NMR solution structure of a cold-adapted thiol-disulfide oxidoreductase

Psychrophilic enzymes produced by cold-adapted micro-organisms have successfully overcome the low temperature challenge and adapted to maintain high catalytic rates in their permanently cold environments. The current consensus is that this high activity at low temperatures is mainly achieved through an increase in the flexibility of the protein structure, thereby allowing for the molecular motions necessary for activity in their low thermal energy environment. The actual molecular basis for the adaptation is still however only poorly understood and direct evidence of the proposed increased flexibility is scant, with previous attempts to demonstrate this leading to conflicting results. In an attempt to better understand strategies of cold adaptation we have determined the NMR solution structure of the reduced form of a cold adapted thiol disulphide oxidoreductase (DsbA) isolated from an Antarctic bacterium. While a number of crystal structures for cold adapted enzymes have been published, this is the first report of an NMR structure of these enzymes and thereby opens up a new dimension in the study of cold adaptation. In particular, the potential power of NMR to monitor both local and global motions over a large range of time scales should allow for a better understanding of the role of dynamics in protein adaptation to temperature. The gene encoding the cold-adapted enzyme has been isolated and the protein overexpressed in E. coli with both unlabelled and labelled (15N13C, 15N) protein being purified from the periplasmic extracts. NMR data were acquired on a Bruker AvanceII+ 800 MHz spectrometer and the solution structure of the reduced form of this cold adapted oxidoreductase determined and compared to that of its mesophilic homolog from Vibrio cholerae. In addition, the temperature dependence of activity and stability of both the psychrophile and mesophile have been ascertained and compared. Here, the results of the NMR structure determination and the comparative structural and physicochemical studies of the cold adapted DsbA with its mesophilic homolog will be presented.European Molecular Biology Organisation (EMBO). Fundação para a Ciência e a Tecnologia (FCT) via Programa Ciência 2008

Biochemical characterization of the maltokinase from Mycobacterium bovis BCG

<p>Abstract</p> <p>Background</p> <p>Maltose-1-phosphate was detected in <it>Mycobacterium bovis </it>BCG extracts in the 1960's but a maltose-1-phosphate synthetase (maltokinase, Mak) was only much later purified from <it>Actinoplanes missouriensis</it>, allowing the identification of the <it>mak </it>gene. Recently, this metabolite was proposed to be the intermediate in a pathway linking trehalose with the synthesis of glycogen in <it>M. smegmatis</it>. Although the <it>M. tuberculosis </it>H37Rv <it>mak </it>gene (Rv0127) was considered essential for growth, no mycobacterial Mak has, to date, been characterized.</p> <p>Results</p> <p>The sequence of the Mak from <it>M. bovis </it>BCG was identical to that from <it>M. tuberculosis </it>strains (99-100% amino acid identity). The enzyme was dependent on maltose and ATP, although GTP and UTP could be used to produce maltose-1-phosphate, which we identified by TLC and characterized by NMR. The K_{<it>m </it>}for maltose was 2.52 ± 0.40 mM and 0.74 ± 0.12 mM for ATP; the <it>V</it>_maxwas 21.05 ± 0.89 μmol/min.mg^-1. Divalent cations were required for activity and Mg²⁺was the best activator. The enzyme was a monomer in solution, had maximal activity at 60°C, between pH 7 and 9 (at 37°C) and was unstable on ice and upon freeze/thawing. The addition of 50 mM NaCl markedly enhanced Mak stability.</p> <p>Conclusions</p> <p>The unknown role of maltokinases in mycobacterial metabolism and the lack of biochemical data led us to express the <it>mak </it>gene from <it>M. bovis </it>BCG for biochemical characterization. This is the first mycobacterial Mak to be characterized and its properties represent essential knowledge towards deeper understanding of mycobacterial physiology. Since Mak may be a potential drug target in <it>M. tuberculosis</it>, its high-level production and purification in bioactive form provide important tools for further functional and structural studies.</p

Expanding the toolbox for Synechocystis sp. PCC 6803 : validation of replicative vectors and characterization of a novel set of promoters

Cyanobacteria are promising ‘low-cost’ cell factories since they have minimal nutritional requirements, high metabolic plasticity and can use sunlight and CO2 as energy and carbon sources. The unicellular Synechocystis sp. PCC 6803, already considered the ‘green’ Escherichia coli, is the best studied cyanobacterium but to be used as an efficient and robust photoautotrophic chassis it requires a customized and well-characterized toolbox. In this context, we evaluated the possibility of using three self-replicative vectors from the Standard European Vector Architecture (SEVA) repository to transform Synechocystis. Our results demonstrated that the presence of the plasmid does not lead to an evident phenotype or hindered Synechocystis growth, being the vast majority of the cells able to retain the replicative plasmid even in the absence of selective pressure. In addition, a set of heterologous and redesigned promoters were characterized exhibiting a wide range of activities compared to the reference PrnpB, three of which could be efficiently repressed. As a proof-of-concept, from the expanded toolbox, one promoter was selected and assembled with the ggpS gene [encoding one of the proteins involved in the synthesis of the native compatible solute glucosylglycerol (GG)] and the synthetic device was introduced into Synechocystis using one of the SEVA plasmids. The presence of this device restored the production of the GG in a ggpS deficient mutant validating the functionality of the tools/device developed in this study

Phosphate and polyphosphate anion recognition by a dinuclear copper(ii) complex of an unsymmetrical squaramide

[Abstract] In the search for receptors suitable for the recognition of phosphate or polyphosphate anions, a new unsymmetrical squaramide-based ligand bearing dipicolylamine (dpa) and ethylpiperazine units (L) was designed and prepared. The acid–base reactions of L, its copper(II) complexation behaviour and the binding of phosphate and polyphosphate anions by the copper(II) complexes used as receptors were evaluated. 1H and 13C NMR titrations of L performed in D2O allowed the determination of its protonation sequence. The ligand L is able to coordinate two copper(II) cations forming thermodynamically stable dinuclear complexes likely having two water molecules bound to each metal centre, as supported by DFT calculations. Coordinated water molecules can be replaced by the O-donors of the phosphate/polyphosphate anions. The potentiometric studies showed that at 2 : 1 Cu2+ : L ratio the dinuclear [Cu2LH−1]3+ species predominates from pH ∼ 5 to ∼7, and hydroxodinuclear species prevail at pH > 7. 1H NMR experiments in both H2O/D2O 9 : 1 v/v and in DMSO proved that copper(II) coordination provokes deprotonation of the squaramide NH bound to the ethylpiperazine moiety, resulting in [Cu2LH−1]3+ species. The dicopper(II) complexes of L, [Cu2LH−i]4−i, were used as the receptor for the uptake of some phosphate and polyphosphate anions. The receptor presents very high association constants with HPPi3− and ATP4− and the determined Keff showed that at physiological pH ATP4− is selectively taken from an aqueous solution containing phenylphosphate (PhPO42−), aminoethylphosphate (Haep−), AMP2– and ADP3−, but HPPi3− strongly interferes. DFT calculations suggest that the strong interaction with HPPi3− and ATP4− is related to the simultaneous coordination of the polyphosphate unit to the two copper(II) centres.This work was partially supported by Project LISBOA-01-0145-FEDER-007660 (Microbiologia Molecular, Estrutural e Celular) funded by FEDER funds through COMPETE2020 – Programa Operacional Competitividade e Internacionalização (POCI) and by national funds through FCT. The NMR data were acquired at CERMAX, ITQB-NOVA, Oeiras, Portugal with equipment funded by FCT, project AAC 01/SAICT/2016. C. V. Esteves thanks FCT for the PhD fellowship SFRH/BD/89501/2012 and Project LISBOA-01-0145-FEDER-007660 for a last fellowshipPortugal. Fundação para a Ciência e a Tecnologia; LISBOA-01-0145-FEDER-007660Portugal. Fundação para a Ciência e a Tecnologia; AAC 01/SAICT/2016Portugal. Fundação para a Ciência e a Tecnologia; SFRH/BD/89501/201

Structure and properties of the metastable bacteriocin Lcn972 from Lactococcus lactis

Lactococcus lactis subsp. lactis IPLA 972 produces a polypeptide bacteriocin of 7.5 kDa which has a bactericidal effect on sensitive lactococci, inhibiting septum formation in dividing cells. The active form is a monomer that is metastable under normal conditions but is stabilised by glycerol. The NMR structure of Lcn972 shows a β-sandwich comprising two three-stranded antiparallel β-sheets. Detaching the final strand could allow the sandwich to open, and the irreversible unfolding leads to a loss of antibacterial activity. Covalent linkage of the final strand should increase the stability of Lcn972 and facilitate the study of its interaction with lipid II. © 2012 Elsevier B.V. All rights reserved.This work was supported by Fundação para a Ciência e a Tecnologia (Portugal) through Grant No. PEst-OE/EQB/LA0004/2011, Contract REDE/1517/RMN/2005 and PTDC/QUI-BIQ/114904/2009 and Grant BIO2010-17414 of the Ministerio de Economía y Competitividad (Spain).Peer Reviewe

NMR structure of Desulfovibrio gigas rubredoxin: a model for studying protein stabilization by compatible solutes

Rubredoxins are small, soluble proteins that display a wide variation in thermostability, despite having a high degree of sequence similarity. They also vary in the extent to which they are stabilized by solutes such as diglycerol phosphate. Hence, they provide excellent models for studying the mechanisms of thermostabilization. Nuclear magnetic resonance (NMR) spectroscopy can be used to investigate interactions between molecules, as well as subtle changes in conformation in solution, and also provides a means to measure protein stability. The assignment of the proton NMR spectrum of the zinc rubredoxin from Desulfovibrio gigas is presented, together with its structure in solution. The stabilizing effect of diglycerol phosphate on rubredoxin is demonstrated and assessed by determining selected amide proton exchange rates; diglycerol phosphate at 100 mM concentration caused an additional structural stabilization of 1.2 +/- 0.4 kJ/mol. The pattern of effects on the exchange rates is discussed in relation to the protein structure