Detrimental effect of the 6 His C-terminal tag on YedY enzymatic activity and influence of the TAT signal sequence on YedY synthesis

Background

YedY, a molybdoenzyme belonging to the sulfite oxidase family, is found in most Gram-negative bacteria. It contains a twin-arginine signal sequence that is cleaved after its translocation into the periplasm. Despite a weak reductase activity with substrates such as dimethyl sulfoxide or trimethylamine N-oxide, its natural substrate and its role in the cell remain unknown. Although sequence conservation of the YedY family displays a strictly conserved hydrophobic C-terminal residue, all known studies on Escherichia coli YedY have been performed with an enzyme containing a 6 histidine-tag at the C-terminus which could hamper enzyme activity.

Results

In this study, we demonstrate that the tag fused to the C-terminus of Rhodobacter sphaeroides YedY is detrimental to the enzyme’s reductase activity and results in an eight-fold decrease in catalytic efficiency. Nonetheless this C-terminal tag does not influence the properties of the molybdenum active site, as assayed by EPR spectroscopy. When a cleavable His-tag was fused to the N-terminus of the mature enzyme in the absence of the signal sequence, YedY was expressed and folded with its cofactor. However, when the signal sequence was added upstream of the N-ter tag, the amount of enzyme produced was approximately ten-fold higher.

Conclusion

Our study thus underscores the risk of using a C-terminus tagged enzyme while studying YedY, and presents an alternative strategy to express signal sequence-containing enzymes with an N-terminal tag. It brings new insights into molybdoenzyme maturation in R. sphaeroides showing that for some enzymes, maturation can occur in the absence of the signal sequence but that its presence is required for high expression of active enzyme.

     

Introduction of a Carboxymethylamino(or oxy Or thio) Group in the 3 Position of 2-Aryl(or Arylmethyl)isoindol-1-ones from α Hydroxylactams

The preparation of 3-carboxymethylamino (or oxy or thio)-2-aryl (or arylmethyl)-isoindol-1-ones are described from hydroxylactams according to three different pathways.     

Synthesis and Separation of New Dibenz[b,d]azonine Atropisomers

A synthesis of the dibenz[b,d]azonine atropisomers (14) from 2-phenylbenzoic acid is described. This heterocycle belongs to a new series of biaryl compounds with potential antitubulin activity.     

EFFECT OF MOLYBDENUM PROMOTER ON THE Cu-Co BASED CATALYSTS

The synthesis of mixed alcohols (C1-C5) from syngas has been studied at 6. 0 MPa over modified Co/CuLaZr catalysts. The molybdenum addition enhanced greatly the activity and the selectivity to alcohols. The improvement of hydrogenation capacity of the system via a reversible spillover effect of hydrogen could explain this effect, together with a great capacity of CO insertion, illustrated by the effect of C2H4 addition as a probe to syngas. Under our experimental conditions, a mixed alcohol production of 147.1g/kg. cat/h containing a proportion of 33. 1% of higher alcohols (C2 OH) was obtained with a selectivity to ROH of 52. 8%.     

Dual Gold Catalysis: σ,π‐Propyne Acetylide and Hydroxyl‐Bridged Digold Complexes as Easy‐To‐Prepare and Easy‐To‐Handle Precatalysts

A series of dinuclear gold σ,π‐propyne acetylide complexes were prepared and tested for their catalytic ability in dual gold catalysis that was based on the reaction of an electrophilic π‐complex of gold with a gold acetylide. The air‐stable and storable catalysts can be isolated as silver‐free catalysts in their activated form. These dual catalysts allow a fast initiation phase for the dual catalytic cycles without the need for additional additives for acetylide formation. Because propyne serves as a throw‐away ligand, no traces of the precatalyst are generated. Based on the fast initiation process, side products are minimized and reaction rates are higher for these catalysts. A series of test reactions were used to demonstrate the general applicability of these catalysts. Lower catalyst loadings, faster reaction rates, and better selectivity, combined with the practicability of these catalysts, make them ideal catalysts for dual gold catalysis.     

A printed nanolitre-scale bacterial sensor array

The last decade has witnessed a significant increase in interest in whole-cell biosensors for diverse applications, as well as a rapid and continuous expansion of array technologies. The combination of these two disciplines has yielded the notion of whole-cell array biosensors. We present a potential manifestation of this idea by describing the printing of a whole-cell bacterial bioreporters array. Exploiting natural bacterial tendency to adhere to positively charged abiotic surfaces, we describe immobilization and patterning of bacterial "spots" in the nanolitre volume range by a non-contact robotic printer. We show that the printed Escherichia coli-based sensor bacteria are immobilized on the surface, and retain their viability and biosensing activity for at least 2 months when kept at 4 °C. Immobilization efficiency was improved by manipulating the bacterial genetics (overproducing curli protein), the growth and the printing media (osmotic stress and osmoprotectants) and by a chemical modification of the inanimate surface (self-assembled layers of 3-aminopropyl-triethoxysilane). We suggest that the methodology presented herein may be applicable to the manufacturing of whole-cell sensor arrays for diverse high throughput applications.     

Inclusion complexes of trivalent lutetium cations with an acidic derivative of per(3,6-anhydro)-alpha-cyclodextrin

The cyclodextrin derivative (hexakis (2-O-carboxymethyl-3,6-anhydro)-alpha-cyclodextrin) forms mono- and bimetallic complexes with lutetium(III) in aqueous solution; the X-ray structure of the binuclear complex [Lu2(ACX)(H2O)2] is the first example of a lanthanide-cyclodextrin inclusion complex.     

A theoretical study of the formation of the parent phosphinine C5H5P from the flash vacuum thermolysis of diallylvinylphosphine

The gas-phase decomposition of diallylvinylphosphine 1 into C5H5P 12 is studied by DFT/6-311+G(d,p) calculations with the B3LYP functional, followed by single-point energy-only calculations at the CCSD(T)/6-311+G(d,p) level. According to these calculations, the first step involves a retro-ene elimination that yields 3-phosphahexatrienes 2Z and 2E. Both compounds equilibrate through the formation of 1,2- and 3,4-dihydrophosphetes 3 and 4, and it is shown that the formation of 2Z is favored by the exothermic formation of the 3,4-dihydrophosphinine 5 through a 6pi-electrocyclization. Though 5 can easily isomerize into 2,3- (6) and 1,2-diyhydrophosphinines (7) by successive 1,5-hydrogen shifts, the formation of 12 from 5, 6, or 7 through an elimination of H2 is found to be a high energy process. It is also shown that the elimination of H2 from lambda5-phosphinine 8 following a C2v pathway is a symmetry-forbidden process. Finally, 1,4-dihydrophosphinine 9, which can be formed through a 1,4-hydrogen shift from lambda5-phosphinine 8, is found to be a convenient precursor of 12 through a 1,4-elimination of H2. The formation of 9 from 5 involves the intermediary formation of 3-phosphabicyclo[3.1.0]hex-2-ene 10. The mechanism eventually proposed for the formation of 12 from 2Z is given in Scheme 16 at the CCSD(T) level.     

In situ layer-by-layer film formation kinetics under an applied voltage measured by optical waveguide lightmode spectroscopy

Layer-by-layer (LbL) thin film assembly occurs via the alternate adsorption of positively and negatively charged macromolecular species. We investigate here the control of LbL film growth through the electric potential of the underlying substrate. We employ optical waveguide lightmode spectroscopy (OWLS) to obtain in situ kinetic measurements of poly(allylamine hydrochloride)/poly(sodium 4-styrenesulfonate) (PAH/PSS) and poly(L-lysine)/dextran sulfate (PLL/DXS) multilayer film formation in the presence of an applied voltage difference (deltaV) between the adsorbing substrate, an indium tin oxide- (ITO-) coated waveguiding sensor chip, and a parallel platinum counterelectrode. We find initial layer adsorption to be significantly enhanced by an applied potential for both polyelectrolyte systems: the mass and thickness of (positively charged) PAH and PLL layers on ITO are about 60% and 500% larger, respectively, at deltaV = 2 V than at open circuit potential (OCP), in apparent violation of electrostatics. A kinetic analysis reveals the initial attachment rate constant to decrease with voltage, in agreement with electrostatics. To reconcile these results, we propose a more coiled and loosely bound adsorbed polymer conformation at higher applied potential. Following 10 adsorption steps, the mass and thickness of a PAH/PSS film grown under deltaV = 2 V are about 15% less than those of a comparable film grown under OCP, reflecting a lower degree of complexation between adsorbing polyanions and more highly coiled adsorbed polycations. Following 14 adsorption steps, the mass and thickness of a PLL/DXS film grown under deltaV = 2 V are about 70% greater than those of a comparable film grown under OCP, reflecting the increased charge overcompensation in the initial layer. We find the scaling of film mass () with the number of adsorption steps (n) to be linear in the PAH/PSS system and exponential (i.e., approximately eyn) in the PLL/DXS system, irrespective of applied voltage. We observe to decrease with applied voltage and to exhibit a crossover to a smaller value around n = 5. Extrapolation reveals PLL/DXS multilayer films to be suppressed by increased voltage in the limit of large n: the mass of films grown at OCP and deltaV = 1 V would surpass that of a film grown under deltaV = 2 V at about the 23rd and 18th adsorption steps, respectively. The formation kinetics of PLL/DXS, but not PAH/PSS, change qualitatively under voltage: PLL adsorption is slow to reach a plateau, possibly due to the formation of secondary structure, and a decrease in film mass occurs toward the end of each DXS adsorption step, suggesting spontaneous removal of some PLL/DXS complexes from the film.     

Cobalt-catalyzed hydrohydrazination of dienes and enynes: access to allylic and propargylic hydrazides

[reaction: see text] The cobalt-catalyzed hydrohydrazination reaction of dienes and enynes is presented. Allylic and propargylic hydrazines were obtained in synthetically useful yields (allylic amines, 60-90%; propargylic amines, 47-83%) and good chemo- and regioselectivity.