Engineering protein filaments with enhanced thermostability for nanomaterials

Self-assembling protein templates have enormous potential as biomaterials for the fabrication of multifunctional nanostructures that require precise positioning of individual molecules in regular patterns over large surface areas. Furthermore, the development of protein templates that are stable under extreme conditions of heat or chemical denaturants will expand processing conditions and end-use applications for biomaterials that require exceptional stability and robustness. In the present work, we characterized the high thermal stability of a filamentous protein template, the γ-prefoldin (γPFD) from the hyperthermophile Methanocaldococcus jannaschii, and subsequently used rational design to further enhance the filament's thermal stability for application as a biotemplate in the creation of platinum nanowires. The γPFD assembles into long fibers with lengths that exceed 2 μm, which when heated to various temperatures and examined by transmission electron microscopy, revealed a T_m of 93°C for the quaternary filament structure. Subsequently, we increased the hydrophobicity of the α-helices of the γPFD's coiled-coil, which appeared to strengthen the filamentous structure, leading to filaments of greater length at elevated temperatures. These enhanced filaments functioned as templates for the synthesis of platinum nanowires at unprecedented temperatures, and may create new opportunities for other applications of nanoscale biotemplates that require exceptional thermal stability. See accompanying commentary by Jonathan S. Dordick DOI: 10.1002/biot.201200338     

N-linked oligosaccharides of Aspergillus awamori feruloyl esterase are important for thermostability and catalysis

A unique N-linked glycosylation motif (Asn(79)-Tyr-Thr) was found in the sequence of type-A feruloyl esterases from Aspergillus spp. To clarify the function of the flap, the role of N-linked oligosaccharides located in the flap region on the biochemical properties of feruloyl esterase (AwFAEA) from Aspergillus awamori expressed in Pichia pastoris was analyzed by removing the N-linked glycosylation recognition site by site-directed mutagenesis. N79 was replaced with A or Q. N-glycosylation-free N79A and N79Q mutant enzymes had lower activity than that of the glycosylated recombinant AwFAEA wild-type enzyme toward alpha-naphthylbutyrate (C4), alpha-naphthylcaprylate (C8), and phenolic acid methyl esters. Kinetic analysis of the mutant enzymes indicated that the lower catalytic efficiency was due to a combination of increased Km and decreased k(cat) for N79A, and to a considerably decreased k(cat) for N79Q. N79A and N79Q mutant enzymes also exhibited considerably reduced thermostability relative to the wild-type.     

A mutant subtilisin E with enhanced thermostability

A mutant subtilisin E with remarkably thermostability is reported. It is more active against the typical substrate s-AAPF-pna than the wild-type subtilisin E. The time required for getting 50% residual activity of Ser236Cys subtilisin E at 60 °C in aqueous solution was approximately 80 min which is 4 times longer than that of wild-type subtilisin E. Similar to the wild-type subtilisin E, the amidase activity of Ser236Cys subtilisin E is dramatically reduced in the presence of dimethylformamide (DMF).     

Important amino acid properties for enhanced thermostability from mesophilic to thermophilic proteins

Understanding the role of various interactions in enhancing the thermostability of proteins is important not only for clarifying the mechanism of protein stability but also for designing stable proteins. In this work, we have analyzed the thermostability of 16 different families by comparing mesophilic and thermophilic proteins with 48 various physicochemical, energetic and conformational properties. We found that the increase in shape, s (location of branch point in side chain) increases the thermostability, whereas, an opposite trend is observed for Gibbs free energy change of hydration for native proteins, GhN, in 14 families. A good correlation is observed between these two properties and the simultaneous increases of -GhN and s is necessary to enhance the thermostability from mesophile to thermophile. The increase in shape, which tends to increase with increasing number of carbon atoms both for polar and non-polar residues, may generate more packing and compactness, and the position of beta and higher order branches may be important for better packing. On the other hand, the increase in -GhN in thermophilic proteins increases the solubility of the proteins. This tendency counterbalances the increases in insolubility and unfolding heat capacity change due to the increase in the number of carbon atoms. Thus, the present results suggest that the stability of thermophilic proteins may be achieved by a balance between better packing and solubility.     

Structures of l-fuculose-1-phosphate aldolase mutants outlining motions during catalysis

The crystal structures of l-fuculose-1-phosphate aldolase (FucA) with and without a ligated analogue of dihydroxyacetone phosphate (DHAP) and of a number of active center mutants have resulted in a model of the catalytic mechanism. This model has now been confirmed by structural analyses of further mutations at the zinc coordination sphere and at the phosphate site. In addition, these mutants have revealed new aspects of the catalysis: the hydroxyl group of Tyr113' (from a neighboring subunit), which sits just outside the zinc coordination sphere, steers DHAP towards a productive binding mode at the zinc ion; Glu73 contacts zinc in between the two ligand positions intended for the DHAP oxygen atoms and thus avoids blocking of these positions by a tetrahedrally coordinated hydroxy ion; the FucA polypeptide does not assume its minimum energy state but oscillates between two states of elevated energy as demonstrated by a mutant in a minimum energy state. The back and forth motion involves a mobile loop connecting the phosphate site with intersubunit motions and thus with the Brownian motion of the solvent. The phosphate group is bound strongly at a given distance to the zinc ion, which prevents the formation of too tight a DHAP:zinc complex. This observation explains our failure to find mutants that accept phosphate-free substitutes for DHAP. The FucA zinc coordination sphere is compared with that of carbonic anhydrase.     

Role of mono- and divalent metal cations in the catalysis by yeast aldolase

The rate of deuterium exchange between [1-(S)-2H]dihydroxyacetone 3-phosphate and the solvent catalyzed by native and metal-substituted yeast aldolases has been measured. In the presence of 0.1 M potassium acetate at 15 degrees C, pH 7.3, the deuterium exchange reaction catalyzed by native yeast aldolase has a kcat of 95 s-1. In contrast to the 7-fold activity enhancement by 0.1 M potassium ion (relative to 0.1 M sodium ion) of the cleavage of D-fructose 1,6-bisphosphate catalyzed by native yeast aldolase, a negligible (1.1-fold) activation by 0.1 M potassium ion is observed in the rate of dedeuteration of [1(S)-2H]dihydroxyacetone 3-phosphate. The order of reactivity of the yeast metalloaldolases in the deuterium exchange roughly parallels that seen in the fructose bisphosphate cleavage reaction. These findings suggest that the carbonyl groups of enzyme-bound D-fructose 1,6-bisphosphate and dihydroxyacetone phosphate are both polarized by the active site divalent metal cation. A mechanistic formulation consistent with the results of this and the previous paper is presented.     

Immobilization of glucose oxidase onto gold nanoparticles with enhanced thermostability

Immobilized proteins and enzymes were widely investigated in medical field as well as in food and environmental fields. In this paper, glucose oxidase (GOD) monolayer was covalently immobilized on the surface of gold nanoparticles (AuNPs) to fabricate bioconjugate complex. The citrate-stabilized AuNPs were first functionalized by a carboxyl-terminated alkanethiol and the terminal carboxyl groups were subsequently bonded with side-chain amino groups of protein surface through EDC/NHS coupling reaction. The enzyme activity assays of the obtained bioconjugates display an enhanced thermostability and similar pH-dependence behavior in contrast with that of free enzyme. Such GOD/AuNPs bioconjugates can be considered as a catalytic nanodevice to construct nanoreactor based on glucose oxidation reaction for biotechnological purpose.     

A mutant phospholipase D with enhanced thermostability from Streptomyces sp

To investigate the contribution of amino acid residues to the thermostability of phospholipase D (PLD), a chimeric form of two Streptomyces PLDs (thermolabile K1PLD and thermostable TH-2PLD) was constructed. K/T/KPLD, in which residues 329-441 of K1PLD were recombined with the homologous region of TH-2PLD, showed a thermostability midway between those of K1PLD and TH-2PLD. By comparing the primary structures of Streptomyces PLDs, the seven candidates of thermostability-related amino acid residues of K1PLD were identified. The K1E346DPLD mutant, in which Glu346 of K1PLD was substituted with Asp by site-directed mutagenesis, exhibited enhanced thermostability, which was almost the same as that of TH-2PLD.     

Pseudomonas putida KT2440低特异性L-苏氨酸醛缩酶的表达、酶学性质及温度稳定性提高

【目的】从Pseudomonas putida KT2440基因组中,钓取低特异性L-苏氨酸醛缩酶基因(lta E),构建重组大肠杆菌。研究目标酶的酶学性质,和关键氨基酸位点突变对酶活和温度稳定性的影响。【方法】以P. putida KT2440基因组DNA为模板,PCR扩增出lta E基因,构建重组表达质粒p ET28a-KT2440并转化Escherichia coli BL21 (DE3),获得重组菌E. coli BL21 (DE3)/p ET-KT2440,利用Ni~(2+)柱亲和层析纯化低特异性L-苏氨酸醛缩酶(LTA),对关键氨基酸位点Thr206和Lys207实施定点突变。【结果】SDS-PAGE结果表明LTA在大肠杆菌中获得高效表达,分子量为40k Da左右,与理论值大小相符。Ni~(2+)柱亲和层析纯化LTA,获得单一条带。利用双酶耦联法测得LTA酶活为5577.3U/mg,最适反应温度为50°C,最适p H为8.0。在温度低于45°C,p H 5.0-9.0时,重组酶较稳定。LTA酶的Km和kcat值为23.95 mmol/L和19216.6 s–1。Mg~(2+)、Ca~(2+)金属离子对LTA有明显的促进作用,而Ni~(~(2+))、Cu~(2+)、Zn~(2+)、Fe~(2+)等对酶有明显的抑制作用。该酶在叔丁基甲基醚溶剂中具有良好的耐受性,在叔丁基甲基醚中保存1h后仍保留90%以上的酶活。Thr206Ser突变明显提高了酶对温度的稳定性。Lys207对酶催化功能是必需的,该位点突变对酶活都是致死的。【结论】克隆并表达P. putida KT2440的LTA酶,研究了酶学性质,通过定点改造提高了酶的温度稳定性,筛选获得一种酶耐受性好的有机溶剂,为LTA酶在有机溶剂中高效稳定催化β-羟基-α-氨基酸奠定了较坚实的研究基础。     

Protein-alginate gel for enzyme immobilisation

Summary Propylene glycol alginate forms strong, covalently bonded gel when mixed with certain proteins in alkaline conditions. Enzymes can be immobilised onto the alginate ester before gelating with protein to form the above gel. Some data on the immobilisation of -glucosidase are presented.     

Amino acid residues essential for catalysis by peptidyl dipeptidase-4 from Pseudomonas maltophilia

To assess residues essential for catalysis by prokaryotic peptidyl dipeptidase-4, the enzyme was subjected to chemical modification by a series of reagents. Treatment with either tetranitromethane or N-acetylimidazole abolished catalytic activity. Hydroxylamine reversed inactivation by acetylimidazole only. Thus, an essential tyrosine is indicated. Enzymatic activity also was quenched by either trinitrobenzenesulfonic acid or diethyl pyrocarbonate. Inactivation by these reagents was not reversed by hydroxylamine. These data suggest an essential lysine. The competitive inhibitor Phe-Arg protected partially against inactivation by tetranitromethane, and fully against inactivation by N-acetylimidazole. The substrate Hip-Phe-Arg protected against inactivation by trinitrobenzenesulfonic acid and diethyl pyrocarbonate. Thus, both tyrosine and lysine are located at the catalytic site.     

Kinetics of enhanced thermostability of an extracellular glucoamylase from Arachniotus sp.

Purified extracelluar glucoamylase from Arachniotus sp. was used for kinetic and thermodynamic characterization. Thermal inactivation followed first order kinetics. The denaturation/activation energies of enzyme were 57 and 89 kJ mol^–1, respectively. Both enthalpy and entropy of activation for inactivation were lower than those for glucoamylases reported in literature. It is suggested that the enzyme is highly thermostable and is suitable for industrial applications.     

Amino acid sequence of an invertebrate FBP aldolase (from Drosophila melanogaster)

The complete amino acid sequence of FBP aldolase from Drosophila melanogaster has been determined. The enzyme contains four identical subunits of 360 amino acid residues. The primary structure of the monomer was established using automated Edman degradation on fragments prepared by CNBr-cleavage, by partial acid cleavage at the unique Asp-Pro bond and by oxidative cleavage at the three tryptophan residues. Manual Edman-Chang degradation was used on smaller peptides obtained by digestion with Staphylococcus aureus V8 protease, trypsin or chymotrypsin. The primary structure of Drosophila aldolase exhibits very extensive homology with the sequence of rabbit muscle aldolase (71% identity), thus explaining the early observation that Drosophila and mammalian aldolases form active interspecies hybrid quaternary structures (Brenner-Holzach, O. and Leuthardt, F., Eur. J. Biochem. (1972) 31, 423-426).     

An oleic acid-mediated pathway induces constitutive defense signaling and enhanced resistance to multiple pathogens in soybean

Stearoyl-acyl carrier protein-desaturase (SACPD)-catalyzed synthesis of oleic acid (18:1) is an essential step in fatty acid biosynthesis. Arabidopsis mutants (ssi2) with reduced SACPD activity accumulate salicylic acid (SA) and exhibit enhanced resistance to multiple pathogens. We show that reduced levels of 18:1 induce similar defense-related phenotypes in soybean. A Bean pod mottle virus (BPMV)-based vector was employed to effectively silence soybean SACPDs. The silenced plants contained reduced 18:1 and increased stearic acid, developed spontaneous cell death lesions, increased SA accumulation, and constitutively expressed pathogenesis-related genes. These plants also expressed elevated levels of resistance-like genes and showed resistance to bacterial and oomycete pathogens. Exogenous application of glycerol induced similar phenotypes, mimicking the effect of silencing SACPDs in healthy soybean plants. Overexpression of a soybean SACPD increased 18:1 levels in ssi2 but not in wild-type Arabidopsis plants, suggesting that the soybean enzyme was under feedback regulation similar to that of the Arabidopsis isozymes. These results suggest that soybean and Arabidopsis respond similarly to 18:1-derived cues by inducing a novel broad-spectrum resistance-conferring pathway, even though they differ significantly in their lipid biosynthetic pathways. We also demonstrate the efficacy of BPMV-induced gene silencing as a tool for functional studies in soybean.