the handle compared to ecRNH. Increased activation barriers, coupled with motion between alpha-helices B and C not present in ecRNH, may be associated with the reduced catalytic activity of the thermophilic enzyme at 310 K.     

中高温污泥厌氧消化系统中微生物群落比较

【目的】结合中温与高温消化两者优势的两相厌氧消化工艺可能是推进污泥厌氧消化发展的重要方向,因此,探究和比较中温和高温污泥厌氧消化系统中微生物群落组成的异同具有重要意义。【方法】利用高通量测序技术检测中温和高温厌氧消化系统中细菌与古菌的16S r RNA基因序列信息和真菌的内转录间隔(ITS)序列信息,利用基因芯片(Geo Chip 5.0)检测病毒和病原菌致病基因的信息,以对比中温和高温条件下微生物群落在物种组成和功能基因层面上的异同。【结果】中温和高温条件下细菌和古菌在群落物种组成上存在显著差异,病毒和病原菌毒性基因也显著不同,而两种系统中真菌群落的物种组成相似且丰度相对较低。中温条件下产甲烷古菌和未分类微生物相对丰度较高,而高温条件下产酸及嗜热菌相对丰度较高,且高温消化后病毒和病原菌毒性基因相对丰度下降。微生物群落结构与COD、TS和VS有着显著相关性。【结论】微生物群落组成和功能基因在中高温的污泥厌氧消化系统中显著不同,从而解释了两个系统功能的差异。微生物群落的形成与进水参数相关,说明微生物对进水条件敏感。     

Xylan-hydrolysing enzymes from thermophilic and mesophilic fungi

Screening of 40 mesophilic and 13 thermophilic fungi indicated that enzyme activities capable of degrading oat spelt xylan extensively were produced by only a few of the mesophilic species investigated. The relatively low degree of hydrolysis effected by the enzymes from thermophilic organisms could be explained, in part, by their lack of -xylosidase. Several strains of Aspergillus awamori and Aspergillus phoenicis were notable in producing high xylanase and -xylosidase and low protease activities. Of the fungl tested, 13 produced activities capable of removing O-acetyl, arabinosyl, 4-O-methylglucuronyl, feruloyl and coumaroyl substituents from the backbone of xylan polysaccharides as well as endo-1,4--d-xylanase and -1,4-xylosidase. When the growth medium contained oat spelt xylan as carbon source, higher levels of xylanase, -xylosidase and acetyl xylan esterase were found than in cultures containing meadow fescue grass but the latter were richer in ferulic acid and coumaric acid esterases and 4-O-methylglucuronidase. No single organism or carbon source used was capabie of producing high levels of all the debranching enzymes as well as high levels of enzymes capable of cleaving the glycosidic linkages of the xylan backbone. The best ballnce of enzymes was obtained in cultures of A. awamori IMI 142717 and NRRL 2276 and A. phoenicis IMI 214827. Either of these would be suitable for strain improvement studies.The authors are with The Rowett Research Institute. Bucksburn, Aberdeen, AB2 9SB, UK.T.M. Wood is the corresponding author.     

Biochemistry and biotechnology of mesophilic and thermophilic nitrile metabolizing enzymes

Mesophilic nitrile-degrading enzymes are widely dispersed in the Bacteria and lower orders of the eukaryotic kingdom. Two distinct enzyme systems, a nitrilase catalyzing the direct conversion of nitriles to carboxylic acids and separate but cotranscribed nitrile hydratase and amidase activities, are now well known. Nitrile hydratases are metalloenzymes, incorporating Fe^III or Co^II ions in thiolate ligand networks where they function as Lewis acids. In comparison, nitrilases are thiol-enzymes and the two enzyme groups have little or no apparent sequence or structural homology. The hydratases typically exist as αβ dimers or tetramers in which the α- and β-subunits are similar in size but otherwise unrelated. Nitrilases however, are usually found as homomultimers with as many as 16 subunits. Until recently, the two nitrile-degrading enzyme classes were clearly separated by functional differences, the nitrile hydratases being aliphatic substrate specific and lacking stereoselectivity, whereas the nitrilases are enantioselective and aromatic substrate specific. The recent discovery of novel enzymes in both classes (including thermophilic representatives) has blurred these functional distinctions. Purified mesophilic nitrile-degrading enzymes are typically thermolabile in buffered solution, rarely withstanding exposure to temperatures above 50°C without rapid inactivation. However, operational thermostability is often increased by addition of aliphatic acids or by use of immobilized whole cells. Low molecular stability has frequently been cited as a reason for the limited industrial application of "nitrilases"; such statements notwithstanding, these enzymes have been successfully applied for more than a decade to the kiloton production of acrylamide and more recently to the smaller-scale production of nicotinic acid, R-(−)-mandelic acid and S-(+)-ibuprofen. There is also a rapidly growing catalog of other potentially useful conversions of complex nitriles in which the regioselectivity of the enzyme coupled with the ability to achieve high conversion efficiencies without detriment to other sensitive functionalities is a distinct process advantage. Received: January 22, 1998 / Accepted: February 16, 1998     

Role of loop dynamics in thermal stability of mesophilic and thermophilic adenylosuccinate synthetase: a molecular dynamics and normal mode analysis study

Enzymes from thermophiles are poorly active at temperatures at which their mesophilic homologs exhibit high activity and attain corresponding active states at high temperatures. In this study, comparative molecular dynamics (MD) simulations, supplemented by normal mode analysis, have been performed on an enzyme Adenylosuccinate synthetase (AdSS) from E. coli (mesophilic) and P. horikoshii (thermophilic) systems to understand the effects of loop dynamics on thermal stability of AdSS. In mesophilic AdSS, both ligand binding and catalysis are facilitated through the coordinated movement of five loops on the protein. The simulation results suggest that thermophilic P. horikoshii preserves structure and catalytic function at high temperatures by using the movement of only a subset of loops (two out of five) for ligand binding and catalysis unlike its mesophilic counterpart in E. coli. The pre-arrangement of the catalytic residues in P. horikoshii is well-preserved and salt bridges remain stable at high temperature (363K). The simulations suggest a general mechanism (including pre-arrangement of catalytic residues, increased polar residue content, stable salt bridges, increased rigidity, and fewer loop movements) used by thermophilic enzymes to preserve structure and be catalytically active at elevated temperatures.     

Cold-active enzymes studied by comparative molecular dynamics simulation

Enzymes from cold-adapted species are significantly more active at low temperatures, even those close to zero Celsius, but the rationale of this adaptation is complex and relatively poorly understood. It is commonly stated that there is a relationship between the flexibility of an enzyme and its catalytic activity at low temperature. This paper gives the results of a study using molecular dynamics simulations performed for five pairs of enzymes, each pair comprising a cold-active enzyme plus its mesophilic or thermophilic counterpart. The enzyme pairs included α-amylase, citrate synthase, malate dehydrogenase, alkaline protease and xylanase. Numerous sites with elevated flexibility were observed in all enzymes; however, differences in flexibilities were not striking. Nevertheless, amino acid residues common in both enzymes of a pair (not present in insertions of a structure alignment) are generally more flexible in the cold-active enzymes. The further application of principle component analysis to the protein dynamics revealed that there are differences in the rate and/or extent of opening and closing of the active sites. The results indicate that protein dynamics play an important role in catalytic processes where structural rearrangements, such as those required for active site access by substrate, are involved. They also support the notion that cold adaptation may have evolved by selective changes in regions of enzyme structure rather than in global change to the whole protein. Figure Collective motions in C^α atoms of the active site of cold-active xylanase Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Global and local motions in ribonuclease A: a molecular dynamics study

The understanding of protein dynamics is one of the major goals of structural biology. A direct link between protein dynamics and function has been provided by x-ray studies performed on ribonuclease A (RNase A) (B. F. Rasmussen et al., Nature, 1992, Vol. 357, pp. 423-424; L. Vitagliano et al., Proteins: Structure, Function, and Genetics, 2002, Vol. 46, pp. 97-104). Here we report a 3 ns molecular dynamics simulation of RNase A in water aimed at characterizing the dynamical behavior of the enzyme. The analysis of local and global motions provides interesting insight on the dynamics/function relationship of RNase A. In agreement with previous crystallographic reports, the present study confirms that the RNase A active site is constituted by rigid (His12, Asn44, Thr45) and flexible (Lys41, Asp83, His119, Asp121) residues. The analysis of the global motions, performed using essential dynamics, shows that the two beta-sheet regions of RNase A move coherently in opposite directions, thus modifying solvent accessibility of the active site, and that the mixed alpha/3(10)-helix (residues 50-60) behaves as a mechanical hinge during the breathing motion of the protein. These data demonstrate that this motion, essential for RNase A substrate binding and release, is an intrinsic dynamical property of the ligand-free enzyme.     

Protein-water interactions in ribonuclease A and angiogenin: a molecular dynamics study

It is known that water molecules play an important role in the biological functioning of proteins. The members of the ribonuclease A (RNase A) family of proteins, which are sequentially and structurally similar, are known to carry out the obligatory function of cleaving RNA and individually perform other diverse biological functions. Our focus is on elucidating whether the sequence and structural similarity lead to common hydration patterns, what the common hydration sites are and what the differences are. Extensive molecular dynamics simulations followed by a detailed analysis of protein-water interactions have been carried out on two members of the ribonuclease A superfamily-RNase A and angiogenin. The water residence times are analyzed and their relationship with the characteristic properties of the protein polar atoms, such as their accessible surface area and mean hydration, is studied. The capacity of the polar atoms to form hydrogen bonds with water molecules and participate in protein-water networks are investigated. The locations of such networks are identified for both proteins.     

Similarity and difference in the unfolding of thermophilic and mesophilic cold shock proteins studied by molecular dynamics simulations

Molecular dynamics simulations were performed to unfold a homologous pair of thermophilic and mesophilic cold shock proteins at high temperatures. The two proteins differ in just 11 of 66 residues and have very similar structures with a closed five-stranded antiparallel beta-barrel. A long flexible loop connects the N-terminal side of the barrel, formed by three strands (beta1-beta3), with the C-terminal side, formed by two strands (beta4-beta5). The two proteins were found to follow the same unfolding pathway, but with the thermophilic protein showing much slower unfolding. Unfolding started with the melting of C-terminal strands, leading to exposure of the hydrophobic core. Subsequent melting of beta3 and the beta-hairpin formed by the first two strands then resulted in unfolding of the whole protein. The slower unfolding of the thermophilic protein could be attributed to ion pair formation of Arg-3 with Glu-46, Glu-21, and the C-terminal. These ion pairs were also found to be important for the difference in folding stability between the pair of proteins. Thus electrostatic interactions appear to play similar roles in the difference in folding stability and kinetics between the pair of proteins.     

Protein dynamics. A time-resolved fluorescence, energetic and molecular dynamics study of ribonuclease T1

Studies using time-resolved fluorescence depolarization were performed on the internal motion of Trp 59 of ribonuclease T1 (EC 3.1.27.3) in the free enzyme, 2'-GMP-enzyme complex and 3'-GMP-enzyme complex. The Trp 59 motion was also studied in the free enzyme using molecular dynamics simulations. Energetic analysis of activation barriers to the Trp 59 motion was performed using both the transition state theory and Kramers' theory. The activation parameters showed a dependence on solvent viscosity indicating the transition state approach in aqueous solution to be inadequate. When taking solvent viscosity contributions into account agreement between the transition state and Kramers' theories was obtained. The results indicate the three enzyme forms to have different conformations with the free enzyme and 3'-GMP-enzyme complex being similar. Comparison of the experimental and theoretical results showed a good agreement on the Trp 59 motion in the free enzyme. Trp 59 appears to vibrate rapidly, with a relaxation time of the order of 1 ps, within free space in the protein matrix and to have a slower motion, with a relaxation time of the order of 100 ps, which is related to breathing of the surrounding protein matrix. Molecular dynamics results indicate high mobility in regions of the enzyme involved in the interaction with the guanine base of the inhibitor or substrate while much lower mobility occurred in residues involved in the catalytic mechanism of ribonuclease T1.     

Identifying and engineering ion pairs in adenylate kinases. Insights from molecular dynamics simulations of thermophilic and mesophilic homologues

Molecular dynamics simulations were performed to study thermal stabilization of proteins via electrostatic interactions of ion pairs. Dynamic motions of four ion pairs previously proposed to be important in thermal stability of adenylate kinase from the thermophile Bacillus stearothermophilus were monitored during the simulation. One of the four ion pairs identified in the crystal structure, Lys180-Asp114, was not maintained in close contact suggesting that the ion pair does not contribute to thermal stability. Among the other three ion pairs, the ion pair Arg116-Glu198 was proposed to be the most important for stability. To predict behaviors of the ion pairs when engineered into a mesophilic homologue to increase stability, in silico mutants of adenylate kinase from the mesophile Bacillus subtilis were generated, and their molecular dynamics simulations were carried out. The ion pairs in the mutant simulations displayed similar behaviors to those in the simulation of the thermophilic protein. To validate the results of the simulations experimentally, the same mutants were produced in vitro and their thermal stabilities were measured using differential scanning calorimetry. In agreement with the simulations, the Lys180-Asp114 did not result in any increase in stability by itself or additive effect with other ion pairs, whereas a mutant with the Arg116-Glu198 exhibited the highest stability among the mutants having one of the four ion pairs. These results provide specific knowledge about stability in adenylate kinases and more generally suggest that molecular dynamics simulations can provide valuable information for identifying and engineering ion pairs.     

A comparative study of the thermal stability of plastocyanin, cytochrome c6 and Photosystem I in thermophilic and mesophilic cyanobacteria

Cytochrome c₆ (Cyt) from the thermophilic cyanobacterium Phormidium laminosum has been purified and characterized. It is a mildly acidic protein, with physicochemical properties very similar to those of plastocyanin (Pc). This is in agreement with the functional interchangeability of the two metalloproteins as electron donors to Photosystem I (PS I). The kinetic analyses of the interaction of Pc and Cyt with Photosystem I show that both metalloproteins reduce PS I with similar efficiencies, according to an oriented collisional kinetic model involving repulsive electrostatic interactions. The thermostability study of the Phormidium Pc/PS I system compared with those from mesophilic cyanobacteria (Synechocystis, Anabaena and Pseudanabaena) reveals that Pc is the partner limiting the thermostability of the Phormidium couple. The cross-reactions between Pc and PS I from different organisms demonstrate not only that Phormidium Pc enhances the stability of the Pc/PS I system using PS I from mesophilic cyanobacteria, but also that Phormidium PS I possesses a higher thermostability than the other photosystems. This revised version was published online in June 2006 with corrections to the Cover Date.     

Spectral densities of nitrogen nuclei in Escherichia coli ribonuclease HI obtained by 15N NMR relaxation and molecular dynamics

Summary Spectral densities of the ¹⁵N amide in Escherichia coli ribonuclease HI, obtained from NMR relaxation experiments, were compared with those calculated using a molecular dynamics (MD) simulation. All calculations and comparisons assumed that the auto-correlation function describing the internal motions of the molecule was independent of the auto-correlation function associated with overall rotational diffusion. Comparisons were limited to those residues for which the auto-correlation function of internal motions rapidly relaxed and reached a steady state within 205 ps. The results show the importance of frequency components as well as amplitudes of internal motions in order to obtain a meaningful comparison of MD simulations with NMR data.     

Discrimination of thermophilic and mesophilic proteins

Background

There is a considerable literature on the source of the thermostability of proteins from thermophilic organisms. Understanding the mechanisms for this thermostability would provide insights into proteins generally and permit the design of synthetic hyperstable biocatalysts.

Results

We have systematically tested a large number of sequence and structure derived quantities for their ability to discriminate thermostable proteins from their non-thermostable orthologs using sets of mesophile-thermophile ortholog pairs. Most of the quantities tested correspond to properties previously reported to be associated with thermostability. Many of the structure related properties were derived from the Delaunay tessellation of protein structures.

Conclusions

Carefully selected sequence based indices discriminate better than purely structure based indices. Combined sequence and structure based indices improve performance somewhat further. Based on our analysis, the strongest contributors to thermostability are an increase in ion pairs on the protein surface and a more strongly hydrophobic interior.

     

A thermodynamic comparison of mesophilic and thermophilic ribonucleases H

The mechanisms by which thermophilic proteins attain their increased thermostability remain unclear, as usually the sequence and structure of these proteins are very similar to those of their mesophilic homologues. To gain insight into the basis of thermostability, we have determined protein stability curves describing the temperature dependence of the free energy of unfolding for two ribonucleases H, one from the mesophile Escherichia coli and one from the thermophile Thermus thermophilus. The circular dichroism signal was monitored as a function of temperature and guanidinium chloride concentration, and the resulting free energies of unfolding were fit to the Gibbs-Helmholtz equation to obtain a set of thermodynamic parameters for these proteins. Although the maximal stabilities for these proteins occur at similar temperatures, the heat capacity of unfolding for T. thermophilus RNase H is lower, resulting in a smaller temperature dependence of the free energy of unfolding and therefore a higher thermal melting temperature. In addition, the stabilities of these proteins are similar at the optimal growth temperatures for their respective organisms, suggesting that a balance of thermodynamic stability and flexibility is important for function.