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.

Large scale changes in the transcriptome of Eisenia fetida during regeneration.

Earthworms show a wide spectrum of regenerative potential with certain species like Eisenia fetida capable of regenerating more than two-thirds of their body while other closely related species, such as Paranais litoralis seem to have lost this ability. Earthworms belong to the phylum Annelida, in which the genomes of the marine oligochaete Capitella telata and the freshwater leech Helobdella robusta have been sequenced and studied. Herein, we report the transcriptomic changes in Eisenia fetida (Indian isolate) during regeneration. Following injury, E. fetida regenerates the posterior segments in a time spanning several weeks. We analyzed gene expression changes both in the newly regenerating cells and in the adjacent tissue, at early (15days post amputation), intermediate (20days post amputation) and late (30 days post amputation) by RNAseq based de novo assembly and comparison of transcriptomes. We also generated a draft genome sequence of this terrestrial red worm using short reads and mate-pair reads. An in-depth analysis of the miRNome of the worm showed that many miRNA gene families have undergone extensive duplications. Sox4, a master regulator of TGF-beta mediated epithelial-mesenchymal transition was induced in the newly regenerated tissue. Genes for several proteins such as sialidases and neurotrophins were identified amongst the differentially expressed transcripts. The regeneration of the ventral nerve cord was also accompanied by the induction of nerve growth factor and neurofilament genes. We identified 315 novel differentially expressed transcripts in the transcriptome, that have no homolog in any other species. Surprisingly, 82% of these novel differentially expressed transcripts showed poor potential for coding proteins, suggesting that novel ncRNAs may play a critical role in regeneration of earthworm.

Introduction of a thermophile-sourced ion pair network in the fourth beta/alpha unit of a psychophile-derived triosephosphate isomerase from Methanococcoides burtonii significantly increases its kinetic thermal stability.

Hyperthermophile proteins commonly have higher numbers of surface ionic interactions than homologous proteins from other domains of life. PfuTIM, a triosephosphate isomerase (TIM) from the hyperthermophile archaeon, Pyrococcus furiosus, contains an intricate network of 4 ion pairs in its 4th beta/alpha unit, (ß/α)4, whereas MbuTIM, a triosephosphate isomerase from a psychrophile archaeon, Methanococcoides burtonii, lacks this network. Notably, (ß/α)4 is the first element of the structure formed during folding of certain TIM-type (beta/alpha)8 barrel proteins. Previously, we have shown that elimination of PfuTIM's ion pair network in PfuTIM significantly decreases its kinetic structural stability. Here, we describe the reciprocal experiment in which this ion pair network is introduced into MbuTIM, to produce MutMbuTIM. Recombinant MbuTIM displays multi-state unfolding with apparent Tm values of autonomous structural elements approaching, or above, 70°C, when a temperature scanning rate of 90°C/h is used. The protein displays significant intrinsic kinetic stability, i.e., there is a marked temperature scan rate-dependence of the Tm values associated with unfolding transitions. The Tm values drop by as much as ~10°C when the temperature scanning rate is lowered to 5°C/h. MutMbuTIM, incorporating PfuTIM's ion pair network, shows significantly higher apparent Tm values (raised by 4-6°C over those displayed by MbuTIM). MutMbuTIM also displays significantly higher kinetic thermal stability. Thus, it appears that the thermal stability of triosephosphate isomerase can be increased, or decreased, by either enhancing, or reducing, the strength of ion pair interactions stabilizing (ß/α)4, presumably through reduced cooperativity (and increased autonomy) in unfolding transitions.

N-Terminal sequencing by mass spectrometry through specific fluorescamine labeling of α-amino groups before tryptic digestion.

We present a single-step procedure for the specific mass labeling of unblocked protein N termini. We show that the dye fluorescamine, which is commonly assumed to require mildly alkaline conditions for undergoing a nonspecific reaction with α- and ε-amino groups associated with amino acids, in fact shows a specific reaction only with α-amino groups present at protein N termini when mildly acidic conditions are used. We use this finding to label, identify, and sequence the trypsinolysis-derived N-terminal peptide of lysozyme, using only mass spectrometry, to illustrate how this method could be used with other proteins.

Expression, purification, refolding and characterization of a putative lysophospholipase from Pyrococcus furiosus: retention of structure and lipase/esterase activity in the presence of water-miscible organic solvents at high temperatures.

A putative lysophospholipase (PF0480) encoded by the Pyrococcus furiosus genome has previously been cloned and expressed in Escherichia coli. Studies involving crude extracts established the enzyme to be an esterase; however, owing presumably to its tendency to precipitate into inclusion bodies, purification and characterization have thus far not been reported. Here, we report the overexpression and successful recovery and refolding of the enzyme from inclusion bodies. Dynamic light scattering suggests that the enzyme is a dimer, or trimer, in aqueous solution. Circular dichroism and fluorescence spectroscopy show, respectively, that it has mixed beta/alpha structure and well-buried tryptophan residues. Conformational changes are negligible over the temperature range of 30-80 degrees C, and over the concentration range of 0-50% (v/v) of water mixtures with organic solvents such as methanol, ethanol and acetonitrile. The enzyme is confirmed to be an esterase (hydrolyzing p-NP-acetate and p-NP-butyrate) and also shown to be a lipase (hydrolyzing p-NP-palmitate), with lipolytic activity being overall about 18- to 20-fold lower than esterase activity. Against p-NP-palmitate the enzyme displays optimally activity at pH 7.0 and 70 degrees C. Remarkably, over 50% activity is retained at 70 degrees C in the presence of 25% acetonitrile. The high organic solvent stability and thermal stability suggest that this enzyme may have useful biodiesel-related applications, or applications in the pharmaceutical industry, once yields are optimized.