On the enzymatic provision of redox power in synthetic cells

The development of artificial systems mimicking the essential properties of living cells is one of the most intriguing areas of research of the last decades. By rationally assembling the fundamental molecular building blocks through a bottom-up approach, it should be possible to create a cell-like system capable of self-sustaining, growing and replicating. Among the hundreds of enzymatic biochemical reactions necessary for an effective minimal metabolism, are the redox reactions. The reducing equivalents are transferred from one reactant to another through the action of soluble coenzymes, namely the nicotinamide adenine dinucleotides NAD(H) and NAD(P)H. Strategies that ensure the lasting regeneration of the redox status of these cofactors are therefore crucial for the construction of a functioning compartmentalized metabolic network. This thesis focuses on the reactants and in particular the enzymes suitable for the provision of reducing power in synthetic cells

A conserved sequence motif in the E. coli soluble FAD-containing pyridine nucleotide transhydrogenase is important for reaction efficiency

Soluble pyridine nucleotide transhydrogenases (STHs) are flavoenzymes involved in the redox homeostasis of the essential cofactors NAD(H) and NADP(H). They catalyze the reversible transfer of reducing equivalents between the two nicotinamide cofactors. The soluble transhydrogenase from Escherichia coli (SthA) has found wide use in both in vivo and in vitro applications to steer reducing equivalents towards NADPH-requiring reactions. However, mechanistic insight into SthA function is still lacking. In this work, we present a biochemical characterization of SthA, focusing for the first time on the reactivity of the flavoenzyme with molecular oxygen. We report on oxidase activity of SthA that takes place both during transhydrogenation and in the absence of an oxidized nicotinamide cofactor as an electron acceptor. We find that this reaction produces the reactive oxygen species (ROS) hydrogen peroxide and superoxide anion. Furthermore, we explore the evolutionary significance of the well conserved CXXXXT motif that distinguishes STHs from the related family of flavoprotein disulfide reductases in which a CXXXXC motif is conserved. Our mutational analysis revealed the cysteine and threonine combination in SthA leads to better coupling efficiency of transhydrogenation and reduced ROS release compared to enzyme variants with mutated motifs. These results expand our mechanistic understanding of SthA by highlighting reactivity with molecular oxygen and the importance of the evolutionarily conserved sequence motif

Biochemical and structural insight into the chemical resistance and cofactor specificity of the formate dehydrogenase from Starkeya novella

Formate dehydrogenases (Fdhs) mediate the oxidation of formate to carbon dioxide and concomitant reduction of nicotinamide adenine dinucleotide (NAD + ). The low cost of the substrate formate and importance of the product NADH as a cellular source of reducing power make this reaction attractive for biotechnological applications. However, the majority of Fdhs are sensitive to inactivation by thiol-modifying reagents. In this study, we report a chemically resistant Fdh (Fdh SNO ) from the soil bacterium Starkeya novella strictly specific for NAD + . We present its recombinant overproduction, purification and biochemical characterization. The mechanistic basis of chemical resistance was found to be a valine in position 255 (rather than a cysteine as in other Fdhs) preventing the inactivation by thiol-modifying compounds. To further improve the usefulness of Fdh SNO as for generating reducing power, we rationally engineered the protein to reduce the coenzyme nicotinamide adenine dinucleotide phosphate (NADP + ) with better catalytic efficiency than NAD + . The single mutation D221Q enabled the reduction of NADP + with a catalytic efficiency k CAT /K M of 0.4 s -1 mM -1 at 200 mM formate, while a quadruple mutant (A198G/D221Q/H379K/S380V) resulted in a 5-fold increase in catalytic efficiency for NADP + compared to the single mutant. We determined the cofactor-bound structure of the quadruple mutant to gain mechanistic evidence behind the improved specificity for NADP + . Our efforts to unravel the key residues for the chemical resistance and cofactor specificity of Fdh SNO may lead to wider use of this enzymatic group in a more sustainable (bio)manufacture of value-added chemicals, as for instance the biosynthesis of chiral compounds. </p

Minimal Pathway for the Regeneration of Redox Cofactors

[Image: see text] Effective metabolic pathways are essential for the construction of in vitro systems mimicking the biochemical complexity of living cells. Such pathways require the inclusion of a metabolic branch that ensures the availability of reducing equivalents. Here, we built a minimal enzymatic pathway confinable in the lumen of liposomes, in which the redox status of the nicotinamide cofactors NADH and NADPH is controlled by an externally provided formate. Formic acid permeates the membrane where a luminal formate dehydrogenase uses NAD(+) to form NADH and carbon dioxide. Carbon dioxide diffuses out of the liposomes, leaving only the reducing equivalents in the lumen. A soluble transhydrogenase subsequently utilizes NADH for reduction of NADP(+) thereby making NAD(+) available again for the first reaction. The pathway is functional in liposomes ranging from a few hundred nanometers in diameter (large unilamellar vesicles) up to several tens of micrometers (giant unilamellar vesicles) and remains active over a period of 7 days. We demonstrate that the downstream biochemical process of reduction of glutathione disulfide can be driven by the transfer of reducing equivalents from formate via NAD(P)H, thereby providing a versatile set of electron donors for reductive metabolism

A conserved sequence motif in the Escherichia coli soluble FAD-containing pyridine nucleotide transhydrogenase is important for reaction efficiency

Soluble pyridine nucleotide transhydrogenases (STHs) are flavoenzymes involved in the redox homeostasis of the essential cofactors NAD(H) and NADP(H). They catalyze the reversible transfer of reducing equivalents between the two nicotinamide cofactors. The soluble transhydrogenase from Escherichia coli (SthA) has found wide use in both in vivo and in vitro applications to steer reducing equivalents toward NADPH-requiring reactions. However, mechanistic insight into SthA function is still lacking. In this work, we present a biochemical characterization of SthA, focusing for the first time on the reactivity of the flavoenzyme with molecular oxygen. We report on oxidase activity of SthA that takes place both during transhydrogenation and in the absence of an oxidized nicotinamide cofactor as an electron acceptor. We find that this reaction produces the reactive oxygen species hydrogen peroxide and superoxide anion. Furthermore, we explore the evolutionary significance of the well-conserved CXXXXT motif that distinguishes STHs from the related family of flavoprotein disulfide reductases in which a CXXXXC motif is conserved. Our mutational analysis revealed the cysteine and threonine combination in SthA leads to better coupling efficiency of transhydrogenation and reduced reactive oxygen species release compared to enzyme variants with mutated motifs. These results expand our mechanistic understanding of SthA by highlighting reactivity with molecular oxygen and the importance of the evolutionarily conserved sequence motif.Fil: Partipilo, Michele. University of Groningen; Países BajosFil: Yang, Guang. University of Groningen; Países BajosFil: Mascotti, María Laura. University of Groningen; Países Bajos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis. Universidad Nacional de San Luis. Facultad de Ciencias Físico Matemáticas y Naturales. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis; ArgentinaFil: Wijma, Hein J.. University of Groningen; Países BajosFil: Slotboom, Dirk Jan. University of Groningen; Países BajosFil: Fraaije, Marco Wilhelmus. University of Groningen; Países Bajo

A Hitchhiker’s Guide to Supplying Enzymatic Reducing Power into Synthetic Cells

The construction from scratch of synthetic cells by assembling molecular building blocks is unquestionably an ambitious goal from a scientific and technological point of view. To realize functional life-like systems, minimal enzymatic modules are required to sustain the processes underlying the out-of-equilibrium thermodynamic status hallmarking life, including the essential supply of energy in the form of electrons. The nicotinamide cofactors NAD(H) and NADP(H) are the main electron carriers fueling reductive redox reactions of the metabolic network of living cells. One way to ensure the continuous availability of reduced nicotinamide cofactors in a synthetic cell is to build a minimal enzymatic module that can oxidize an external electron donor and reduce NAD(P)+. In the diverse world of metabolism there is a plethora of potential electron donors and enzymes known from living organisms to provide reducing power to NAD(P)+ coenzymes. This perspective proposes guidelines to enable the reduction of nicotinamide cofactors enclosed in phospholipid vesicles, while avoiding high burdens of or cross-talk with other encapsulated metabolic modules. By determining key requirements, such as the feasibility of the reaction and transport of the electron donor into the cell-like compartment, we select a shortlist of potentially suitable electron donors. We review the most convenient proteins for the use of these reducing agents, highlighting their main biochemical and structural features. Noting that specificity toward either NAD(H) or NADP(H) imposes a limitation common to most of the analyzed enzymes, we discuss the need for specific enzymes─transhydrogenases─to overcome this potential bottleneck

Minimal Out-of-Equilibrium Metabolism for Synthetic Cells: A Membrane Perspective

Life-like systems need to maintain a basal metabolism, which includes importing a variety of building blocks required for macromolecule synthesis, exporting dead-end products, and recycling cofactors and metabolic intermediates, while maintaining steady internal physical and chemical conditions (physicochemical homeostasis). A compartment, such as a unilamellar vesicle, functionalized with membrane-embedded transport proteins and metabolic enzymes encapsulated in the lumen meets these requirements. Here, we identify four modules designed for a minimal metabolism in a synthetic cell with a lipid bilayer boundary: energy provision and conversion, physicochemical homeostasis, metabolite transport, and membrane expansion. We review design strategies that can be used to fulfill these functions with a focus on the lipid and membrane protein composition of a cell. We compare our bottom-up design with the equivalent essential modules of JCVI-syn3a, a top-down genome-minimized living cell with a size comparable to that of large unilamellar vesicles. Finally, we discuss the bottlenecks related to the insertion of a complex mixture of membrane proteins into lipid bilayers and provide a semiquantitative estimate of the relative surface area and lipid-to-protein mass ratios (i.e., the minimal number of membrane proteins) that are required for the construction of a synthetic cell

Impatto dei fattori ostetrici sulla donazione del sangue cordonale: due anni di attività

Scopo. L’obiettivo di questo studio è analizzare due anni di attività del nostro Centro di Raccolta del Sangue Placentare tra ottobre 2008 e settembre 2010 e verificare l’impatto dei fattori clinici ed ostetrici sul numero delle unità di sangue del cordone ombelicale (SCO) bancate. Materiale e metodi. Il consenso informato per il reclutamento alla donazione di SCO è stato raccolto a 35 settimane di gravidanza, previo counselling della coppia, attraverso un’accurata anamnesi materna e paterna alla ricerca di malattie genetiche, immunologiche ed infettive trasmissibili. Le unità di sangue cordonale sono state raccolte ogni giorno della settimana al momento del parto spontaneo o del taglio cesareo tra 37-41+6 settimane di gravidanza presso la UOC Ostetricia-Ginecologia Ospedale “Di Venere”, Bari, tra ottobre 2008 e settembre 2010. Le unità di SCO sono state raccolte in sala parto o in sala operatoria con placenta inserita in utero. Il cordone ombelicale è stato clampato non prima di 60 secondi dall’espulsione del feto. Le unità di SCO sono state accettate dalla Banca Cordonale Regione Puglia (PUCBB, presso l’IRCCS “Casa Sollievo della Sofferenza” in San Giovanni Rotondo) per la crioconservazione quando il numero totale delle cellule nucleate (TCN) era ≥80¥107. I dati sono stati analizzati mediante il coefficiente di correlazione di Pearson, il t-test di Student, il test di Mann-Whitne e analisi chi quadro. Risultati. Il numero dei parti presso il nostro punto nascita durante il periodo di studio è stato di 2.971. Le pazienti sensibilizzate alla raccolta del sangue cordonale sono state 1.117 (37,6%). Le pazienti escluse dalla donazione del sangue cordonale al momento della raccolta del Consenso Informato sono state 244; le unità di SCO escluse al momento della raccolta in sala parto o in sala operatoria per fattori ostetrici (materno-fetali) sono state 160; il numero totale di SCO inviate alla PUCBB è stato di 713 (1.117-404), cioè il 24% di 2.971 parti totali. Il numero delle sacche escluse dalla PUCBB sono state 445 per bassa cellularità e 62 per altri motivi ostetrici e tecnici. Possiamo affermare che le unità di SCO scartate prima della crioconservazione sono state 507. Se consideriamo che altre 22 unità cordonali sono state scartate dopo la crioconservazione per patologie materne e neonatali, il numero totale di unità eliminate è stato di 529, cioè il 74,2% delle 713 inviate. Le unità bancate sono state, quindi, 184 (713-529), cioè il 25,8% delle 713 unità di sangue cordonale raccolte. Prendendo in considerazione le analisi condotte mediante il coefficiente di correlazione di Pearson: il peso neonatale correla positivamente con le settimane di gestazione al momento del parto (r=0,304; p=0,000), con il volume di SCO raccolto (r=0,191; p=0,000), con il numero TCN (r=0,252; p=0,000) e con il numero di CD34+ raccolte (r=0,222; p=0,001). Il volume dell’unità di SCO raccolta correla positivamente con la raccolta di CD34+(r=0,433; p=0,000). L’età materna correla in maniera inversamente proporzionale con le settimane di gestazione (r=0,155; p=0,000). Le donatrici al primo parto in assoluto rispetto a tutte le altre (almeno una gravidanza pregressa) presentano l’età materna significativamente più bassa. Anche la durata del travaglio e le settimane di gestazione sono maggiori per le nullipare, che peraltro raccolgono mediamente 100 milioni di TNC in più, oltre che un più alto valore di concentrazione di TNC per unità di volume. solo nelle nullipare il volume raccolto, seppur maggiore e significativamente diverso nel cesareo, non basta a compensare la maggiore raccolta di cellule che è assolutamente superiore nello spontaneo: 2,81¥103/ul TNC e ben 190 milioni di TNC in più. Discussione. Dati i nuovi tetti di bancaggio, ≥1,2 miliardi di TNC al congelamento, abbiamo analizzato quali fossero le caratteristiche delle donatrici che nel nostro centro hanno raccolto almeno 1,2 miliardi di cellule. Esse rappresentano il 18,4% delle 713 unità di SCO raccolte, cioè 131. Questo gruppo è composto per il 68,5% da donne al loro primo parto e per il restante 31,5% da pluripare. Nasce, quindi, l’esigenza di reclutare più primigravide. Inoltre, le unità che hanno almeno 1,2 miliardi di cellule nell’89,3% dei casi sono provenienti da nati di peso >3.000 g e solo il 10,7% da nati di peso <3.000 g. In altre parole possiamo affermare che nel nostro centro è inutile raccogliere da nati di peso <3.000 g. Conclusioni. Secondo la nostra esperienza una raccolta ottimale di SCO deve rispettare le seguenti caratteristiche: reclutare più primigravide, con epoca gestazionale al prelievo di SCO tra 37-40 settimane, con peso fetale superiore a 3.000 g, tempo di clampaggio del cordone ombelicale entro 30 secondi, raccolta di sangue cordonale con placenta inserita ancora in utero, e da preferire il parto spontaneo, di solito associato a lungo travaglio (aumento dello stress fetale)