Citrate is an intermediate in catabolic as well as biosynthetic pathways and is an important regulatory molecule in the control of glycolysis and lipid metabolism. Mass spectrometric and NMR based metabolomics allow measuring citrate concentrations, but only with limited spatial and temporal resolution. Methods are so far lacking to monitor citrate levels in real-time in-vivo. Here, we present a series of genetically encoded citrate sensors based on Förster resonance energy transfer (FRET). We screened databases for citrate-binding proteins and tested three candidates in vitro. The citrate binding domain of the Klebsiella pneumoniae histidine sensor kinase CitA, inserted between the FRET pair Venus/CFP, yielded a sensor highly specific for citrate. We optimized the peptide linkers to achieve maximal FRET change upon citrate binding. By modifying residues in the citrate binding pocket, we were able to construct seven sensors with different affinities spanning a concentration range of three orders of magnitude without losing specificity. In a first in vivo application we show that E. coli maintains the capacity to take up glucose or acetate within seconds even after long-term starvation.
The crystal structure of the "ene" nicotinamide-dependent cyclohexenone reductase (NCR) from Zymomonas mobilis (PDB ID: 4A3U) has been determined in complex with acetate ion, FMN, and nicotinamide, to a resolution of 1.95 Å. To study the activity and enantioselectivity of this enzyme in the bioreduction of activated α,β-unsaturated alkenes, the rational design methods site- and loop-directed mutagenesis were applied. Based on a multiple sequence alignment of various members of the Old Yellow Enzyme family, eight single-residue variants were generated and investigated in asymmetric bioreduction. Furthermore, a structural alignment of various ene reductases predicted four surface loop regions that are located near the entrance of the active site. Four NCR loop variants, derived from loop-swapping experiments with OYE1 from Saccharomyces pastorianus, were analysed for bioreduction. The three enzyme variants, P245Q, D337Y and F314Y, displayed increased activity compared to wild-type NCR towards the set of substrates tested. The active-site mutation Y177A demonstrated a clear influence on the enantioselectivity. The loop-swapping variants retained reduction efficiency, but demonstrated decreased enzyme activity compared with the wild-type NCR ene reductase enzyme.
The enzymatic reduction of C=C bonds in allylic alcohols with Old Yellow Enzymes represents a challenging task, due to insufficient activation through the hydroxy group. In our work, we coupled an alcohol dehydrogenase with three wild-type ene reductases-namely nicotinamide-dependent cyclohex-2-en-1-one reductase (NCR) from Zymomonas mobilis, OYE1 from Saccharomyces pastorianus and morphinone reductase (MR) from Pseudomonas putida M10-and four rationally designed β/α loop variants of NCR in the bienzymatic cascade hydrogenation of allylic alcohols. Remarkably, the wild type of NCR was not able to catalyse the cascade reaction whereas MR and OYE1 demonstrated high to excellent activities. Through the rational loop grafting of two intrinsic β/α surface loop regions near the entrance of the active site of NCR with the corresponding loops from OYE1 or MR we successfully transferred the cascade reduction activity from one family member to another. Further we observed that loop grafting revealed certain influences on the interaction with the nicotinamide cofactor.
SummaryThrombus formation in the circulation is accompanied by covalent linkage of fibronectin (FN) through transglutamination of glutamine no. 3 in the fibrin binding amino terminal domain (FBD) of FN. We have exploited this phenomenon for thrombus detection by the employment of radioactively-labelled recombinant polypeptide molecules derived from the 5-finger FBD of human FN. Three recombinant FBD polypeptides, 12 kDa (“2 fingers”), 18.5 kDa (“3 fingers”) and 31 kDa FBD (“5 fingers”), were prepared and compared to native FN-derived 31 kDa-FBD with respect to their ability to attach to fibrin clots in vitro and in vivo. The accessibility of Gln-3 in these molecules was demonstrated by the incorporation of stoichiometric amounts of 14C- putrescine in the presence of plasma transglutaminase. Competitive binding experiments to fibrin have indicated that, although the binding affinities of the FBD molecules are lower than that of FN, substantial covalent linkage was obtained in the presence of transglutaminase, and even in the presence of excess FN or heparin. The biological clearance rates of radioactively labelled FBD molecules in rats and rabbits were much higher than those of FN and fibrinogen, thus indicating their potential advantage for use as a diagnostic imaging tool. Of the three molecules, the 12 kDa FBD exhibited the highest rate of clearance. The potential of the 12 kDa and 31 kDa FBDs as imaging agents was examined in a stainless steel coil-induced thrombus model in rats and in a jugular vein thrombus model in rabbits, using either [125I] or [111ln]-labelled materials. At 24 h, clot-to-blood ratios ranged between 10 and 22 for [125I]-12 kDa FBD and 40 and 60 for [luIn]-12 kDa FBD. In the rat model, heparin did not inhibit the uptake of FBD. Taken together, the results indicate that recombinant 12 kDa FBD is a good candidate for the diagnosis of venous thrombosis.
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