Crystal structure of an Fe-S cluster-containing fumarate hydratase enzyme from

Fumarate hydratases (FHs) are essential metabolic enzymes grouped into two classes. Here, we present the crystal structure of a class I FH, the cytosolic FH from Leishmania major, which reveals a previously undiscovered protein fold that coordinates a catalytically essential [4Fe-4S] cluster. Our 2.05 Å resolution data further reveal a dimeric architecture for this FH that resembles a heart, with each lobe comprised of two domains that are arranged around the active site. Besides the active site, where the substrate S-malate is bound bidentate to the unique iron of the [4Fe-4S] cluster, other binding pockets are found near the dimeric enzyme interface, some of which are occupied by malonate, shown here to be a weak inhibitor of this enzyme. Taken together, these data provide a framework both for investigations of the class I FH catalytic mechanism and for drug design aimed at fighting neglected tropical diseases

Enhanced dynamic flux variability analysis for improving growth and production rate in microbial strains

Metabolic engineering is highly demanded currently for the production of various useful compounds such as succinate and lactate that are very useful in food, pharmaceutical, fossil fuels, and energy industries. Gene or reaction deletion known as knockout is one of the strategies used in in silico metabolic engineering to change the metabolism of the chosen microbial cells to obtain the desired phenotypes. However, the size and complexity of the metabolic network are a challenge in determining the near-optimal set of genes to be knocked out in the metabolism due to the presence of competing pathway that interrupts the high production of desired metabolite, leading to low production rate and growth rate of the required microorganisms. In addition, the inefficiency of existing algorithms in reconstructing high growth rate and production rate becomes one of the issues to be solved. Therefore, this research proposes Dynamic Flux Variability Analysis (DFVA) algorithm to identify the best knockout reaction combination to improve the production of desired metabolites in microorganisms. Based on the experimental results, DFVA shows an improvement of growth rate of succinate and lactate by 12.06% and 47.16% respectively in E. coli and by 4.62% and 47.98% respectively in S. Cerevisae. Suggested reactions to be knocked out to improve the production of succinate and lactate have been identified and validated through the biological database

A unique way of energy conservation in glutamate fermenting clostridia

Genetic analysis revealed that Rhodobacter capsulatus contains six rnfABCDEG-genes that are responsible for the electron flow in nitrogen fixation (rnf = Rhodobacter nitrogen fixation). Homolgous genes have been detected in Clostridium tetani. In this work, a membrane complex has been purified from the related Clostridium tetanomorphum that catalyses the reduction of NAD + (E°' = −320 mV) with ferredoxin (E°' ≤ −420 mV). The difference in the redox potential of ≥ 100 mV could be useful for additional energy conservation in the fermentation of glutamate to ammonia, CO 2 , acetate, butyrate, and H 2 . The complex consists of six subunits (RnfABCDEG), of which four N-termini (RnfCDEG) could be sequenced. The sequences are 60-80% identical to the deduced sequences of the Rnf-subunits from C. tetani. The rnf operon has been completely sequenced and aligned with the sequences of C. tetani. The complex contains both non-covalently bound flavin as well as covalently bound flavin. The non-covalently bound flavin was identified as FMN and riboflavin in 1:1 stochiometric ratio, each 0.3 mol/mol Rnf complex (180 kDa). The subunits RnfG and RnfD contain covalently bound flavin linked via phosphodiester bond. The iron was determined as 25±1 mol per Rnf complex. Usually, Rnf activity was measured with NADH and ferricyanide at 420 nm. In order to measure NAD + reduction with reduced ferredoxin catalysed by Rnf complex, the ferredoxin was purified from C. tetanomorphum and reduced by Ti(III)citrate at pH 7.0. High Rnf activities were observed in the membrane preparations of Clostridium aminobutyricum, Clostridium pascui and Clostridium propionicum. Thus, additional energy conservation can be explained in these bacteria. However Rnf activity was absent in Eubacterium barkeri, a nicotinate fermenting bacteria. The soluble butyryl-CoA-dehydrogenase/electron transferring flavoprotein (Bcd/Etf) complex was purified from C. pascui as well as from C. tetanomorphum. The N- terminal sequences of the three subunits (αβγ) showed high identities with the deduced sequences of C. tetani. The Bcd/Etf complex purified from C. tetanomorphum was shown to catalyze the endergonic reduction of ferredoxin with NADH coupled to the exergonic reduction of crotonyl-CoA to butyryl-CoA (E°' = -10 mV) with NADH. The12 reduced ferredoxin could be used for H 2 production catalysed by a hydrogenase or probably used for additional energy conservation via Rnf (about 0.3 mol ATP/ mol glutamate). Experiments with [2,4,4- 2 H] glutamate and detection of citramalate-lyase activity showed that C. pascui and C. tetanomorphum ferment glutamate via the methylaspartate pathway

In vitro Realisation of the Hydroxypropionyl-CoA/Acrylyl-CoA Cycle

The birth of the industrial revolution initiated a significant shift in the global carbon cycle. In the intervening centuries, the production of anthropogenic atmospheric carbon rose dramatically and has resulted in a pronounced climactic shift. The rate of this change is accelerating, largely irreversible in the short-term, and is expected to have a profound negative impact on nearly every aspect of human life from culture and economics to mental and physical health. It is now generally recognised that past practices are unsustainable and that we must take immediate action if we are to ameliorate this problem. Global efforts to reduce carbon emissions have begun, and many novel technologies are currently being developed both to make manufacturing more efficient and to actively remove carbon from the atmosphere. Despite these efforts, average atmospheric CO2 concentrations have continued to rise, and the climate has continued to change. While a multitude of different methods will be required if we are to be successful in reducing global atmospheric carbon, an intriguing approach is generating designer organisms capable of fixing CO2. Natural carbon fixation is the cornerstone of organic life, but there are potential improvements that could be made to generate more efficient carbon-fixing organisms. In addition to removing atmospheric CO2, the carbon can potentially be funnelled into any number of value-added products. In this work, we have pursued the artificial Hydroxypropionyl-CoA/Acrylyl-CoA Cycle in an in vitro system. We aim to demonstrate that the cycle is functional at ambient temperature and in the presence of oxygen making it an appealing candidate for future in vivo engineering efforts. In the first part, we investigate the oxidative portion of the cycle which involves the conversion of (2S)-methylmalonyl-CoA to malonyl-CoA. Originally this involved chemistry analogous to the TCA cycle, however due to difficulties with multiple steps of this pathway, we introduced a novel bypass that directly oxidises succinyl-CoA to the metabolically unusual fumaryl-CoA. This portion of the cycle also includes the first carbon-fixing reaction, the ATP-dependent carboxylation of acetyl-CoA. In the second part, we evaluate the reductive portion of the cycle which involves the conversion of malonyl-CoA back to (2S)-methylmalonyl-CoA. This portion involves three reduction reactions and we explored two potential pathways going through either 3-hydroxypropionyl-CoA or β-alanyl-CoA. While both versions are functional, the lack of β-alanine-specific enzymes, especially a β-alanyl-CoA synthetase made the 3-hydroxypropionyl-CoA pathway more practical. In either case, this portion terminates with the reductive carboxylation of acrylyl-CoA to (2S)-methylmalonyl-CoA. Finally, these pathways were combined to yield a continuous cycle. After flux through the cycle was achieved, we sought to resolve a variety of resultant issues including regeneration of ATP and NADPH, elimination of reactive oxygen species, protection from coenzyme B12 radical inactivation, regeneration of FAD-dependent enzymes, and the repair of chemically modified cofactors. The current HOPAC cycle was found to produce ~500 µM glycolate, or five CO2-equivalents per molecule of acetyl-CoA. Overall, in this work we established the HOPAC cycle, a new-to-nature synthetic CO2-fixation pathway, in vitro and further optimised its functioning. This works provides another proof-of-principle for synthetic CO2-fixation and opens the path for implementation of HOPAC in natural and synthetic cells in the future

Synthesis and evaluation of enzyme inhibitors based on amino- and cyclopropane carboxylic acids

The coenzyme B12-dependent enzyme, glutamate mutase (E. C. 5.4.99.1), catalyses the reversible carbon-skeleton rearrangement of (2S)-glutamic acid to (25.35)-3-methylaspartic acid. Glutamate mutase is the first enzyme on the mesaconate pathway. A variety of glutamate and 3-methylaspartate analogues (which also include isotopically labelled molecules), were synthesised as molecular probes of the enzyme. Synthesis of stereospecifically labelled 3-ethylaspartic acid: (2S,3S)-[3'-C2H3], and (2S,3S)-[C2H2C2H3]-ethylaspartic acids were constructed using appropriately labelled iodoethane. (2S,3S)-2-Bromo-3-methylsuccinic acid was synthesised via the diazotization of (2S,3S)-3-methylaspartic acid, in the presence of bromide ion. (2S)-Methylsuccinic acid was synthesised by the catalytic hydrogenation of (2S,3S)-2-bromo-3-methylsucdnic acid. Biological studies of the synthesised compounds (including the labelled isotopomers) displayed no activity against glutamate mutase. 3-Methylaspartate ammonia-lyase, the second enzyme in the mesaconate pathway, catalyses the deamination of (2S,3S)-3-methylaspartic acid to mesaconic acid. A range of 1-substituted cyclopropane 1,2-dicarboxylic acids were synthesised using short efficient routes and were found to be good to potent inhibitors of 3-methylaspartase. X-ray crystallographic studies have determined the absolute stereochemistry. The mode of action of the most potent inhibitor, (1S,2S)-1-methylcyclopropane 1,2-dicarboxylic acid (20 mumol dm-3), is consistent with it acting as a transition state analogue for the central substrate deamination reaction catalysed by the enzyme. beta-Amino acids are constituents of many biologically active peptides. A general procedure for the synthesis of alpha-substituted-beta-amino acids has been developed. The synthesis involves a Baylis-Hillman amine catalysed conversion of methyl acrylate, with an appropriate aldehyde, to give the alpha-(hydroxyalkyl) acrylate. Bromination and subsequent azide displacement furnishes the azido alkene, which is catalytically hydrogenated, to furnish the beta-amino ester

Investigations into the mechanism of the coenzyme B12 dependent reaction catalyzed by glutamate mutase from Clostridium cochlearium

Aims of this study were the search for inhibitors of the coenzyme B12-dependent glutamate mutase and for insight into the first step of its catalytic mechanism, the homolytic cleavage of the cobalt-carbon bond. Glutamate mutase is composed of two separately isolated protein components S and E2, which in the presence of coenzyme B12 assemble to the active holo-glutamate mutase E2S2-B12 that catalyzes the reversible conversion of (S)-glutamate to (2S,3S)-3-methylaspartate. This reaction has been coupled with methylaspartase, which deaminates (2S,3S)-3-methylaspartate to mesaconate absorbing at 240 nm, to allow activity assays for glutamate mutase by UV-spectrophotometry. As potential inhibitors, compounds with sp2-centers and structural analogies to the intermediate radicals in the proposed mechanism were selected. Analogues to the 4-glutamyl radical were (E)- and (Z)-glutaconates, whereas analogues to the (2S,3S)-3-methyleneaspartate radical included itaconate, buta-1,3-diene-2,3-dicarboxylate, fumarate, maleate and mesaconate. Because all these compounds inhibited the auxiliary enzyme methylaspartase, glutamate mutase was incubated with these compounds for a certain time, followed by gelfitration on Sephadex G25. The residual activity of the inactivator-free enzyme was then determined by the coupled assay described above, whereby unexpectedly fumarate, maleate and mesaconate caused inactivation of the mutase. To check whether the other compounds acted as reversible inhibitors, a new assay with (2S,3S)-3-methylaspartate and pyruvate as substrates involving glutamate-pyruvate aminotransferase and the NADH-dependent (R)-2-hydroxyglutarate dehydrogenase was developed. Application of this assay showed that 2.5 mM itaconate and 8 mM (E)-glutaconate inhibited glutamate mutase in the presence of 200 mM (2S,3S)-3-methylaspartate by 50%. Furthermore, the kinetic constants of (2S,3S)-3-methylaspartate in the reaction of glutamate mutase were determined as Km= 7 ± 0.07 mM, kcat= 0.54 ± 0.06 s-1and kcatKm-1= 77 s-1M-1. Together with the kinetic constants of (S)-glutamate determined with the methylaspartase assay (Km = 2.25 ± 0.03 mM, kcat = 2.85 ± 0.5 s-1 and kcatKm-1 = 1.3 × 10-3 s-1M-1), an equilibrium constant of Keq = [glutamate] × [methylaspartate]-1 = 16 was calculated by the Briggs-Haldane equation close to that described in the literature (Keq = 12). ... The mutL gene from Clostridium tetanomorphum is located between the structural genes of glutamate mutase. We speculate that MutL acts as chaperone, which removes cob(II)alamin from inactive glutamate mutase complexes in an ATP dependent manner. The liberated components E2 and S recombine with coenzyme B12 to form a new active enzyme. To check this hypothesis, mutL was successful cloned on pASG-IBA3 and pASG-IBA 5 expression vectors via the pre-entry vector IBA-20. The MutL chaperone was produced in E. coli Rossetta in good yield

Activation of UDP-glucose Pyrophosphorylase and Evaluation of the Lactose Synthetase Assay

Chemistr