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Highlights • Increase of intracellular FAD concentration resulting in electroactive E. coli. • Neutral red-mediated electrosynthesis is applied to the MES reaction. • More reductive products are achieved by E. coli 8739(pRibAB-YbjI-YigB, pRibECF). • The succinate yield is improved by E. coli T110(pRibAB-YbjI-YigB, pRibECF). Abstract In this study, the FAD synthesis pathway was manipulated to increase its cellular concentration and thereby improve the electroactivity of E. coli. In a microbial electrosynthesis (MES) system with neutral red as electron carrier and fumarate as the sole electron acceptor, the engineered strains derived from three E. coli lines displayed increased electric current in the reaction system, indicating improved electroactivity. Furthermore, the production of succinate from fumarate increased by around 60% compared with that of the parent strains, confirming the improvement of E. coli electroactivity by manipulating the FAD synthesis pathway. An MES reaction was performed with engineered E. coli 8739, and an altered metabolic profile with more reductive fermentation products was obtained. When the electroactive succinate-producing strain E. coli T110 was used in the MES, a yield of 0.97 ± 0.02 mol/mol glucose was achieved, which corresponds to an approximately 1.4-fold increase compared with the fermentation with no electricity supply or non-electroactive T110. In addition, a carbon concentration mechanism (CCM) was employed to further improve succinate production and yield in the MES, which produced a succinate yield of 1.16 mol/mol glucose, a 1.7-fold increase compared with that of the parent strain T110, indicating that the electroactive E. coli could be used in MES to produce specific fermentation products with improved yield. [ABSTRACT FROM AUTHOR]

Renalase is a recently discovered secretory protein involved in the regulation of blood pressure. Cells synthesize all known isoforms of human renalase (1 and 2) as flavoproteins. Accommodation of FAD in the renalase protein requires the presence of its N-terminal peptide. However, in secretory proteins, such peptides are usually cleaved during their export from the cell. In the present study, we have isolated human renalase from urinary samples of healthy volunteers and human recombinant renalases 1 and 2 expressed in Escherichia coli cells. In these proteins, we investigated the presence of the renalase N-terminal peptide and the FAD cofactor and performed computer-aided molecular analysis of the renalase crystal structure to evaluate possible consequences of removal of the N-terminal peptide. In contrast to human recombinant renalase isoforms 1 and 2 containing non-covalently bound FAD and clearly detectable N-terminal peptide, renalase purified from human urine lacks both the N-terminal signal peptide and FAD. The computer-aided analysis indicates that the removal of this peptide results in inability of the truncated renalase to bind the FAD cofactor. Thus, our results indicate that human renalase secreted in urine lacks its N-terminal peptide, and therefore catalytic activities of urinary renalase reported in the literature cannot be attributed to FAD-dependent mechanisms. We suggest that FAD-dependent catalytic functions are intrinsic properties of intracellular renalases, whereas extracellular renalases act in FAD- and possibly catalytic-independent manner. [ABSTRACT FROM AUTHOR]

Abstract: Methylenetetrahydrofolate reductase (MTHFR) family of proteins catalyze the conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate. They contain a flavin adenine dinucleotide (FAD) as the cofactor and the enzyme in eukaryotes, except in yeast, is known to be allosterically regulated by S-adenosylmethionine. Some cardiovascular diseases, neural tube defects, neuropsychiatric diseases and certain type of cancers in humans are associated with certain polymorphisms of MTHFR. Here, we analyzed 57 of MTHFR polypeptide sequences by multiple sequence alignment and determined previously unrecognized conserved residues that may have a functional or structural importance. A previously unrecognized ATP synthase motif was found in all of the examined plant MTHFRs, suggesting a different functional capability to the plant MTHFRs in addition to the known function. On a phylogenetic tree built, eukaryotic MTHFR proteins formed a clear cluster separated from prokaryotic and archeal relatives. The sequence identities among the eukaryotic MTHFRs were less divergent than the bacterial MTHFRs. [Copyright &y& Elsevier]

Protoporphyrinogen IX oxidase (PPO), the last common enzyme of haem and chlorophyll biosynthesis, catalyses the oxidation of protoporphyrinogen IX to protoporphyrin IX. The membrane-embedded flavoprotein is the target of a large class of herbicides. In humans, a defect in PPO is responsible for the dominantly inherited disease variegate porphyria. Here we present the crystal structure of mitochondrial PPO from tobacco complexed with a phenyl-pyrazol inhibitor. PPO forms a loosely associated dimer and folds into an FAD-binding domain of the p-hydroxybenzoate-hydrolase fold and a substrate-binding domain that enclose a narrow active site cavity beneath the FAD and an a-helical membrane-binding domain. The active site architecture suggests a specific substrate-binding mode compatible with the unusual six-electron oxidation. The membrane-binding domains can be docked onto the dimeric structure of human ferrochelatase, the next enzyme in haem biosynthesis, embedded in the opposite side of the membrane. This modelled transmembrane complex provides a structural explanation for the uncoupling of haem biosynthesis observed in variegate porphyria patients and in plants after inhibiting PPO. [ABSTRACT FROM AUTHOR]

In the diazotroph Klebsiella pneumoniae, NifL and NifA regulate transcription of the nitrogen fixation genes in response to molecular oxygen and combined nitrogen. We recently showed that Fnr is the primary oxygen sensor. Fnr transduces the oxygen signal towards the negative regulator NifL by activating genes whose products reduce the FAD moiety of NifL under anoxic conditions. These Fnr-dependent gene products could be membrane-bound components of the anaerobic electron transport chain. Consequently, in this study we examined the localization of NifL within the cell under various growth conditions. In K. pneumoniae grown under oxygen- and nitrogen-limited conditions, approximately 55% of the total NifL protein was found in the membrane fraction. However, when the cells were grown aerobically or shifted to nitrogen sufficiency, less than 10% of total NifL was membrane-associated. In contrast to NifL, NifA was located in the cytoplasm under all growth conditions tested. Further studies using K. pneumoniae mutant strains showed that, under derepressing conditions but in the absence of either the primary oxygen sensor Fnr or the primary nitrogen sensor GlnK and the ammonium transporter AmtB, NifL was located in the cytoplasm and inhibited NifA activity. These findings suggest that under nitrogen- and oxygen-limitation, a significantly higher membrane affinity of NifL might create a spatial gap between NifL and its cytoplasmic target protein NifA, thereby impairing inhibition of NifA by NifL. Localization of GlnK further showed that, under nitrogen-limited conditions but independent of the presence of oxygen, 15–20% of the total GlnK is membrane-associated. [ABSTRACT FROM AUTHOR]