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Background: Glycogen and starch branching enzymes catalyze the formation of α(1→6) linkages in storage polysaccharides by rearrangement of preexisting α-glucans. This reaction occurs through the cleavage of α(1→4) linkage and transfer in α(1→6) of the fragment in non-reducing position. These enzymes define major elements that control the structure of both glycogen and starch., Methods: The kinetic parameters of the branching enzyme of Rhodothermus obamensis (RoBE) were established after in vitro incubation with different branched or unbranched α-glucans of controlled structure., Results: A minimal chain length of ten glucosyl units was required for the donor substrate to be recognized by RoBE that essentially produces branches of DP 3-8. We show that RoBE preferentially creates new branches by intermolecular mechanism. Branched glucans define better substrates for the enzyme leading to the formation of hyper-branched particles of 30-70nm in diameter (dextrins). Interestingly, RoBE catalyzes an additional α-4-glucanotransferase activity not described so far for a member of the GH13 family., Conclusions: RoBE is able to transfer α(1→4)-linked-glucan in C4 position (instead of C6 position for the branching activity) of a glucan to create new α(1→4) linkages yielding to the elongation of linear chains subsequently used for further branching. This result is a novel case for the thin border that exists between enzymes of the GH13 family., General Significance: This work reveals the original catalytic properties of the thermostable branching enzyme of R. obamensis. It defines new approach to produce highly branched α-glucan particles in vitro., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The research on complex I has gained recently a new enthusiasm, especially after the resolution of the crystallographic structures of bacterial and mitochondrial complexes. Most attention is now dedicated to the investigation of the energy coupling mechanism(s). The proton has been identified as the coupling ion, although in the case of some bacterial complexes I Na(+) has been proposed to have that role. We have addressed the relation of some complexes I with Na(+) and developed an innovative methodology using (23)Na NMR spectroscopy. This allowed the investigation of Na(+) transport taking the advantage of directly monitoring changes in Na(+) concentration. Methodological aspects concerning the use of (23)Na NMR spectroscopy to measure accurately sodium transport in bacterial membrane vesicles are discussed here. External-vesicle Na(+) concentrations were determined by two different methods: 1) by integration of the resonance frequency peak and 2) using calibration curves of resonance frequency shift dependence on Na(+) concentration. Although the calibration curves are a suitable way to determine Na(+) concentration changes under conditions of fast exchange, it was shown not to be applicable to the bacterial membrane vesicle systems. In this case, the integration of the resonance frequency peak is the most appropriate analysis for the quantification of external-vesicle Na(+) concentration. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012)., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Until recently cytochrome bc(1) complexes were the only enzymes known to be able to transfer electrons from reduced quinones to cytochrome c. However, a complex with the same activity and with a unique subunit composition was purified from the membranes of Rhodothermus marinus. This complex, named alternative complex III (ACIII) was then biochemical, spectroscopic and genetically characterized. Later it was observed that the presence of ACIII was not exclusive of R. marinus being the genes coding for ACIII widespread, at least in the Bacteria domain. In this work, a comprehensive description of the current knowledge on ACIII is presented. The relation of ACIII with members of the complex iron-sulfur molybdoenzyme family is investigated by analyzing all the available completely sequenced genomes. It is concluded that ACIII is a new complex composed by a novel combination of modules already identified in other respiratory complexes., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Background: The twin phenomena of aggregation and degradation are classically associated with protein storage. However, although aggregation has been thought to be a possible consequence of protein degradation, it has never before been proposed to be a cause of degradation., Methods: Proteins stored under physiological conditions and electrophoresed on SDS-PAGE were examined zymographically for the presence of detergent-resistant high molecular weight (HMW) forms, and association of such HMW forms with time-correlated, seeding-dependent gelatinolytic activity, under various conditions., Results: Eight different proteins aggregate naturally during storage at near-neutral pH, with concomitant development of 'gelatinolytic' activity diminished greatly by storage at low temperatures, extremes of pH, arginine, imidazole, BSA, azide, EDTA, DTT, PMSF (but not AEBSF), and diisopropyl fluorophosphate (DFP), suggesting involvement of surface serine residues in a novel aggregate-borne proteolytic activity., Conclusions: Naturally-formed aggregates of proteins appear to use surface serines to perform peptide bond hydrolysis, explaining degradation of proteins during storage, and indicating why aggregates are cytotoxic., General Significance: The study suggests that a bi-directional cause-effect relationship operates between protein aggregation, and protein degradation, providing clues to the design of better conditions for long-term protein storage.

Using several tens of rationally-selected substitutions, insertions and deletions of predominantly non-contiguous residues, we have remodeled the solvent-exposed face of a beta sheet functioning as the substrate-binding and catalytically-active groove of a thermophile cellulase (Rhodothermus marinus Cel12A) to cause it to resemble, both in its structure and function, the equivalent groove of a mesophile homolog (Trichoderma reesei Cel12A). The engineered protein, a mesoactive-thermostable cellulase (MT Cel12A) displays the temperature of optimal function of its mesophile ancestor and the temperature of melting of its thermophile ancestor, suggesting that such 'grafting' of a mesophile-derived surface onto a thermophile-derived structural scaffold can potentially help generate novel enzymes that recombine structural and functional features of homologous proteins sourced from different domains of life.