RESUMEN
Protein glycosylation is important in many organisms for proper protein folding, signaling, cell adhesion, protein-protein interactions, and immune responses. Thus, effectively determining the extent of glycosylation in glycoprotein therapeutics is crucial. Up to now, characterizing protein glycosylation has been carried out mostly by liquid chromatography mass spectrometry (LC-MS), which requires careful sample processing, e.g., glycan removal or protein digestion and glycopeptide enrichment. Herein, we introduce an NMR-based method to better characterize intact glycoproteins in natural abundance. This non-destructive method relies on exploiting differences in nuclear relaxation to suppress the NMR signals of the protein while maintaining glycan signals. Using RNase B Man5 and RNase B Man9, we establish reference spectra that can be used to determine the different glycoforms present in heterogeneously glycosylated commercial RNase B.
Asunto(s)
Glicoproteínas/química , Manosa/química , Resonancia Magnética Nuclear Biomolecular , Ribonucleasas/química , GlicosilaciónRESUMEN
Therapeutic monoclonal antibodies (mAbs) are largely based on the immunoglobulin G1 (IgG1) scaffold, and many elicit a cytotoxic cell-mediated response by binding Fc γ receptors. Core fucosylation, a prevalent modification to the asparagine (N)-linked carbohydrate on the IgG1 crystallizable fragment (Fc), decreases the Fc γ receptor IIIa (CD16a) binding affinity and mAb efficacy. We determined IgG1 Fc fucosylation reduced the CD16a affinity by 1.7 ± 0.1 kcal/mol when compared to that of afucosylated IgG1 Fc; however, CD16a N-glycan truncation decreased this penalty by 1.2 ± 0.1 kcal/mol or 70%. Fc fucosylation restricted the manifold of conformations sampled by displacing the CD16a Asn162-glycan that impinges upon the linkage between the α-mannose(1-6)ß-mannose residues and promoted contacts with the IgG Tyr296 residue. Fucosylation also impacted the IgG1 Fc structure as indicated by changes in resonance frequencies and nuclear spin relaxation observed by solution nuclear magnetic resonance spectroscopy. The effects of fucosylation on IgG1 Fc may account for the remaining 0.5 ± 0.1 kcal/mol penalty of fucosylated IgG1 Fc binding CD16a when compared to that of afucosylated IgG1 Fc. Our results indicated the CD16a Asn162-glycan modulates the antibody affinity indirectly by reducing the volume sampled, as opposed to a direct mechanism with intermolecular glycan-glycan contacts previously proposed to stabilize this system. Thus, antibody engineering to enhance intermolecular glycan-glycan contacts will likely provide limited improvement, and future designs should maximize the affinity by maintaining the CD16a Asn162-glycan conformational heterogeneity.
Asunto(s)
Afinidad de Anticuerpos , Fucosa/inmunología , Fragmentos Fc de Inmunoglobulinas/inmunología , Inmunoglobulina G/inmunología , Receptores de IgG/inmunología , Conformación de Carbohidratos , Cristalografía por Rayos X , Fucosa/análisis , Glicosilación , Células HEK293 , Humanos , Fragmentos Fc de Inmunoglobulinas/química , Inmunoglobulina G/química , Simulación del Acoplamiento Molecular , Polisacáridos/análisis , Polisacáridos/inmunología , Receptores de IgG/químicaRESUMEN
This review will cover the structure, enzymology, and related aspects that are important for structure-based engineering of the transacylase enzymes from fatty acid biosynthesis and polyketide synthesis. Furthermore, this review will focus on in vitro characteristics and not cover engineering of the upstream or downstream reactions or strategies to manipulate metabolic flux in vivo. The malonyl-coenzyme A(CoA)-holo-acyl-carrier protein (holo-ACP) transacylase (FabD) from Escherichia coli serves as a model for this enzyme with thorough descriptions of structure, enzyme mechanism, and effects of mutation on substrate binding presented in the literature. Here, we discuss multiple practical and theoretical considerations regarding engineering transacylase enzymes to accept non-cognate substrates and form novel acyl-ACPs for downstream reactions.
Asunto(s)
Acilcoenzima A/metabolismo , S-Maloniltransferasa de la Proteína Transportadora de Grupos Acilo/genética , S-Maloniltransferasa de la Proteína Transportadora de Grupos Acilo/metabolismo , Bioingeniería/tendencias , Microbiología Industrial/métodos , Acilcoenzima A/genética , S-Maloniltransferasa de la Proteína Transportadora de Grupos Acilo/química , Aciltransferasas/química , Aciltransferasas/genética , Aciltransferasas/metabolismo , Microbiología Industrial/tendenciasRESUMEN
The Escherichia coli holo-(acyl carrier protein) synthase (ACPS) catalyzes the coenzyme A-dependent activation of apo-ACPP to generate holo-(acyl carrier protein) (holo-ACPP) in an early step of fatty acid biosynthesis. E. coli ACPS is sufficiently different from the human fatty acid synthase to justify the development of novel ACPS-targeting antibiotics. Models of E. coli ACPS in unliganded and holo-ACPP-bound forms solved by X-ray crystallography to 2.05and 4.10Å, respectively, revealed that ACPS bound three product holo-ACPP molecules to form a 3:3 hexamer. Solution NMR spectroscopy experiments validated the ACPS binding interface on holo-ACPP using chemical shift perturbations and by determining the relative orientation of holo-ACPP to ACPS by fitting residual dipolar couplings. The binding interface is organized to arrange contacts between positively charged ACPS residues and the holo-ACPP phosphopantetheine moiety, indicating product contains more stabilizing interactions than expected in the enzyme:substrate complex. Indeed, holo-ACPP bound the enzyme with greater affinity than the substrate, apo-ACPP, and with negative cooperativity. The first equivalent of holo-ACPP bound with a KD=62±13nM, followed by the binding of two more equivalents of holo-ACPP with KD=1.2±0.2µM. Cooperativity was not observed for apo-ACPP which bound with KD=2.4±0.1µM. Strong product binding and high levels of holo-ACPP in the cell identify a potential regulatory role of ACPS in fatty acid biosynthesis.
Asunto(s)
Proteína Transportadora de Acilo/química , Proteína Transportadora de Acilo/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimología , Acido Graso Sintasa Tipo II/química , Acido Graso Sintasa Tipo II/metabolismo , Transferasas/química , Transferasas/metabolismo , Clonación Molecular , Cristalografía por Rayos X , Escherichia coli/crecimiento & desarrollo , Modelos Moleculares , Unión Proteica , Conformación ProteicaRESUMEN
The commercial impact of fermentation systems producing novel and biorenewable chemicals will flourish with the expansion of enzymes engineered to synthesize new molecules. Though a small degree of natural variability exists in fatty acid biosynthesis, the molecular space accessible through enzyme engineering is fundamentally limitless. Prokaryotic fatty acid biosynthesis enzymes build carbon chains on a functionalized acyl carrier protein (ACP) that provides solubility, stability, and a scaffold for interactions with the synthetic enzymes. Here, we identify the malonyl-coenzyme A (CoA)/holo-ACP transacylase (FabD) from Escherichia coli as a platform enzyme for engineering to diversify microbial fatty acid biosynthesis. The FabD R117A variant produced novel ACP-based primer and extender units for fatty acid biosynthesis. Unlike the wild-type enzyme that is highly specific for malonyl-CoA to produce malonyl-ACP, the R117A variant synthesized acetyl-ACP, succinyl-ACP, isobutyryl-ACP, 2-butenoyl-ACP, and ß-hydroxybutyryl-ACP among others from holo-ACP and the corresponding acyl-CoAs with specific activities from 3.7 to 120 nmol min-1 mg-1. FabD R117A maintained K M values for holo-ACP (~ 40 µM) and displayed small changes in K M for acetoacetyl-CoA (110 ± 30 µM) and acetyl-CoA (200 ± 70 µM) when compared to malonyl-CoA (80 ± 30 µM). FabD R117A represents a novel catalyst that synthesizes a broad range of acyl-acyl-ACPs.
Asunto(s)
Proteína Transportadora de Acilo/biosíntesis , S-Maloniltransferasa de la Proteína Transportadora de Grupos Acilo/genética , S-Maloniltransferasa de la Proteína Transportadora de Grupos Acilo/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimología , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutación Missense , Acido Graso Sintasa Tipo II/genética , Acido Graso Sintasa Tipo II/metabolismo , Ácidos Grasos/biosíntesis , CinéticaRESUMEN
The development of biorenewable chemicals will support green chemistry initiatives and supplement the catalog of starting materials available to the chemical industry. Bacterial fatty acid biosynthesis is being pursued as a source of protein catalysts to synthesize novel reduced carbon molecules in fermentation systems. The availability of methods to measure enzyme catalysis for native and engineered enzymes from this pathway remains a bottleneck because a simple quantitative enzyme assay for numerous enzymes does not exist. Here we present two variations of a fluorescence assay that is readily extendable to high-throughput screening and is appropriate for thiol consuming and generating enzymes including the Escherichia coli enzymes malonyl-coenzyme A transacylase (FabD) and keto-acylsynthase III (FabH). We measured KM values of 60 ± 20 µM (acetyl-CoA) and 20 ± 10 µM (malonyl-ACP) and a kcat of 7.4-9.0 s-1 with FabH. Assays of FabD included a precipitation step to remove the thiol-containing substrate holo-ACP from the reaction product coenzyme-A to estimate reaction rates. Analysis of initial velocity measurements revealed KM values of 60 ± 20 µM (malonyl-CoA) and 40 ± 10 µM (holo-ACP) and a kcat of 2100-2600 s-1for the FabD enzyme. Our data show similar results when compared to existing radioactive and continuous coupled assays in terms of sensitivity while providing the benefit of simplicity, scalability and repeatability. Fluorescence detection also eliminates the need for radioactive substrates traditionally used to assay these enzymes.
RESUMEN
The fatty acid biosynthetic pathway generates highly reduced carbon based molecules. For this reason fatty acid synthesis is a target of pathway engineering to produce novel specialty or commodity chemicals using renewable techniques to supplant molecules currently derived from petroleum. Malonyl-[acyl carrier protein] (malonyl-ACP) is a key metabolite in the fatty acid pathway and donates two carbon units to the growing fatty acid chain during each step of biosynthesis. Attempts to test engineered fatty acid biosynthesis enzymes in vitro will require malonyl-ACP or malonyl-ACP analogs. Malonyl-ACP is challenging to prepare due to the instability of the carboxylate leaving group and the multiple steps of post-translational modification required to activate ACP. Here we report the expression and purification of holo- and malonyl-ACP from Escherichia coli with high yields (>15 mg per L of expression). The malonyl-ACP is efficiently recognized by the E. coli keto-acyl synthase enzyme, FabH. A FabH assay using malonyl-ACP and a coumarin-based fluorescent reagent is described that provides a high throughput alternative to reported radioactive assays.