RESUMEN
For the last two decades, synthetic biologists have been able to unlock and expand the genetic code, generating proteins with unique properties through the incorporation of noncanonical amino acids (ncAAs). These evolved biomaterials have shown great potential for applications in industrial biocatalysis, therapeutics, bioremediation, bioconjugation, and other areas. Our ability to continue developing such technologies depends on having relatively easy access to ncAAs. However, the synthesis of enantiomerically pure ncAAs in practical quantitates for large-scale processes remains a challenge. Biocatalytic ncAA production has emerged as an excellent alternative to traditional organic synthesis in terms of cost, enantioselectivity, and sustainability. Moreover, biocatalytic synthesis offers the opportunity of coupling the intracellular generation of ncAAs with genetic-code expansion to overcome the limitations of an external supply of amino acid. In this minireview, we examine some of the most relevant achievements of this approach and its implications for improving technological applications derived from synthetic biology.
Asunto(s)
Aminoácidos/síntesis química , Biotecnología/métodos , Código Genético , Biosíntesis de Proteínas , Proteínas/química , Biología Sintética , BiocatálisisRESUMEN
Electrostatic interactions are essential for controlling the protein structure and function. Whereas so far experimental and theoretical efforts focused on the effect of local electrostatics, this work aims at elucidating the long-range modulation of electric fields in proteins upon binding to charged surfaces. The study is based on cytochrome c (Cytc) variants carrying nitrile reporters for the vibrational Stark effect that are incorporated into the protein via genetic engineering and chemical modification. The Cytc variants were thoroughly characterized with respect to possible structural perturbations due to labeling. For the proteins in solution, the relative hydrogen bond occupancy and the calculated electric fields, both obtained from molecular dynamics (MD) simulations, and the experimental nitrile stretching frequencies were used to develop a relationship for separating hydrogen-bonding and non-hydrogen-bonding electric field effects. This relationship provides an excellent description for the stable Cytc variants in solution. For the proteins bound to Au electrodes coated with charged self-assembled monolayers (SAMs), the underlying MD simulations can only account for the electric field changes Δ Eads due to the formation of the electrostatic SAM-Cytc complexes but not for the additional contribution, Δ Eint, representing the consequences of the potential drops over the electrode/SAM/protein interfaces. Both Δ Eads and Δ Eint, determined at distances between 20 and 30 Å with respect to the SAM surface, are comparable in magnitude to the non-hydrogen-bonding electric field in the unbound protein. This long-range modulation of the internal electric field may be of functional relevance for proteins in complexes with partner proteins (Δ Eads) and attached to membranes (Δ Eads + Δ Eint).
Asunto(s)
Citocromos c/química , Campos Electromagnéticos , Animales , Citocromos c/genética , Técnicas Electroquímicas , Electrodos , Oro/química , Caballos , Enlace de Hidrógeno , Proteínas Inmovilizadas/química , Proteínas Inmovilizadas/genética , Simulación de Dinámica Molecular , Mutación , Nitrilos/química , Electricidad EstáticaRESUMEN
Global substitution of canonical amino acids (cAAs) with noncanonical (ncAAs) counterparts in proteins whose function is dependent on post-translational events such as cofactor binding is still a methodically challenging and difficult task as ncAA insertion generally interferes with the cofactor biosynthesis machinery. Here, we report a technology for the expression of fully substituted and functionally active cofactor-containing hemeproteins. The maturation process which yields an intact cofactor is timely separated from cAAâncAA substitutions. This is achieved by an optimised expression and fermentation procedure which includes pre-induction of the heme cofactor biosynthesis followed by an incorporation experiment at multiple positions in the protein sequence. This simple strategy can be potentially applied for engineering of other cofactor-containing enzymes.
Asunto(s)
Sustitución de Aminoácidos , Escherichia coli/genética , Escherichia coli/metabolismo , Hemoproteínas/biosíntesis , Hemoproteínas/genética , Secuencia de Aminoácidos , Fermentación , Hemo/biosíntesis , Hemoproteínas/química , Ingeniería de Proteínas/métodos , Modificación Traduccional de las Proteínas , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMEN
Fluorine being not substantially present in the chemistry of living beings is an attractive element in tailoring novel chemical, biophysical, and pharmacokinetic properties of peptides and proteins. The hallmark of ribosome-mediated artificial amino acid incorporation into peptides and proteins is a broad substrate tolerance, which is assumed to rely on the absence of evolutionary pressure for efficient editing of artificial amino acids. We used the well-characterized editing proficient isoleucyl-tRNA synthetase (IleRS) from Escherichia coli to investigate the crosstalk of aminoacylation and editing activities against fluorinated amino acids. We show that translation of trifluoroethylglycine (TfeGly) into proteins is prevented by hydrolysis of TfeGly-tRNAIle in the IleRS post-transfer editing domain. The remarkable observation is that dissociation of TfeGly-tRNAIle from IleRS is significantly slowed down. This finding is in sharp contrast to natural editing reactions by tRNA synthetases wherein fast editing rates for the noncognate substrates are essential to outcompete fast aa-tRNA dissociation rates. Using a post-transfer editing deficient mutant of IleRS (IleRSAla10), we were able to achieve ribosomal incorporation of TfeGly in vivo. Our work expands the knowledge of ribosome-mediated artificial amino acid translation with detailed analysis of natural editing function against an artificial amino acid providing an impulse for further systematic investigations and engineering of the translation and editing of unusual amino acids.
RESUMEN
BACKGROUND: To find experimental validation for electrostatic interactions essential for catalytic reactions represents a challenge due to practical limitations in assessing electric fields within protein structures. SCOPE OF REVIEW: This review examines the applications of non-canonical amino acids (ncAAs) as genetically encoded probes for studying the role of electrostatic interactions in enzyme catalysis. MAJOR CONCLUSIONS: ncAAs constitute sensitive spectroscopic probes to detect local electric fields by exploiting the vibrational Stark effect (VSE) and thus have the potential to map the protein electrostatics. GENERAL SIGNIFICANCE: Mapping the electrostatics in proteins will improve our understanding of natural catalytic processes and, in beyond, will be helpful for biocatalyst engineering. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue.
Asunto(s)
Código Genético , Proteínas/química , Análisis Espectral/métodos , Electricidad Estática , Vibración , Catálisis , Simulación de Dinámica Molecular , Ingeniería de Proteínas/métodos , Proteínas/genética , Proteínas/metabolismoRESUMEN
Cytochrome c (cyt c), a redox protein involved in diverse fundamental biological processes, is among the most traditional model proteins for analyzing biological electron transfer and protein dynamics both in solution and at membranes. Studying the role of electric fields in energy transduction mediated by cyt c relies upon appropriate reporter groups. Up to now these had to be introduced into cyt c by in vitro chemical modification. Here, we have overcome this restriction by incorporating the noncanonical amino acid p-cyanophenylalanine (pCNF) into cyt c in vivo. UV and CD spectroscopy indicate preservation of the overall protein fold, stability, and heme coordination, whereas a small shift of the redox potential was observed by cyclic voltammetry. The C≡N stretching mode of the incorporated pCNF detected in the IR spectra reveals a surprising difference, which is related to the oxidation state of the heme iron, thus indicating high sensitivity to changes in the electrostatics of cyt c.
Asunto(s)
Citocromos c/química , Electricidad Estática , Alanina/análogos & derivados , Alanina/química , Alanina/metabolismo , Citocromos c/metabolismo , Transporte de Electrón , Modelos Moleculares , Nitrilos/química , Nitrilos/metabolismoRESUMEN
In this paper, we present a novel, "single experiment" methodology based on genetic engineering of metabolic pathways for direct intracellular production of non-canonical amino acids from simple precursors, coupled with expanded genetic code. In particular, we engineered the intracellular biosynthesis of L-azidohomoalanine from O-acetyl-L-homoserine and NaN3, and achieved its direct incorporation into recombinant target proteins by AUG codon reassignment in a methionine-auxotroph E. coli strain. In our system, the host's methionine biosynthetic pathway was first diverted towards the production of the desired non-canonical amino acid by exploiting the broad reaction specificity of recombinant pyridoxal phosphate-dependent O-acetylhomoserine sulfhydrylase from Corynebacterium glutamicum. Then, the expression of the target protein barstar, accompanied with efficient L-azidohomoalanine incorporation in place of L-methionine, was accomplished. This work stands as proof-of-principle and paves the way for additional work towards intracellular production and site-specific incorporation of biotechnologically relevant non-canonical amino acids directly from common fermentable sources.
Asunto(s)
Alanina/análogos & derivados , Proteínas Bacterianas/biosíntesis , Alanina/biosíntesis , Química Clic , Escherichia coli , Fermentación , Ingeniería Genética , Redes y Vías Metabólicas , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes/biosíntesis , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
New diMn(III) complexes of general formula [Mn(2)L(mu-OR)(mu-OAc)]BPh(4) (H(3)L = 1,5-bis[(2-hydroxy-5-X-benzyl)(2-pyridylmethyl)amino]pentan-3-ol, 1: X = H, R = Me, 2: X = OMe, R = Me, 3: X = Br, R = Me, 4: X = Br, R = Et) have been prepared and structurally characterized. The synthesized complexes possess a triply bridged (mu-alkoxo)(2)(mu-acetato)Mn(2)(3+) core, a short intermetallic distance of 2.95/6 A modulated by the aliphatic spacers between the central alcoholato and N-amino donor sites, and the remaining coordination sites of the two Mn(III) centers occupied by the six donor atoms of the polydentate ligand. In dimethylformamide, complexes 1-3 are able to disproportionate more than 1500 equiv of H(2)O(2) without significant decomposition, with first-order dependence on catalyst and saturation kinetic on [H(2)O(2)]. Spectroscopic monitoring of the reaction mixtures revealed that the catalyst converts into [Mn(2)(III)(mu-O)(mu-OAc)L], which is the major active form during cycling. Overall, kinetics and spectroscopic studies of H(2)O(2) dismutation by these complexes converge at a catalytic cycle between Mn(III)(2) and Mn(II)(2) oxidation levels. Comparison to other alkoxo-bridged complexes suggests that the binding mode of peroxide to the metal center of the Mn(III)(2) form of the catalyst is a key factor for tuning the Mn oxidation states involved in the H(2)O(2) dismutation mechanism.
Asunto(s)
Manganeso/química , Compuestos Organometálicos/química , Compuestos Organometálicos/síntesis química , Pentanoles/química , Piridinas/química , Catalasa/metabolismo , Cristalografía por Rayos X , Electroquímica , Cinética , Ligandos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Espectrometría de Masa por Ionización de Electrospray , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
Two new diMn(III) complexes [Mn(2)(III)L(1)(mu-AcO)(mu-MeO)(methanol)(2)]Br (1) and [Mn(2)(III)L(2)(mu-AcO)(mu-MeO)(methanol)(ClO(4))] (2) (L(1)H(3)=1,5-bis(2-hydroxybenzophenylideneamino)pentan-3-ol; L(2)H(3)=1,5-bis(2-hydroxynaphtylideneamino)pentan-3-ol) were synthesized and structurally characterized. Structural studies evidence that these complexes have a bis(mu-alkoxo)(mu-carboxylato) triply bridged diMn(III) core in the solid state and in solution, with two substitution-labile sites--one on each Mn ion--in cis-position. The two complexes show catalytic activity toward disproportionation of H(2)O(2), with saturation kinetics on [H(2)O(2)], in methanol and dimethyl formamide at 25 degrees C. Spectroscopic monitoring of the H(2)O(2) disproportionation reaction suggests that (i) complexes 1 and 2 dismutate H(2)O(2) by a mechanism involving redox cycling between Mn(2)(III) and Mn(2)(IV), (ii) the complexes retain the dinuclearity during catalysis, (iii) the active form of the catalyst contains bound acetate, and (iv) protons favors the formation of inactive Mn(II) species. Comparison to other dimanganese complexes of the same family shows that the rate of catalase reaction is not critically dependent on the redox potential of the catalyst, that substitution of phenolate by naphtolate in the Schiff base ligand favors formation of the catalyst-substrate adduct, and that, in the non-protic solvent, the bulkier substituent at the imine proton position hampers the binding to the substrate.
Asunto(s)
Catalasa/química , Peróxido de Hidrógeno/química , Compuestos de Manganeso/química , Manganeso/química , Catalasa/metabolismo , Química Inorgánica/métodos , Electroquímica/métodos , Espectroscopía de Resonancia por Spin del Electrón , Peróxido de Hidrógeno/metabolismo , Cinética , Ligandos , Espectroscopía de Resonancia Magnética , Magnetismo , Manganeso/metabolismo , Compuestos de Manganeso/metabolismo , Metanol , Estructura Molecular , Bases de Schiff , Espectrometría de Masa por Ionización de Electrospray , Espectrofotometría InfrarrojaRESUMEN
The dimanganese(III,III) complexes [Mn(2)(III)(5-NO(2)-salpentO)(mu-AcO)(mu-MeO)(methanol)(2)]Y (1: Y=Br, 2a: Y=I, 2b: Y=I(3)), [Mn(2)(III)(5-NO(2)-salpentO)(mu-AcO)(mu-MeO)(methanol)(ClO(4))] (3) and [Mn(2)(III)(5-Cl-salpentO)(mu-AcO)(mu-MeO)(methanol)(2)]Br (4), where salpentOH is the symmetrical Schiff base ligand 1,5-bis(salicylidenamino)pentan-3-ol, were synthesised and structurally characterized. Complex 2b crystallises in the monoclinic system, space group P2(1)/c, and exhibits Mn. . .Mn separation of 2.911 A. This Mn. . .Mn separation is very close to the other characterized (mu-alkoxo)(2)(mu-acetato)Mn(2)(III) complexes of X-salpentOH (X=MeO, Br and H) and reveals that the aromatic substituent has little influence on the geometric parameters of the bimetallic core. A correlation between the electronic character of the different ring substituents, the redox potentials of the dinuclear complexes and their catalase activity was evidenced. Complexes 1-4 show saturation kinetics with [H(2)O(2)] and the H(2)O(2) disproportionation involves redox cycling between the Mn(2)(III)/Mn(2)(IV) levels. The catalytic activity studies show that bound acetate is required for catalase activity and that the acetato and alkoxo bridges serve as internal bases facilitating the proton transfer coupled to oxidation of the metal centre.