The flavoprotein methylenetetrahydrofolate reductase from Escherichia coli catalyzes the reduction of 5,10-methylenetetrahydrofolate (CH 2 -H 4 folate) by NADH via a ping-pong reaction mechanism. Structures of the reduced enzyme in complex with NADH and of the oxidized Glu28Gln enzyme in complex with CH 3 -H 4 folate [Pejchal, R., Sargeant, R., and Ludwig, M. L. (2005) Biochemistry 44, 11447-11457] have revealed Phe223 as a conformationally mobile active site residue. In the NADH complex, the NADH adopts an unusual hairpin conformation and is wedged between the isoalloxazine ring of the FAD and the side chain of Phe223. In the folate complex, Phe223 swings out from its position in the NADH complex in order to stack against the p-aminobenzoate ring of the folate. Although Phe223 contacts each substrate in E. coli MTHFR, this residue is not invariant; for example, a leucine occurs at this site in the human enzyme. To examine the role of Phe223 in substrate binding and catalysis, we have constructed mutants Phe223Ala and Phe223Leu. As predicted, our results indicate that Phe223 participates in the binding of both substrates. The Phe223Ala mutation impairs NADH and CH 2 -H 4 folate binding each 40-fold, yet slows catalysis of both half-reactions less than 2-fold. Affinity for CH 2 -H 4 folate is unaffected by the Phe223Leu mutation, and the variant catalyzes the oxidative half-reaction 3-fold faster than the wild-type enzyme. Structures of ligandfree Phe223Leu and Phe223Leu/Glu28Gln MTHFR in complex with CH 3 -H 4 folate have been determined at 1.65 Å and 1.70 Å resolution, respectively. The structures show that the folate is bound in a catalytically competent conformation, and Leu223 undergoes a conformational change similar to that observed for Phe223 in the Glu28Gln-CH 3 -H 4 folate structure. Taken together, our results suggest that Leu may be a suitable replacement for Phe223 in the oxidative half-reaction of E. coli MTHFR. † This work was supported in part by the American Chemical Society Petroleum Research Fund Grant 39599-GB4 (E.E. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 August 18. Published in final edited form as:Biochemistry. Methylenetetrahydrofolate reductase (MTHFR) 1 is a flavoprotein that catalyzes the NAD(P) H-dependent reduction of 5,10-methylenetetrahydrofolate (CH 2 -H 4 folate), as shown in eq 1.(1)The reaction provides CH 3 -H 4 folate, which is the sole methyl donor to homocysteine in the production of methionine by methionine synthase. MTHFR plays a significant role in the homeostasis of homocysteine; mutations in the enzyme lead to hyperhomocyst(e)inemia, (1, 2), which is associated with an increased risk for the development of cardiovascular disease (reviewed in (3)) and Alzheimer's disease (4-6) in adults, and of neural tube defects in the fetus (reviewed in (7)).MTHFRs from pig liver, human, E. coli, yeast, Arabidopsis, and Leishmania have been characterized (reviewed in (8); (9-12)). Whereas the mammalian and yeast MTHFRs are h...
For improved cost-effectiveness and temperaturestability, a ready to use lateral flow assay (LFA) is developed in this work for detecting inflammation/infection biomarker C-reactive protein (CRP) in human patient samples on the basis of aptamers. In prescreening investigations, an aptamer with CRP affinity was immobilized on microarray chips in forward and sandwich formats to optimize assay conditions. We suggest these microarray techniques as a resource-sparing and fast-screening instrument for evaluation of various conditions. The capability of the aptamer to detect CRP was shown. Optimized assay conditions were consequently transferred to the LFA-platform. Here we could demonstrate for the first time an aptamer-based LFA for the detection of CRP in human patient samples in pathologically relevant concentrations. The cutoff for CRP detection is set at 10 mg/L, providing a distinctive "yes" (≥10 mg/L CRP) or "no" (<10 mg/L CRP) answer for the patient. The resulting aptamer-based LFA is promising with regard to its application as point-of-care testing (POCT) for efficient monitoring, especially of patients affected by frequent infections or inflammations.
The selection of aptamers represents a promising route in the development of high affinity ligands. In these processes the formation of by-products is a common problem during the PCR-based amplification of complex oligonucleotide libraries. One approach to overcome this drawback is to separate each template oligonucleotide into an individual reaction compartment provided by a droplet. This method, termed emulsion PCR (ePCR), has already emerged to a standard method in sample preparation for 2nd generation sequencing. In this work, we compare different literature protocols that have been developed to generate stable emulsions for ePCR. We investigate different emulsification methods and evaluate the importance of the initial template concentration. We demonstrate that emulsion stability is of utmost importance for the successful inhibition of by-product formation and give an optimized protocol for generation of an emulsified PCR.
Many 1,2,4-benzotriazine 1,4-dioxides display the ability to selectively kill the oxygen-poor cells found in solid tumors. As a result, there is a desire for synthetic routes that afford access to substituted 1,2,4-benzotriazine 1-oxides that can be used as direct precursors in the synthesis of 1,2,4-benzotriazine 1,4-dioxides. Here we describe the use of Suzuki-Miyaura and Buchwald-Hartwig cross-coupling reactions for the construction of various 1,2,4-benzotriazine 1-oxide analogs bearing substituents at the 3-, 6-, and 7-positions.
Renal rejection is a major incidence in patients after kidney transplantation and associated with allograft scarring and function loss, especially in antibody-mediated rejection. Regular clinical monitoring of kidney-transplanted patients is thus necessary, but measuring donor-specific antibodies is not always predictive, and graft biopsies are time-consuming and costly and may come up with a histological result unsuspicious for rejection. Therefore, a noninvasive diagnostic approach to estimate an increased probability of kidney graft rejection by measuring specific biomarkers is highly desired. The chemokine CXCL9 is described as an early indicator of rejection. In this work, we identified clickmers and an aptamer by split−combine click-SELEX (systematic evolution of ligands by exponential enrichment) that bind CXLC9 with high affinity. The aptamers recognize native CXCL9 and maintain binding properties under urine conditions. These features render the molecules as potential binding and detector probes for developing point-of-care devices, e.g., lateral flow assays, enabling the noninvasive monitoring of CXCL9 in renal allograft patients.
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