'Nunchuck' proteins: Short flexible linkers resist proteolysis by facilitating motions in flanking domains to inhibit the approach of proteases.

Peptides linking well-folded and non-interacting domains in fusion proteins can undergo proteolytic degradation. This leads to physical separation of the domains that were originally sought to be joined. In order to identify characteristics that determine linker degradation propensity, we selected a pair of thermostable, proteolytically-resistant domains, and joined them using five different linkers. We then assessed linker degradation propensities through size-exclusion chromatography, and denaturing and non-denaturing electrophoresis. The domains used were Coh2, an all-beta cohesin from C. thermocellum CipA, and BSX, a beta/alpha barrel xylanase from Bacillus sp. NG-27, while the linkers used were Rigid (3 repeats of N-EAAAK-C), Flexible (two repeats of N-SGGGG-C), Nat-full (42 residues of a Coh2-adjacent linker from CipA), Nat-half (a 21 residues-long derivative of Nat-full) and Nat-quarter (a 9 residues-long derivative of Nat-full). Both with proteolysis effected by proteases present in the environment, and with an exogenously-added protease (Subtilisin A), we found that Flexible underwent little or no degradation, whereas linkers of comparable length like Nat-quarter or Rigid underwent extensive degradation, as did longer linkers like Nat-Half and Nat-Full. Our analyses disfavor the likelihood of the sequence of Flexible being naturally resistant to proteolysis, and instead favor the explanation that the flexibility of Flexible facilitates movements of Coh2 relative to BSX which then serve to sterically prevent the approach of proteases. Thus, the construct incorporating Flexible appears to behave like a 'nunchuck' in which rods/spheres flanking a chain collide with approaching swords that are capable of severing the chain, to prevent severance.

Use of discarded corneo-scleral rims to create cornea-like tissue.

BACKGROUND: Corneal disease is a major cause of blindness. Transplantation of cadaver-derived corneas (keratoplasty) is still the current therapy of choice; however, the global shortage of donor corneas continues to drive a search for alternatives. To this end, biosynthetic corneal substitutes have recently begun to gain importance. Here, we present a novel method for the generation of a cornea-like tissue (CLT), using corneo-scleral rims discarded after keratoplasty. METHODS AND RESULTS: Type I collagen was polymerized within the corneo-scleral rim, which functioned as a 'host' mould, directing the 'guest' collagen to polymerize into disc-shaped cornea-like material (CLM), displaying the shape, curvature, thickness, and transparency of normal cornea. This polymerization of collagen appears to derive from some morphogenetic influence exerted by the corneo-scleral rim. Once the CLM had formed naturally, we used collagen crosslinking to fortify it, and then introduced cells to generate a stratified epithelial layer to create cornea-like tissue (CLT) displaying characteristics of native cornea. Through the excision and reuse of rims, each rim turned out to be useful for the generation of multiple cornea-shaped CLTs. CONCLUSIONS: The approach effectively helps to shorten the gap between demand and supply of CLMs/CLTs for transplantation. We are exploring the surgical transplantation of this CLT into animal eyes, as keratoprostheses, as a precursor to future applications involving human eyes. It is possible to use either the CLM or CLT, for patients with varying corneal blinding diseases.

Avoidance of the use of tryptophan in buried chromosomal proteins as a mechanism for reducing photo/oxidative damage to genomes.

In cells that are exposed to terrestrial sunlight, the indole moiety in the side chain of tryptophan (Trp) can suffer photo/oxidative damage (POD) by reactive oxygen species (ROS) and/or ultraviolet light (UV-B). Trp is oxidized to produce N-formylkynurenine (NFK), a UV-A-responsive photosensitizer that further degenerates into photosensitizers capable of generating ROS through exposure to visible light. Thus, Trp-containing proteins function as both victims, and perpetrators, of POD if they are not rapidly replaced through protein turnover. The literature indicates that protein turnover and DNA repair occur poorly in chromosomal interiors. We contend, therefore, that basic chromosomal proteins (BCPs) that are enveloped by DNA should have evolved to lack Trp residues in their amino acid sequences, since these could otherwise function as 'Trojan horse-type' DNA-damaging agents. Our global analyses of protein sequences demonstrates that BCPs consistently lack Trp residues, although DNA-binding proteins in general do not display such a lack. We employ HU-B (a wild-type, Trp-lacking bacterial BCP) and HU-B F47W (a mutant, Trp-containing form of the same bacterial BCP) to demonstrate that the possession of Trp is deleterious to BCPs and associated chromosomal DNA. Basically, we show that UV-B and UV-A (a) cause no POD in HU-B, but cause extensive POD in HU-B F47W (in vitro), as well as (b) only nominal DNA damage in bacteria expressing HU-B, but extensive DNA damage in bacteria expressing F47W HU-B (in vivo). Our results suggest that Trp-lacking BCPs could have evolved to reduce scope for protein-facilitated, sunlight-mediated damage of DNA by UV-A and visible light, within chromosomal interiors that are poorly serviced by protein turnover and DNA repair machinery.

The bacterial nucleoid-associated proteins, HU and Dps, condense DNA into context-dependent biphasic or multiphasic complex coacervates.

The bacterial chromosome, known as its nucleoid, is an amorphous assemblage of globular nucleoprotein domains. It exists in a state of phase separation from the cell's cytoplasm, as an irregularly-shaped, membrane-less, intracellular compartment. This state (the nature of which remains largely unknown) is maintained through bacterial generations ad infinitum. Here, we show that HU and Dps, two of the most abundant nucleoid-associated proteins (NAPs) of Escherichia coli, undergo spontaneous complex coacervation with different forms of DNA/RNA, both individually and in each other's presence, to cause accretion and compaction of DNA/RNA into liquid-liquid phase separated condensates in vitro. Upon mixing with nucleic acids, HU-A and HU-B form (a) biphasic heterotypic mixed condensates in which HU-B helps to lower the Csat of HU-A and also (b) multiphasic heterotypic condensates, with Dps, in which demixed domains display different contents of HU and Dps. We believe that these modes of complex coacervation that are seen in vitro can serve as models for the in vivo relationships among NAPs in nucleoids, involving local and global variations in the relative abundances of the different NAPs, especially in demixed subdomains that are characterized by differing grades of phase separation. Our results clearly demonstrate some quantitative, and some qualitative, differences in the coacervating abilities of different NAPs with DNA, potentially explaining (i) why E. coli has two isoforms of HU, and (ii) why changes in the abundances of HU and Dps facilitate the lag, logarithmic, and stationary phases of E. coli growth.

Counter-intuitive enhancement of degradation of polyethylene terephthalate through engineering of lowered enzyme binding to solid plastic.

Degradation of solid polyethylene terephthalate (PET) by leaf branch compost cutinase (LCC) produces various PET-derived degradation intermediates (DIs), in addition to terephthalic acid (TPA), which is the recyclable terminal product of all PET degradation. Although DIs can also be converted into TPA, in solution, by LCC, the TPA that is obtained through enzymatic degradation of PET, in practice, is always contaminated by DIs. Here, we demonstrate that the primary reason for non-degradation of DIs into TPA in solution is the efficient binding of LCC onto the surface of solid PET. Although such binding enhances the degradation of solid PET, it depletes the surrounding solution of enzyme that could otherwise have converted DIs into TPA. To retain a subpopulation of enzyme in solution that would mainly degrade DIs, we introduced mutations to reduce the hydrophobicity of areas surrounding LCC's active site, with the express intention of reducing LCC's binding to solid PET. Despite the consequent reduction in invasion and degradation of solid PET, overall levels of production of TPA were ~3.6-fold higher, due to the partitioning of enzyme between solid PET and the surrounding solution, and the consequent heightened production of TPA from DIs. Further, synergy between such mutated LCC (F125L/F243I LCC) and wild-type LCC resulted in even higher yields, and TPA of nearly ~100% purity.

Rational mutagenesis of Thermobifida fusca cutinase to modulate the enzymatic degradation of polyethylene terephthalate.

Thermobifida fusca cutinase (TfCut2) is a carboxylesterase (CE) which degrades polyethylene terephthalate (PET) as well as its degradation intermediates [such as oligoethylene terephthalate (OET), or bis-/mono-hydroxyethyl terephthalate (BHET/MHET)] into terephthalic acid (TPA). Comparisons of the surfaces of certain CEs (including TfCut2) were combined with docking and molecular dynamics simulations involving 2HE-(MHET)3, a three-terephthalate OET, to support the rational design of 22 variants with potential for improved generation of TPA from PET, comprising 15 single mutants (D12L, E47F, G62A, L90A, L90F, H129W, W155F, ΔV164, A173C, H184A, H184S, F209S, F209I, F249A, and F249R), 6 double mutants [H129W/T136S, A173C/A206C, A173C/A210C, G62A/L90F, G62A/F209I, and G62A/F249R], and 1 triple mutant [G62A/F209I/F249R]. Of these, nine displayed no activity, three displayed decreased activity, three displayed comparable activity, and seven displayed increased (~1.3- to ~7.2-fold) activity against solid PET, while all variants displayed activity against BHET. Of the variants that displayed increased activity against PET, four displayed more activity than G62A, the most-active mutant of TfCut2 known till date. Of these four, three displayed even more activity than LCC (G62A/F209I, G62A/F249R, and G62A/F209I/F249R), a CE known to be ~5-fold more active than wild-type TfCut2. These improvements derived from changes in PET binding and not changes in catalytic efficiency.

Does chronic inflammation cause acute inflammation to spiral into hyper-inflammation in a manner modulated by diet and the gut microbiome, in severe Covid-19?

We propose that hyper-inflammation (HYPi) is a ''runaway'' consequence of acute inflammation (ACUi) that arises more easily (and also abates less easily) in those who host a pre-existing chronic inflammation (CHRi), because (i) most factors involved in generating an ACUi to limit viral proliferation are already present when there is an underlying CHRi, and also because (ii) anti-inflammatory (AI) mechanisms for the abatement of ACUi (following containment of viral proliferation) are suppressed and desensitized where there is an underlying CHRi, with this causing the ACUi to spiral into a HYPi. Stress, pollution, diet, and gut microbiomes (alterable in weeks through dietary changes) have an intimate and bidirectional cause-effect relationship with CHRi. We propose that avoidance of CHRi-promoting foods and adoption of CHRi-suppressing foods could reduce susceptibility to HYPi, in Covid-19 and in other viral diseases, such as influenza, which are characterized by episodic and unpredictable HYPi.

HU-AB simulacrum: Fusion of HU-B and HU-A into HU-B-A, a functional analog of the Escherichia coli HU-AB heterodimer.

In enteric bacteria such as Escherichia coli, there are two homologs of the DNA-binding nucleoid associated protein (NAP) known as HU. The two homologs are known as HU-A and HU-B, and exist either in the form of homodimers (HU-AA, or HU-BB) or as heterodimers (HU-AB), with different propensities to form higher-order oligomers. The three different dimeric forms dominate different stages of bacterial growth, with the HU-AB heterodimer dominating cultures in the stationary phase. Due to similarities in their properties, and the facile equilibrium that exists between the dimeric forms, the dimers are difficult to purify away from each other. Although HU-AA and HU-BB can be purified through extensive ion-exchange chromatography, reestablishment of equilibrium interferes with the purification of the HU-AB heterodimer (which constitutes ∼90% of any population with equal numbers of HU-B and HU-A chains). Here, we report the creation of a functional analog of HU-AB that does not appear to partition to generate any minority populations of HU-AA or HU-BB. The analog was constructed through genetic fusion of the HU-B and HU-A chains into a single polypeptide (HU-B-A) with a glycine/serine-rich linker of 11 amino acids separating HU-B from HU-A, and a histidine tag at the N-terminus of HU-B. HU-B-A folds to bind 4-way junction DNA, and displays a significant tendency to form dimers (i.e., analogs of HU tetramers), and a higher thermodynamic stability than HU-BB or HU-AA, thus explaining why it dominates mixtures of HU-B and HU-A chains.

N-Terminal Extensions Appear to Frustrate HU Heterodimer Formation by Strengthening Intersubunit Contacts and Blocking the Formation of a Heterotetrameric Intermediate.

HU is a bacterial nucleoid-associated protein. Two homologues, known as HU-A, and HU-B, are found in Escherichia coli within which the early, late, and stationary phases of growth are dominated by HU-AA, HU-BB, and HU-AB dimers, respectively. Here, using genetic manipulation, mass spectrometry, spectroscopy, chromatography, and electrophoretic examination of glutaraldehyde-mediated cross-linking of subunits, in combination with experiments involving mixing, co-expression, unfolding, and refolding of HU chains, we show that the spontaneous formation of HU-AB heterodimers that is reported to occur upon mixing of wild-type HU-AA and HU-BB homodimers does not occur if chains possess N-terminal extensions. We show that N-terminal extensions interfere with the conversion of homodimers into heterodimers. We also show that heterodimers are readily formed at anticipated levels by chains possessing N-terminal extensions in vivo, when direct chain-chain interactions are facilitated through production of HU-A and HU-B chains from proximal genes located upon the same plasmid. From the data, two explanations emerge regarding the mechanism by which N-terminal extensions happen to adversely affect the conversion of homodimers into heterodimers. (1) The disappearance of the α-amino group at HU's N-terminus impacts the intersubunit stacking of ß-sheets at HU's dimeric interface, reducing the ease with which subunits dissociate from each other. Simultaneously, (2) the presence of an N-terminal extension appears to sterically prevent the association of HU-AA and HU-BB homodimers into a critically required, heterotetrameric intermediate (within which homodimers could otherwise exchange subunits without releasing monomers into solution, by remaining physically associated with each other).

The DNA-binding protein HU is a molecular glue that attaches bacteria to extracellular DNA in biofilms.

In biofilms, bacteria that possess a negatively charged surface are embedded within a matrix of polymers consisting mainly of negatively charged extracellular DNA (e-DNA). In all likelihood, a multivalent positively charged substance, for example, a basic protein, exists within biofilms to neutralize charge-charge repulsions and act as a 'glue' attaching negatively charged bacteria to negatively charged e-DNA; however, no protein capable of doing so has yet been identified. We decided to investigate whether a highly abundant nucleoid-associated histone-like protein (HU) happens to be the glue in question. In recent years, HU has been shown to possess qualities that could be considered desirable in the proposed glue, for example, (a) availability in association with e-DNA; (b) multivalent DNA binding; (c) non-sequence-specific DNA-binding; (d) enhancement of biofilm formation upon exogenous addition, and (e) disruption of biofilms, upon removal by HU-cognate antibodies. Geometric considerations suggest that basic residues in HU's canonical and noncanonical DNA-binding sites can interact with sugar-linked terminal phosphates in lipopolysaccharide (LPS) molecules in bacterial outer membranes. Here, using genetic, spectroscopic, biophysical-chemical, microscopy-based, and cytometry-based experiments, we demonstrate that HU's DNA-binding sites also bind to LPS, that this facilitates DNA-DNA, DNA-LPS, and LPS-LPS interactions, and that this facilitates bacterial clumping and attachment of bacteria to DNA. Exogenous addition of HU to bacteria in (nonshaken) cultures is shown to cause cells to become engulfed in a matrix of DNA, potentially arising from the lysis of bacteria with vulnerable cell walls (as they strain to grow, divide, and move away from each other, in opposition to the accreting influence of HUs).

A novel protein-engineered dsDNA-binding protein (HU-Simulacrum) inspired by HU, a nucleoid-associated DNABII protein.

HU, a DNA-binding protein, has a helical N-terminal region (NTR) of ∼44 residues and a beta strand- and IDR-rich C-terminal region (CTR) of ∼46 residues. CTR binds to DNA through (i) a clasp (two arginine/lysine-rich, IDR-rich beta hairpins that bind to phosphate groups in the minor groove), (ii) a flat surface (comprising four antiparallel beta strands that abut the major groove), and (iii) a charge cluster (two lysine residues upon a short C-terminal helix). HU forms a dimer displaying extensive inter-subunit CTR-CTR contacts. A single-chain simulacrum of these contacts (HU-Simul) incorporating all DNA-binding elements was created by fusing together the CTRs of Escherichia coli HU-A and Thermus thermophilus HU. HU-Simul is monomeric, binds to dsDNA and cruciform DNA, but not to ssDNA.

An ultra-stable glucanotransferase-cum-exoamylase from the hyperthermophile archaeon Thermococcus onnurineus.

The genome of the hyperthermophile archaeon Thermococcus onnurineus (strain NA1) encodes a 652 residues-long putative 4-α-glucanotransferase of the GH 57 family which we have expressed in Escherichia coli. The enzyme (TonAmyGT) appears to remove glucose from the reducing end of a donor glucan and transfers it to the non-reducing end of an acceptor glucan, creating a pool of oligosaccharides through disproportionation of any substrate maltooligosaccharide, with maltose acting substantively as the smallest donor glucan as well as the smallest acceptor glucan. Additionally, glucose is also cleaved from maltooligosaccharides and released into solution without being transferred to an acceptor, causing the enzyme to function as an exo-amylase (which can digest starch) in addition to its activity as a glucanotransferase. TonAmyGT functions over a broad range of temperature (20-100 °C) and pH (4.0-9.0), and shows extreme resistance to chemical and thermal denaturation, displaying a melting temperature of 104 °C, at a pressure of 35 psi, in a differential scanning calorimeter. An interesting characteristic is that the glucanotransferase activity shows feedback inhibition through glucose (which the enzyme itself generates), indicating that the exo-amylase and glucanotransferase activities regulate each other.

Understanding anomalous mobility of proteins on SDS-PAGE with special reference to the highly acidic extracellular domains of human E- and N-cadherins.

During SDS-PAGE experiments, proteins generally display electrophoretic mobility in keeping with their molecular weights; however, some proteins display anomalies in mobility. Here, we focus attention on the anomalies displayed by the highly acidic ∼110 residues-long, sequence-homologous, structurally-analogous, extracellular domains of human E- and N-cadherin. We report that there is a strong correlation between the acidity of each domain and the degree of the anomaly that it displays. The anomaly is only seen if the ratio of the numbers of negatively-charged and positively-charged residues is equal to or greater than the value of 1.50. The degree of the anomaly rises in proportion with this NC:PC ratio. Greater-than-expected anomalies are observed for domains containing dense clusters of negatively charged residues. A simple explanation for these observations is that highly acidic domains electrostatically repel SDS. This results in insufficient SDS binding, insufficient electromotive incentive and (consequently) lowered electrophoretic mobility. This explanation is in consonance with the current view that initial stages of SDS-protein engagement tend to be dominated by electrostatics. We discuss the current anomalies within the broader context of all conceivable explanations for such anomalies.

Structure-guided mutational evidence and postulates explaining how a glycohydrolase from Pyrococcus furiosus functions simultaneously as an amylase and as a 4-α-glucanotransferase.

Pyrococcus furiosus exoamylase-cum-4-α-glucanotransferase (4-α-GTase; PF0272; PfuAmyGT) is reported to both (i) act upon starch, and (ii) catalyze 'disproportionation' of maltooligosaccharides (with glucose as the smallest product). PfuAmyGT shares ∼65% sequence identity with a homo-dimeric Thermococcus litoralis 4-α-GTase, for which structures are available in complex with a non-hydrolysable analog of maltotetraose (acarbose) bound to one subunit and maltose (of unknown origin) bound to the other subunit. We structurally transposed the maltose onto the acarbose-bound subunit and discovered that the two molecules lie juxtaposed in what could be perfect 'acceptor' and 'donor' substrate-binding sites, respectively. We also discovered that there is a loop between the two sites which could use an available aspartate to excise a glucose from the donor, and an available tryptophan to transfer the glucose to the non-reducing end of the acceptor glucan. We derived a structure for PfuAmyGT through homology-based modeling, identified the potential donor site, acceptor site, glucan-transferring loop, and catalytically important residues, and mutated these to alanine to examine effect(s) upon activity. Mutation D362A abolished creation of shorter, or longer, maltooligosaccharides. Mutation W365A abolished creation of longer oligosaccharides. Mutation H366A had no effect on activity. We propose that D362 facilitates glucose excision, and that W365 facilitates its transfer, either (a) directly into solution (allowing PfuAmyGT to act as an exoamylase), or (b) by glycoside bond formation with an acceptor (allowing PfuAmyGT to act as a 4-α-glucanotransferase), depending upon whether the acceptor site is vacant or occupied in a reaction cycle.

Multiple thermostable enzyme hydrolases on magnetic nanoparticles: An immobilized enzyme-mediated approach to saccharification through simultaneous xylanase, cellulase and amylolytic glucanotransferase action.

Microbe-derived enzymes such as xylanases, cellulases and amylases, are efficient at hydrolyzing plant biomass. Efforts to harness the functionalities of these enzymes towards applications in energy and fuel biosciences, and food and nutrition, continue apace in many laboratories. Given that enzymes derived from mesophile proteomes undergo facile denaturation and/or degradation at ambient temperatures, and require frequent replenishment during bioprocessing, it is desirable that they be replaced by structurally-stable enzymes capable of functioning efficiently and resisting denaturation and degradation, immobilized on solid media to further add to stability and facilitate recovery and reuse. Towards these objectives, we used synthetic magnetic nanoparticles (MNPs) and immobilized upon their surfaces three different structurally-stable hydrolases: a thermostable xylanase (BSX) derived from Bacillus sp. NG-27, a cellulase (RMCel12A) derived from Rhodothermus marinus, and an amylase-cum-glucanotransferase (PfuAmyGT) derived from Pyrococcus furiosus. The MNPs were activated with glutaraldehyde and BSX, RMCel12A, and PfuAmyGT, respectively, were covalently immobilized with efficiencies of ~92%, 45% and 93%. The enzymes and the MNPs were fully characterized before and after immobilization, and the immobilized enzymes were found to be active at 50 °C against synthetic substrates as well as pre-treated biomass derived from corn cob and rice husk. The enzyme-coupled MNPs displayed high stability upon storage properties, high operational stability as well as high reusability (retaining 69, 48, and 50% residual activity after 13 uses for BSX, RMCel12A and PfuAmyGT, respectively). Experiments were also conducted with MNPs loaded simultaneously with all three enzymes. Such immobilized enzyme combinations on MNPs can be used in the saccharification of plant biomass.

Probing the excited state dynamics of Venus: origin of dual-emission in fluorescent proteins.

Fluorescent proteins exhibit interesting excited state photochemistry, leading to bright fluorescence emission that renders their versatile biological role and wide use as biomarkers. A molecular-level mechanism of the excited state dynamics is desirable to pinpoint the origin of the bright fluorescence of these proteins. Here we present studies on a yellow fluorescent protein variant, Venus, and investigate the photophysics behind the dual fluorescence emission upon UV excitation. Based on our studies, we propose that the unique nature of the potential energy surface is responsible for the observation of minor fluorescence in Venus which is not seen in wild type GFP.

Structural-Mechanical and Biochemical Functions of Classical Cadherins at Cellular Junctions: A Review and Some Hypotheses.

This article begins with a general review of cell adhesion molecules (CAMs) and narrows the focus down progressively to the cadherins (calcium binding-dependent CAMs), classifications of subfamilies of the cadherins, type I (E- and N-) cadherins, evolutionary relationships amongst cadherins, structural-mechanical and functional consequences of calcium binding to the cadherins, differential molecular interactions involving the extracellular (ecto) and intracellular (cytoplasmic) domains of the cadherins, multiple adherence-related homophilic and heterophilic interactions and associated functions of E- and N-cadherin in organismal development and disease and cadherin trafficking and membrane rafts. It ends by summarizing multiple perspectives and hypotheses concerning different aspects of cadherin structure, stability and function.

Characterization of a mildly alkalophilic and thermostable recombinant Thermus thermophilus laccase with applications in decolourization of dyes.

OBJECTIVE: To examine the potential for applications of TthLAC, a monomeric (~ 53 kDa) laccase encoded by the genome of Thermus thermophilus (strain HB 27) which can be produced at low cost in Escherichia coli. RESULT: Functional, thermostable and mildly alkalophilic TthLAC of high purity (> 90%) was produced through simple heating of suspended (TthLAC overexpressing) E.coli cells at 65 °C. For reactions of short duration (< 1 h) the temperature for optimal activity is ~ 90 °C. However, TthLAC undergoes slow partial unfolding and thermal inactivation above 65 °C, making it unsuitable for long incubations above this temperature. With different substrates, optimal function was observed from pH 6 to 8. With the substrate, ABTS, catalytic efficiency (K m) and maximum velocity (Vmax) at 60 °C and pH 6.0 were determined to be 2.4 × 103 µM and 0.04 × 103 µM/min respectively. Ultra-pure, affinity-purified TthLAC was used to confirm and characterize the enzyme's ability to oxidize known (laccase) substrates such as ABTS, syringaldazine and 4-fluoro-2-methylphenol. TthLAC decoloured up to six different industrial dyes, with or without the use of redox mediators such as ABTS. CONCLUSIONS: Unlike versatile laccases from most other sources, which tend to be thermolabile as well as acidophilic, TthLAC is a versatile, thermostable, mildly alkalophilic laccase which can be produced at low cost in E.coli for various redox applications.

Endoglucanase activity at a second site in Pyrococcus furiosus triosephosphate isomerase-Promiscuity or compensation for a metabolic handicap?

The eight-stranded (ß/α)8 barrel fold known as the Triosephosphate isomerase (TIM) barrel is the most commonly observed fold in enzymes, displaying an eightfold structural symmetry. The sequences and structures of different TIM barrel enzymes suggest that nature exploits the modularity inherent in the eightfold symmetry to generate enzymes with diverse enzymatic activities and, in certain cases, more than one catalytic activity per enzyme. Here, we report the discovery, verification, and characterization of such an additional activity, a novel endoglucanase/cellulase activity in what is otherwise a triosephosphate isomerase from the hyperthermophile archaeon Pyrococcus furiosus (PfuTIM). The activity is seen in two different ranges of temperatures, with one maximum at 40 °C and a second maximum close to 100 °C. The endoglucanase/cellulase activity is inhibited by norharman, a TIM inhibitor, which is suspected to bind at a site different to that of the regular substrate, glyceraldehyde-3-phosphate (G3P). However, endoglucanase/cellulose activity is not inhibited either by G3P analogs or by glycine-scanning mutations involving residues in loops 1, 4, and 6 of PfuTIM, which are known to be important for TIM activity. It appears, therefore, that two different sites on PfuTIM are responsible for the observed TIM and endoglucanase activities. We discuss possible correlations between this discovery and certain unusual features of the glycolytic pathway in P. furiosus. ENZYME: Pyrococcus furiosus Triosephosphate isomerase (EC:5.3.1.1).

Creation of active TIM barrel enzymes through genetic fusion of half-barrel domain constructs derived from two distantly related glycosyl hydrolases.

Diverse unrelated enzymes that adopt the beta/alpha (or TIM) barrel topology display similar arrangements of beta/alpha units placed in a radial eight-fold symmetry around the barrel's axis. The TIM barrel was originally thought to be a single structural domain; however, it is now thought that TIM barrels arose from duplication and fusion of smaller half-barrels consisting of four beta/alpha units. We describe here the design, expression and purification, as well as characterization of folding, activity and stability, of chimeras of two TIM barrel glycosyl hydrolases, made by fusing different half-barrel domains derived from an endoglucanase from Clostridium cellulolyticum, CelCCA and a beta-glucosidase from Pyrococcus furiosus, CelB. We show that after refolding following purification from inclusion bodies, the two half-barrel fusion chimeras (CelCCACelB and CelBCelCCA) display catalytic activity although they assemble into large soluble oligomeric aggregated species containing chains of mixed beta and alpha structure. CelBCelCCA displays hyperthermophile-like structural stability as well as significant stability to chemical denaturation (Cm of 2.6 m guanidinium hydrochloride), whereas CelCCACelB displays mesophile-like stability (Tm of ~ 71 °C). The endoglucanase activities of both chimeras are an order of magnitude lower than those of CelB or CelCCA, whereas the beta-glucosidase activity of CelBCelCCA is about two orders of magnitude lower than that of CelB. The chimera CelCCACelB shows no beta-glucosidase activity. Our results demonstrate that half-barrel domains from unrelated sources can fold, assemble and function, with scope for improvement. ENZYME: Pyrococcus furiosus beta-glucosidase (CelB, EC: 3.2.1.21). Clostridium cellulolyticum endoglucanase A (CelCCA, EC: 3.2.1.4).