RESUMO
Mevalonate 3,5-bisphosphate decarboxylase is involved in the recently discovered Thermoplasma-type mevalonate pathway. The enzyme catalyzes the elimination of the 3-phosphate group from mevalonate 3,5-bisphosphate as well as concomitant decarboxylation of the substrate. This entire reaction of the enzyme resembles the latter half-reactions of its homologs, diphosphomevalonate decarboxylase and phosphomevalonate decarboxylase, which also catalyze ATP-dependent phosphorylation of the 3-hydroxyl group of their substrates. However, the crystal structure of mevalonate 3,5-bisphosphate decarboxylase and the structural reasons of the difference between reactions catalyzed by the enzyme and its homologs are unknown. In this study, we determined the X-ray crystal structure of mevalonate 3,5-bisphosphate decarboxylase from Picrophilus torridus, a thermoacidophilic archaeon of the order Thermoplasmatales. Structural and mutational analysis demonstrated the importance of a conserved aspartate residue for enzyme activity. In addition, although crystallization was performed in the absence of substrate or ligands, residual electron density having the shape of a fatty acid was observed at a position overlapping the ATP-binding site of the homologous enzyme, diphosphomevalonate decarboxylase. This finding is in agreement with the expected evolutionary route from phosphomevalonate decarboxylase (ATP-dependent) to mevalonate 3,5-bisphosphate decarboxylase (ATP-independent) through the loss of kinase activity. We found that the binding of geranylgeranyl diphosphate, an intermediate of the archeal isoprenoid biosynthesis pathway, evoked significant activation of mevalonate 3,5-bisphosphate decarboxylase, and several mutations at the putative geranylgeranyl diphosphate-binding site impaired this activation, suggesting the physiological importance of ligand binding as well as a possible novel regulatory system employed by the Thermoplasma-type mevalonate pathway.
Assuntos
Carboxiliases/química , Thermoplasmales/enzimologia , Trifosfato de Adenosina/metabolismo , Carboxiliases/metabolismo , Redes e Vias Metabólicas , Ácido Mevalônico/metabolismoRESUMO
Ethanol is a widely available carbon compound that can be increasingly produced with a net negative carbon balance. Carbon-negative ethanol might therefore provide a feedstock for building a wider range of sustainable chemicals. Here we show how ethanol can be converted with a cell free system into acetyl-CoA, a central precursor for myriad biochemicals, and how we can use the energy stored in ethanol to generate ATP, another key molecule important for powering biochemical pathways. The ATP generator produces acetone as a value-added side product. Our ATP generator reached titers of 27 ± 6 mM ATP and 59 ± 15 mM acetone with maximum ATP synthesis rate of 2.8 ± 0.6 mM/h and acetone of 7.8 ± 0.8 mM/h. We illustrated how the ATP generating module can power cell-free biochemical pathways by converting mevalonate into isoprenol at a titer of 12.5 ± 0.8 mM and a maximum productivity of 1.0 ± 0.05 mM/h. These proof-of-principle demonstrations may ultimately find their way to the manufacture of diverse chemicals from ethanol and other simple carbon compounds.
Assuntos
Etanol , Engenharia Metabólica , Acetona , Acetilcoenzima A/metabolismo , Trifosfato de Adenosina , Carbono/metabolismo , Etanol/metabolismoRESUMO
Protein folding is a fundamental process of life with important implications throughout biology. Indeed, tens of thousands of mutations have been associated with diseases, and most of these mutations are believed to affect protein folding rather than function. Correct folding is also a key element of design. These factors have motivated decades of research on protein folding. Unfortunately, knowledge of membrane protein folding lags that of soluble proteins. This gap is partly caused by the greater technical challenges associated with membrane protein studies, but also because of additional complexities. While soluble proteins fold in a homogenous water environment, membrane proteins fold in a setting that ranges from bulk water to highly charged to apolar. Thus, the forces that drive folding vary in different regions of the protein, and this complexity needs to be incorporated into our understanding of the folding process. Here, we review our understanding of membrane protein folding biophysics. Despite the greater challenge, better model systems and new experimental techniques are starting to unravel the forces and pathways in membrane protein folding.
Assuntos
Proteínas de Membrana , Dobramento de Proteína , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismoRESUMO
It is now possible to efficiently fix flue gas CO/CO2 into ethanol using acetogens, thereby making carbon negative ethanol. While the ethanol could be burned as a fuel, returning the CO2 to the atmosphere, it might also be possible to use the fixed carbon in more diverse chemicals, thereby keeping it fixed. Here we describe a simple synthetic biochemistry approach for converting carbon negative ethanol into the synthetic building block chemical 1,3 butanediol (1,3-BDO). The pathway completely conserves carbon from ethanol and can ultimately be powered electrochemically via formate oxidation. Our proof-of-principle system reached a maximum productivity of 0.16 g/L/h and, with replenishment of feedstock and enzymes, achieved a titer of 7.7 g/L. We identify a number of elements that can be addressed in future work to improve both cell-free and cell-based production of 1,3-BDO.
RESUMO
The question of how proteins manage to organize into a unique three-dimensional structure has been a major field of study since the first protein structures were determined. For membrane proteins, the question is made more complex because, unlike water-soluble proteins, the solvent is not homogenous or even unique. Each cell and organelle has a distinct lipid composition that can change in response to environmental stimuli. Thus, the study of membrane protein folding requires not only understanding how the unfolded chain navigates its way to the folded state, but also how changes in bilayer properties can affect that search. Here we review what we know so far about the impact of lipid composition on bilayer physical properties and how those properties can affect folding. A better understanding of the lipid bilayer and its effects on membrane protein folding is not only important for a theoretical understanding of the folding process, but can also have a practical impact on our ability to work with and design membrane proteins.
Assuntos
Membrana Celular , Bicamadas Lipídicas , Proteínas de Membrana , Modelos Moleculares , Dobramento de Proteína , Membrana Celular/química , Membrana Celular/metabolismo , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Estrutura Secundária de ProteínaRESUMO
Moving cannabinoid production away from the vagaries of plant extraction and into engineered microbes could provide a consistent, purer, cheaper and environmentally benign source of these important therapeutic molecules, but microbial production faces notable challenges. An alternative to microbes and plants is to remove the complexity of cellular systems by employing enzymatic biosynthesis. Here we design and implement a new cell-free system for cannabinoid production with the following features: (1) only low-cost inputs are needed; (2) only 12 enzymes are employed; (3) the system does not require oxygen and (4) we use a nonnatural enzyme system to reduce ATP requirements that is generally applicable to malonyl-CoA-dependent pathways such as polyketide biosynthesis. The system produces ~0.5 g l-1 cannabigerolic acid (CBGA) or cannabigerovarinic acid (CBGVA) from low-cost inputs, nearly two orders of magnitude higher than yeast-based production. Cell-free systems such as this may provide a new route to reliable cannabinoid production.
Assuntos
Canabinoides/biossíntese , Sistema Livre de Células/metabolismo , Malonil Coenzima A/metabolismo , Engenharia Metabólica/métodos , Policetídeos/metabolismo , Terpenos/metabolismo , Trifosfato de Adenosina/biossíntese , Benzoatos/isolamento & purificação , Benzoatos/metabolismo , Canabinoides/isolamento & purificação , Sistema Livre de Células/química , Escherichia coli/enzimologia , Escherichia coli/genética , Expressão Gênica , Humanos , Cinética , Engenharia Metabólica/economia , Organofosfatos/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , Policetídeos/química , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Terpenos/química , TermodinâmicaRESUMO
Cost competitive conversion of biomass-derived sugars into biofuel will require high yields, high volumetric productivities and high titers. Suitable production parameters are hard to achieve in cell-based systems because of the need to maintain life processes. As a result, next-generation biofuel production in engineered microbes has yet to match the stringent cost targets set by petroleum fuels. Removing the constraints imposed by having to maintain cell viability might facilitate improved production metrics. Here, we report a cell-free system in a bioreactor with continuous product removal that produces isobutanol from glucose at a maximum productivity of 4 g L-1 h-1, a titer of 275 g L-1 and 95% yield over the course of nearly 5 days. These production metrics exceed even the highly developed ethanol fermentation process. Our results suggest that moving beyond cells has the potential to expand what is possible for bio-based chemical production.
Assuntos
Bioquímica/métodos , Butanóis/metabolismo , Enzimas/metabolismo , Acetolactato Sintase/química , Acetolactato Sintase/metabolismo , Trifosfato de Adenosina , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Bioquímica/instrumentação , Reatores Biológicos , Sistema Livre de Células , Evolução Molecular Direcionada , Enzimas/química , Enzimas/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Glucose/metabolismo , Temperatura , TermodinâmicaRESUMO
Metabolic engineering efforts that harness living organisms to produce natural products and other useful chemicals face inherent difficulties because the maintenance of life processes often runs counter to our desire to maximize important production metrics. These challenges are particularly problematic for commodity chemical manufacturing where cost is critical. A cell-free approach, where biochemical pathways are built by mixing desired enzyme activities outside of cells, can obviate problems associated with cell-based methods. Yet supplanting cell-based methods of chemical production will require the creation of self-sustaining, continuously operating systems where input biomass is converted into desired products at high yields, productivities, and titers. We call the field of designing and implementing reliable and efficient enzyme systems that replace cellular metabolism, synthetic biochemistry.
Assuntos
Bioquímica/tendências , Sistema Livre de Células , Engenharia Metabólica , Biologia Sintética/tendências , BiomassaRESUMO
To understand membrane protein biogenesis, we need to explore folding within a bilayer context. Here, we describe a single-molecule force microscopy technique that monitors the folding of helical membrane proteins in vesicle and bicelle environments. After completely unfolding the protein at high force, we lower the force to initiate folding while transmembrane helices are aligned in a zigzag manner within the bilayer, thereby imposing minimal constraints on folding. We used the approach to characterize the folding pathways of the Escherichia coli rhomboid protease GlpG and the human ß2-adrenergic receptor. Despite their evolutionary distance, both proteins fold in a strict N-to-C-terminal fashion, accruing structures in units of helical hairpins. These common features suggest that integral helical membrane proteins have evolved to maximize their fitness with cotranslational folding.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Endopeptidases/fisiologia , Proteínas de Escherichia coli/fisiologia , Proteínas de Membrana/fisiologia , Dobramento de Proteína , Receptores Adrenérgicos beta 2/fisiologia , Evolução Biológica , Escherichia coli/metabolismo , Humanos , Modelos Moleculares , Conformação Proteica , Modificação Traducional de Proteínas , Imagem Individual de MoléculaRESUMO
In the original version of this Article, the genotype of the M30 mutant presented in Fig. 3b was given incorrectly as Y288V/A232S, and the M31 mutant was given incorrectly as M1/A232S. The correct genotype of the M30 mutant is Y288A/A232S and for M31 it is Y288V/A232S. In addition, to keep consistency in genotype formatting, the genotype of the M27 mutant should be Y288V/G286S. The errors have been corrected in both the PDF and HTML versions of the Article.
RESUMO
Prenylation of natural compounds adds structural diversity, alters biological activity, and enhances therapeutic potential. Because prenylated compounds often have a low natural abundance, alternative production methods are needed. Metabolic engineering enables natural product biosynthesis from inexpensive biomass, but is limited by the complexity of secondary metabolite pathways, intermediate and product toxicities, and substrate accessibility. Alternatively, enzyme catalyzed prenyl transfer provides excellent regio- and stereo-specificity, but requires expensive isoprenyl pyrophosphate substrates. Here we develop a flexible cell-free enzymatic prenylating system that generates isoprenyl pyrophosphate substrates from glucose to prenylate an array of natural products. The system provides an efficient route to cannabinoid precursors cannabigerolic acid (CBGA) and cannabigerovarinic acid (CBGVA) at >1 g/L, and a single enzymatic step converts the precursors into cannabidiolic acid (CBDA) and cannabidivarinic acid (CBDVA). Cell-free methods may provide a powerful alternative to metabolic engineering for chemicals that are hard to produce in living organisms.
Assuntos
Produtos Biológicos/metabolismo , Canabinoides/metabolismo , Proteínas Fúngicas/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Engenharia Metabólica/métodos , Estrutura Molecular , Prenilação/fisiologia , Especificidade por SubstratoRESUMO
ClC chloride channels and transporters are important for chloride homeostasis in species from bacteria to human. Mutations in ClC proteins cause genetically inherited diseases, some of which are likely to involve folding defects. The ClC proteins present a challenging and unusual biological folding problem because they are large membrane proteins possessing a complex architecture, with many reentrant helices that go only partway through membrane and loop back out. Here we were able to examine the unfolding of the Escherichia coli ClC transporter, ClC-ec1, using single-molecule forced unfolding methods. We found that the protein could be separated into two stable halves that unfolded independently. The independence of the two domains is consistent with an evolutionary model in which the two halves arose from independently folding subunits that later fused together. Maintaining smaller folding domains of lesser complexity within large membrane proteins may be an advantageous strategy to avoid misfolding traps.
Assuntos
Canais de Cloreto/química , Cloretos/química , Escherichia coli/química , DNA/química , Dimiristoilfosfatidilcolina/química , Escherichia coli/genética , Proteínas de Escherichia coli/química , Evolução Molecular , Humanos , Proteínas de Membrana Transportadoras/química , Simulação de Dinâmica Molecular , Mutação , Plasmídeos , Desnaturação Proteica , Domínios Proteicos , Dobramento de Proteína , Multimerização Proteica , Estrutura Secundária de ProteínaRESUMO
The computational design of transmembrane proteins with more than one membrane-spanning region remains a major challenge. We report the design of transmembrane monomers, homodimers, trimers, and tetramers with 76 to 215 residue subunits containing two to four membrane-spanning regions and up to 860 total residues that adopt the target oligomerization state in detergent solution. The designed proteins localize to the plasma membrane in bacteria and in mammalian cells, and magnetic tweezer unfolding experiments in the membrane indicate that they are very stable. Crystal structures of the designed dimer and tetramer-a rocket-shaped structure with a wide cytoplasmic base that funnels into eight transmembrane helices-are very close to the design models. Our results pave the way for the design of multispan membrane proteins with new functions.
Assuntos
Proteínas de Membrana/química , Engenharia de Proteínas/métodos , Bioengenharia , Simulação por Computador , Cristalografia por Raios X , Citoplasma/metabolismo , Detergentes , Células HEK293 , Humanos , Proteínas de Membrana/metabolismo , Modelos Químicos , Dobramento de Proteína , Multimerização Proteica , Estabilidade Proteica , Estrutura Secundária de Proteína , Desdobramento de ProteínaRESUMO
Head-to-tail polymers of sterile alpha motifs (SAM) can scaffold large macromolecular complexes. Several SAM-domain proteins that bind each other are mutated in patients with cystic kidneys or laterality defects, including the Ankyrin (ANK) and SAM domain-containing proteins ANKS6 and ANKS3, and the RNA-binding protein Bicc1. To address how their interactions are regulated, we first determined a high-resolution crystal structure of a Bicc1-SAM polymer, revealing a canonical SAM polymer with a high degree of flexibility in the subunit interface orientations. We further mapped interactions between full-length and distinct domains of Bicc1, ANKS3, and ANKS6. Neither ANKS3 nor ANKS6 alone formed macroscopic homopolymers in vivo. However, ANKS3 recruited ANKS6 to Bicc1, and the three proteins together cooperatively generated giant macromolecular complexes. Thus, the giant assemblies are shaped by SAM domains, their flanking sequences, and SAM-independent protein-protein and protein-mRNA interactions.
Assuntos
Proteínas de Transporte/química , Ciliopatias/metabolismo , Proteínas Nucleares/química , Proteínas de Ligação a RNA/química , Cristalografia por Raios X , Células HEK293 , Células HeLa , Humanos , Polímeros , Conformação Proteica , Motivo Estéril alfaRESUMO
Protein folding is a fundamental life process with many implications throughout biology and medicine. Consequently, there have been enormous efforts to understand how proteins fold. Almost all of this effort has focused on water-soluble proteins, however, leaving membrane proteins largely wandering in the wilderness. The neglect has occurred not because membrane proteins are unimportant but rather because they present many theoretical and technical complications. Indeed, quantitative membrane protein folding studies are generally restricted to a handful of well-behaved proteins. Single-molecule methods may greatly alter this picture, however, because the ability to work at or near infinite dilution removes aggregation problems, one of the main technical challenges of membrane protein folding studies.
Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Espectrometria de Massas/métodos , Proteínas de Membrana/química , Microscopia de Força Atômica/métodos , Dobramento de Proteína , Imagem Individual de Molécula/métodos , Animais , Humanos , Lipídeos de Membrana/química , Proteínas de Membrana/isolamento & purificaçãoRESUMO
Synthetic biochemistry seeks to engineer complex metabolic pathways for chemical conversions outside the constraints of the cell. Establishment of effective and flexible cell-free systems requires the development of simple systems to replace the intricate regulatory mechanisms that exist in cells for maintaining high-energy cofactor balance. Here we describe a simple rheostat that regulates ATP levels by controlling the flow down either an ATP-generating or non-ATP-generating pathway according to the free-phosphate concentration. We implemented this concept for the production of isobutanol from glucose. The rheostat maintains adequate ATP concentrations even in the presence of ATPase contamination. The final system including the rheostat produced 24.1 ± 1.8 g/L of isobutanol from glucose in 91% theoretical yield with an initial productivity of 1.3 g/L/h. The molecular rheostat concept can be used in the design of continuously operating, self-sustaining synthetic biochemistry systems.
Assuntos
Trifosfato de Adenosina/metabolismo , Butanóis/metabolismo , Engenharia Metabólica , Sistema Livre de Células , Modelos Moleculares , Transdução de SinaisRESUMO
Although backbone hydrogen bonds in transmembrane (TM) helices have the potential to be very strong due to the low dielectric and low water environment of the membrane, their strength has never been assessed experimentally. Moreover, variations in hydrogen bond strength might be necessary to facilitate the TM helix breaking and bending that is often needed to satisfy functional imperatives. Here we employed equilibrium hydrogen/deuterium fractionation factors to measure backbone hydrogen bond strengths in the TM helix of the amyloid precursor protein (APP). We find an enormous range of hydrogen bond free energies, with some weaker than water-water hydrogen bonds and some over 6 kcal/mol stronger than water-water hydrogen bonds. We find that weak hydrogen bonds are at or near preferred γ-secretase cleavage sites, suggesting that the sequence of APP and possibly other cleaved TM helices may be designed, in part, to make their backbones accessible for cleavage. The finding that hydrogen bond strengths in a TM helix can vary widely has implications for membrane protein function, dynamics, evolution, and design.
Assuntos
Proteínas de Membrana/química , Ligação de Hidrogênio , Ressonância Magnética Nuclear Biomolecular , Espectrofotometria Ultravioleta , TermodinâmicaRESUMO
Cell-free systems designed to perform complex chemical conversions of biomass to biofuels or commodity chemicals are emerging as promising alternatives to the metabolic engineering of living cells. Here we design a system comprises 27 enzymes for the conversion of glucose into monoterpenes that generates both NAD(P)H and ATP in a modified glucose breakdown module and utilizes both cofactors for building terpenes. Different monoterpenes are produced in our system by changing the terpene synthase enzyme. The system is stable for the production of limonene, pinene and sabinene, and can operate continuously for at least 5 days from a single addition of glucose. We obtain conversion yields >95% and titres >15 g l-1. The titres are an order of magnitude over cellular toxicity limits and thus difficult to achieve using cell-based systems. Overall, these results highlight the potential of synthetic biochemistry approaches for producing bio-based chemicals.
Assuntos
Alquil e Aril Transferases/metabolismo , Glucose/metabolismo , Monoterpenos/metabolismo , Bioquímica/métodos , Vias Biossintéticas , Sistema Livre de Células/metabolismo , Biologia Sintética/métodosRESUMO
The topology of helical membrane proteins is generally defined during insertion of the transmembrane helices, yet it is now clear that it is possible for topology to change under unusual circumstances. It remains unclear, however, if topology reorientation is part of normal biogenesis. For dual topology dimer proteins such as the multidrug transporter EmrE, there may be evolutionary pressure to allow topology flipping so that the populations of both orientations can be equalized. We previously demonstrated that when EmrE is forced to insert in a distorted topology, topology flipping of the first transmembrane helix can occur during translation. Here, we show that topological malleability also extends to the C-terminal helix and that even complete topology inversion of the entire EmrE protein can occur after the full protein is translated and inserted. Thus, topology rearrangements are possible during normal biogenesis. Wholesale topology flipping is remarkable given the physical constraints of the membrane and expands the range of possible membrane protein folding pathways, both productive and detrimental.
Assuntos
Proteínas de Transporte/química , Membrana Celular/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Proteínas de Membrana/química , Dobramento de Proteína , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Membrana Celular/genética , Membrana Celular/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Estrutura Secundária de ProteínaRESUMO
Extreme acidophiles are capable of growth at pH values near zero. Sustaining life in acidic environments requires extensive adaptations of membranes, proton pumps, and DNA repair mechanisms. Here we describe an adaptation of a core biochemical pathway, the mevalonate pathway, in extreme acidophiles. Two previously known mevalonate pathways involve ATP dependent decarboxylation of either mevalonate 5-phosphate or mevalonate 5-pyrophosphate, in which a single enzyme carries out two essential steps: (1) phosphorylation of the mevalonate moiety at the 3-OH position and (2) subsequent decarboxylation. We now demonstrate that in extreme acidophiles, decarboxylation is carried out by two separate steps: previously identified enzymes generate mevalonate 3,5-bisphosphate and a new decarboxylase we describe here, mevalonate 3,5-bisphosphate decarboxylase, produces isopentenyl phosphate. Why use two enzymes in acidophiles when one enzyme provides both functionalities in all other organisms examined to date? We find that at low pH, the dual function enzyme, mevalonate 5-phosphate decarboxylase is unable to carry out the first phosphorylation step, yet retains its ability to perform decarboxylation. We therefore propose that extreme acidophiles had to replace the dual-purpose enzyme with two specialized enzymes to efficiently produce isoprenoids in extremely acidic environments.