Real-time detection of enzymatically formed hydrogen sulfide by pathogenic variants of cystathionine beta-synthase using hemoglobin I of Lucina pectinata as a biosensor.

Classical homocystinuria is a rare disease caused by mutations in cystathionine ß-synthase (CBS) gene (OMIM 613381). CBS catalyzes the first step of the transsulfuration pathway that converts homocysteine (Hcy) into cystathionine (Cysta) via a number of co-substrates and mechanisms. Formation of Cysta by condensation of Hcy and cysteine (Cys) produces a molar equivalent of hydrogen sulfide (H2S). H2S plays important roles in cognitive and vascular functions. Clinically, patients with CBS deficiency present with vascular, ocular, neurological and skeletal impairments. Biochemically, CBS deficiency manifests with elevated Hcy and reduced concentration of Cysta in plasma and urine. A number of pathogenic variants of human CBS have been characterized by their residual enzymatic activity, but very few studies have examined H2S production by pathogenic CBS variants, possibly due to technical hurdles in H2S detection and quantification. We describe a method for the real-time, continuous quantification of H2S formed by wild-type and pathogenic variants of human recombinant CBS, as well as by fibroblast extracts from healthy controls and patients diagnosed with CBS deficiency. The method takes advantage of the specificity and high affinity of hemoglobin I of the clam Lucina pectinata toward H2S and is based on UV-visible spectrophotometry. Comparison with the gold-standard, end-point H2S quantification method employing monobromobimane, as well as correlations with CBS enzymatic activity determined by LC-MS/MS showed agreement and correlation, and permitted the direct, time-resolved determination of H2S production rates by purified human recombinant CBS and by CBS present in fibroblast extracts. Rates of H2S production were highest for wild-type CBS, and lower for pathogenic variants. This method enables the examination of structural determinants of CBS that are important for H2S production and its possible relevance to the clinical outcome of patients.

Metabolism-Guided Optimization of Tryptophanol-Derived Isoindolinone p53 Activators.

For the first time, the pharmacokinetic (PK) profile of tryptophanol-derived isoindolinones, previously reported as p53 activators, was investigated. From the metabolites' identification, performed by liquid chromatography coupled to high resolution tandem mass spectrometry (LC-HRMS/MS), followed by their preparation and structural elucidation, it was possible to identify that the indole C2 and C3 are the main target of the cytochrome P450 (CYP)-promoted oxidative metabolism in the tryptophanol-derived isoindolinone scaffold. Based on these findings, to search for novel p53 activators a series of 16 enantiopure tryptophanol-derived isoindolinones substituted with a bromine in indole C2 was prepared, in yields of 62-89%, and their antiproliferative activity evaluated in human colon adenocarcinoma HCT116 cell lines with and without p53. Structural optimization led to the identification of two (S)-tryptophanol-derived isoindolinones 3.9-fold and 1.9-fold more active than hit SLMP53-1, respectively. Compounds' metabolic stability evaluation revealed that this substitution led to a metabolic switch, with the impact of Phase I oxidative metabolism being minimized. Through differential scanning fluorimetry (DSF) experiments, the most active compound of the series in cell assays led to an increase in the protein melting temperature (Tm) of 10.39 °C, suggesting an effective binding to wild-type p53 core domain.

Functional and structural impact of 10 ACADM missense mutations on human medium chain acyl-Coa dehydrogenase.

Medium chain acyl-CoA dehydrogenase (MCAD) deficiency (MCADD) is associated with ACADM gene mutations, leading to an impaired function and/or structure of MCAD. Importantly, after import into the mitochondria, MCAD must incorporate a molecule of flavin adenine dinucleotide (FAD) per subunit and assemble into tetramers. However, the effect of MCAD amino acid substitutions on FAD incorporation has not been investigated. Herein, the commonest MCAD variant (p.K304E) and 11 additional rare variants (p.Y48C, p.R55G, p.A88P, p.Y133C, p.A140T, p.D143V, p.G224R, p.L238F, p.V264I, p.Y372N, and p.G377V) were functionally and structurally characterized. Half of the studied variants presented a FAD content <65 % compared to the wild-type. Most of them were recovered as tetramers, except the p.Y372N (mainly as dimers). No correlation was found between the levels of tetramers and FAD content. However, a correlation between FAD content and the cofactor's affinity, proteolytic stability, thermostability, and thermal inactivation was established. We showed that the studied amino acid changes in MCAD may alter the substrate chain-length dependence and the interaction with electron-transferring-flavoprotein (ETF) necessary for a proper functioning electron transfer thus adding additional layers of complexity to the pathological effect of ACADM missense mutations. Although the majority of the variant MCADs presented an impaired capacity to retain FAD during their synthesis, some of them were structurally rescued by cofactor supplementation, suggesting that in the mitochondrial environment the levels and activity of those variants may be dependent of FAD's availability thus contributing for the heterogeneity of the MCADD phenotype found in patients presenting the same genotype.

Isothermal denaturation fluorimetry vs differential scanning fluorimetry as tools for screening of stabilizers for protein freeze-drying: Human phenylalanine hydroxylase as the case study.

The structural maintenance of therapeutic proteins during formulation and/or storage is a critical aspect, particularly for multi-domain and/or multimeric proteins which usually exhibit intrinsic structural dynamics leading to aggregation with concomitant loss-of-function. Protein freeze-drying is a widely used technique to preserve protein structure and function during storage. To minimize chemical/physical stresses occurring during this process, protein stabilizers are usually included, their effect being strongly dependent on the target protein. Therefore, they should be screened for on a time-consuming case-by-case basis. Herein, differential scanning fluorimetry (DSF) and isothermal denaturation fluorimetry (ITDF) were employed to screen, among different classes of freeze-drying additives, for the most effective stabilizer of the model protein human phenylalanine hydroxylase (hPAH). Correlation studies among retrieved DSF and ITDF parameters with recovered enzyme amount and activity indicated ITDF as the most appropriate screening method. Biochemical and biophysical characterization of hPAH freeze-dried with ITDF-selected stabilizers and a long-term storage study (12 months, 5 ± 3 °C) showed that the selected compounds prevented protein aggregation and preserved hPAH structural and functional properties throughout time storage. Our results provide a solid basis towards the choice of ITDF as a high-throughput screening step for the identification of protein freeze-drying protectors.

Impact of Fluorinated Ionic Liquids on Human Phenylalanine Hydroxylase-A Potential Drug Delivery System.

Phenylketonuria (PKU) is an autosomal recessive disease caused by deficient activity of human phenylalanine hydroxylase (hPAH), which can lead to neurologic impairments in untreated patients. Although some therapies are already available for PKU, these are not without drawbacks. Enzyme-replacement therapy through the delivery of functional hPAH could be a promising strategy. In this work, biophysical methods were used to evaluate the potential of [N1112(OH)][C4F9SO3], a biocompatible fluorinated ionic liquid (FIL), as a delivery system of hPAH. The results herein presented show that [N1112(OH)][C4F9SO3] spontaneously forms micelles in a solution that can encapsulate hPAH. This FIL has no significant effect on the secondary structure of hPAH and is able to increase its enzymatic activity, despite the negative impact on protein thermostability. The influence of [N1112(OH)][C4F9SO3] on the complex oligomerization equilibrium of hPAH was also assessed.

Formulation of spray dried enzymes for dry powder inhalers: An integrated methodology.

One of the main challenges when developing a spray dried formulation of an inhalable enzyme is the generation of a safe and effective aerosol, able to reach the lungs, while preserving protein function and structural levels of the biologic. Hence, an appropriate excipient selection based on enzyme stabilization, inhalation precedence and spray drying (SD) process development is required to meet this balance. Herein, an integrated methodology is presented to expedite the selection of the best dry powder inhaler excipient system to formulate three model enzymes of increasing molecular mass and structural complexity belonging to the oxidoreductase class and often implicated in oxidative stress: superoxide dismutase, glucose oxidase and catalase. Three non-reducing sugars and four amino acids were screened using High Throughput Isothermal Denaturation Fluorimetry (HT-ITDF) for a stabilizing effect on the enzymes quaternary structure. For each tested enzyme, the sugar and amino acid showcasing a stabilizing effect, were spray dried together at fixed process conditions for three different ratios, to assess which formulation would then display the best aerodynamic performance. After SD, using the selected conditions, all powders displayed 65-85% of fine particle fraction (FPF) whilst each enzyme kept the oligomeric state. The present integrated methodology proved to be successful, allowing to narrow down 36 potential formulations (three sugars × four amino acids × three ratios) to only one for each enzyme, within few hours, while requiring a µg range of sample amount.

The Mycobacteriophage Ms6 LysB N-Terminus Displays Peptidoglycan Binding Affinity.

Double-stranded DNA bacteriophages end their lytic cycle by disrupting the host cell envelope, which allows the release of the virion progeny. Each phage must synthesize lysis proteins that target each cell barrier to phage release. In addition to holins, which permeabilize the cytoplasmic membrane, and endolysins, which disrupt the peptidoglycan (PG), mycobacteriophages synthesize a specific lysis protein, LysB, capable of detaching the outer membrane from the complex cell wall of mycobacteria. The family of LysB proteins is highly diverse, with many members presenting an extended N-terminus. The N-terminal region of mycobacteriophage Ms6 LysB shows structural similarity to the PG-binding domain (PGBD) of the φKZ endolysin. A fusion of this region with enhanced green fluorescent protein (Ms6LysBPGBD-EGFP) was shown to bind to Mycobacterium smegmatis, Mycobacterium vaccae, Mycobacterium bovis BGC and Mycobacterium tuberculosis H37Ra cells pretreated with SDS or Ms6 LysB. In pulldown assays, we demonstrate that Ms6 LysB and Ms6LysBPGBD-EGFP bind to purified peptidoglycan of M. smegmatis, Escherichia coli, Pseudomonas aeruginosa and Bacillus subtilis, demonstrating affinity to PG of the A1γ chemotype. An infection assay with an Ms6 mutant producing a truncated version of LysB lacking the first 90 amino acids resulted in an abrupt lysis. These results clearly demonstrate that the N-terminus of Ms6 LysB binds to the PG.

Systematic Modification and Evaluation of Enzyme-Loaded Chitosan Nanoparticles.

Polymeric-based nano drug delivery systems have been widely exploited to overcome protein instability during formulation. Presently, a diverse range of polymeric agents can be used, among which polysaccharides, such as chitosan (CS), hyaluronic acid (HA) and cyclodextrins (CDs), are included. Due to its unique biological and physicochemical properties, CS is one of the most used polysaccharides for development of protein delivery systems. However, CS has been described as potentially immunogenic. By envisaging a biosafe cytocompatible and haemocompatible profile, this paper reports the systematic development of a delivery system based on CS and derived with HA and CDs to nanoencapsulate the model human phenylalanine hydroxylase (hPAH) through ionotropic gelation with tripolyphosphate (TPP), while maintaining protein stability and enzyme activity. By merging the combined set of biopolymers, we were able to effectively entrap hPAH within CS nanoparticles with improvements in hPAH stability and the maintenance of functional activity, while simultaneously achieving strict control of the formulation process. Detailed characterization of the developed nanoparticulate systems showed that the lead formulations were internalized by hepatocytes (HepG2 cell line), did not reveal cell toxicity and presented a safe haemocompatible profile.

Modulation of Human Phenylalanine Hydroxylase by 3-Hydroxyquinolin-2(1H)-One Derivatives.

Phenylketonuria (PKU) is a genetic disease caused by deficient activity of human phenylalanine hydroxylase (hPAH) that, when untreated, can lead to severe psychomotor impairment. Protein misfolding is recognized as the main underlying pathogenic mechanism of PKU. Therefore, the use of stabilizers of protein structure and/or activity is an attractive therapeutic strategy for this condition. Here, we report that 3-hydroxyquinolin-2(1H)-one derivatives can act as protectors of hPAH enzyme activity. Electron paramagnetic resonance spectroscopy demonstrated that the 3-hydroxyquinolin-2(1H)-one compounds affect the coordination of the non-heme ferric center at the enzyme active-site. Moreover, surface plasmon resonance studies showed that these stabilizing compounds can be outcompeted by the natural substrate l-phenylalanine. Two of the designed compounds functionally stabilized hPAH by maintaining protein activity. This effect was observed on the recombinant purified protein and in a cellular model. Besides interacting with the catalytic iron, one of the compounds also binds to the N-terminal regulatory domain, although to a different location from the allosteric l-Phe binding site, as supported by the solution structures obtained by small-angle X-ray scattering.

In Silico and In Vitro Tailoring of a Chitosan Nanoformulation of a Human Metabolic Enzyme.

Enzyme nanoencapsulation holds an enormous potential to develop new therapeutic approaches to a large set of human pathologies including cancer, infectious diseases and inherited metabolic disorders. However, enzyme formulation has been limited by the need to maintain the catalytic function, which is governed by protein conformation. Herein we report the rational design of a delivery system based on chitosan for effective encapsulation of a functionally and structurally complex human metabolic enzyme through ionic gelation with tripolyphosphate. The rationale was to use a mild methodology to entrap the multimeric multidomain 200 kDa human phenylalanine hydroxylase (hPAH) in a polyol-like matrix that would allow an efficient maintenance of protein structure and function, avoiding formulation stress conditions. Through an in silico and in vitro based development, the particulate system was optimized with modulation of nanomaterials protonation status, polymer, counterion and protein ratios, taking into account particle size, polydispersity index, surface charge, particle yield production, protein free energy of folding, electrostatic surface potential, charge, encapsulation efficiency, loading capacity and transmission electron microscopy morphology. Evaluation of the thermal stability, substrate binding profile, relative enzymatic activity, and substrate activation ratio of the encapsulated hPAH suggests that the formulation procedure does not affect protein stability, allowing an effective maintenance of hPAH biological function. Hence, this study provides an important framework for an enzyme formulation process.

Structural and functional impact of clinically relevant E1α variants causing pyruvate dehydrogenase complex deficiency.

Pyruvate dehydrogenase complex (PDC) catalyzes the oxidative decarboxylation of pyruvate to acetyl-coenzyme A, hinging glycolysis and the tricarboxylic acid cycle. PDC deficiency, an inborn error of metabolism, has a broad phenotypic spectrum. Symptoms range from fatal lactic acidosis or progressive neuromuscular impairment in the neonatal period, to chronic neurodegeneration. Most disease-causing mutations in PDC deficiency affect the PDHA1 gene, encoding the α subunit of the PDC-E1 component. Detailed biophysical analysis of pathogenic protein variants is a challenging approach to support the design of therapies based on improving and correcting protein structure and function. Herein, we report the characterization of clinically relevant PDC-E1α variants identified in Portuguese PDC deficient patients. These variants bear amino acid substitutions in different structural regions of PDC-E1α. The structural and functional analyses of recombinant heterotetrameric (αα'ßß') PDC-E1 variants, combined with molecular dynamics (MD) simulations, show a limited impact of the amino acid changes on the conformational stability, apart from the increased propensity for aggregation of the p.R253G variant as compared to wild-type PDC-E1. However, all variants presented a functional impairment in terms of lower residual PDC-E1 enzymatic activity and ≈3-100 × lower affinity for the thiamine pyrophosphate (TPP) cofactor, in comparison with wild-type PDC-E1. MD simulations neatly showed generally decreased stability (increased flexibility) of all variants with respect to the WT heterotetramer, particularly in the TPP binding region. These results are discussed in light of disease severity of the patients bearing such mutations and highlight the difficulty of developing chaperone-based therapies for PDC deficiency.

Engineering Organoids for in vitro Modeling of Phenylketonuria.

Phenylketonuria is a recessive genetic disorder of amino-acid metabolism, where impaired phenylalanine hydroxylase function leads to the accumulation of neurotoxic phenylalanine levels in the brain. Severe cognitive and neuronal impairment are observed in untreated/late-diagnosed patients, and even early treated ones are not safe from life-long sequelae. Despite the wealth of knowledge acquired from available disease models, the chronic effect of Phenylketonuria in the brain is still poorly understood and the consequences to the aging brain remain an open question. Thus, there is the need for better predictive models, able to recapitulate specific mechanisms of this disease. Human induced pluripotent stem cells (hiPSCs), with their ability to differentiate and self-organize in multiple tissues, might provide a new exciting in vitro platform to model specific PKU-derived neuronal impairment. In this review, we gather what is known about the impact of phenylalanine in the brain of patients and highlight where hiPSC-derived organoids could contribute to the understanding of this disease.

Author Correction: Structure of full-length wild-type human phenylalanine hydroxylase by small angle X-ray scattering reveals substrate-induced conformational stability.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Structure of full-length wild-type human phenylalanine hydroxylase by small angle X-ray scattering reveals substrate-induced conformational stability.

Human phenylalanine hydroxylase (hPAH) hydroxylates L-phenylalanine (L-Phe) to L-tyrosine, a precursor for neurotransmitter biosynthesis. Phenylketonuria (PKU), caused by mutations in PAH that impair PAH function, leads to neurological impairment when untreated. Understanding the hPAH structural and regulatory properties is essential to outline PKU pathophysiological mechanisms. Each hPAH monomer comprises an N-terminal regulatory, a central catalytic and a C-terminal oligomerisation domain. To maintain physiological L-Phe levels, hPAH employs complex regulatory mechanisms. Resting PAH adopts an auto-inhibited conformation where regulatory domains block access to the active site. L-Phe-mediated allosteric activation induces a repositioning of the regulatory domains. Since a structure of activated wild-type hPAH is lacking, we addressed hPAH L-Phe-mediated conformational changes and report the first solution structure of the allosterically activated state. Our solution structures obtained by small-angle X-ray scattering support a tetramer with distorted P222 symmetry, where catalytic and oligomerisation domains form a core from which regulatory domains protrude, positioning themselves close to the active site entrance in the absence of L-Phe. Binding of L-Phe induces a large movement and dimerisation of regulatory domains, exposing the active site. Activated hPAH is more resistant to proteolytic cleavage and thermal denaturation, suggesting that the association of regulatory domains stabilises hPAH.

Ichthyological collection of the Museu Oceanográfico D. Carlos I.

The collection of the Museu Oceanográfico D. Carlos I is a historical specimen, instrument, and document collection that has been housed at the Aquário Vasco da Gama since 1935. The collection is largely the result of several scientific campaigns conducted by Dom Carlos de Bragança between 1896 and 1907. Specifically, the ichthyological collection consists of 675 surviving catalogue records of specimens caught, acquired or offered to D. Carlos I between 1892 to 1907, and includes the type specimen for Odontaspis nasutus Bragança, 1904 (junior synonym of Mitsukurina owstoni Jordan, 1898), along with several specimens of deep sea species. All specimens were captured in coastal Portuguese waters, and were preserved in alcohol, formalin, or mounted.

Albumin-binding domain from Streptococcus zooepidemicus protein Zag as a novel strategy to improve the half-life of therapeutic proteins.

Recombinant antibody fragments belong to the promising class of biopharmaceuticals with high potential for future therapeutic applications. However, due to their small size they are rapidly cleared from circulation. Binding to serum proteins can be an effective approach to improve pharmacokinetic properties of short half-life molecules. Herein, we have investigated the Zag albumin-binding domain (ABD) derived from Streptococcus zooepidemicus as a novel strategy to improve the pharmacokinetic properties of therapeutic molecules. To validate our approach, the Zag ABD was fused with an anti-TNFα single-domain antibody (sdAb). Our results demonstrated that the sdAb-Zag fusion protein was highly expressed and specifically recognizes human, rat and mouse serum albumins with affinities in the nanomolar range. Moreover, data also demonstrated that the sdAb activity against the therapeutic target (TNFα) was not affected when fused with Zag ABD. Importantly, the Zag ABD increased the sdAb half-life ∼39-fold (47min for sdAb versus 31h for sdAb-Zag). These findings demonstrate that the Zag ABD fusion is a promising approach to increase the half-life of small recombinant antibodies molecules without affecting their therapeutic efficacy. Moreover, the present study strongly suggests that the Zag ABD fusion strategy can be potentially used as a universal method to improve the pharmokinetics properties of many others therapeutics proteins and peptides in order to improve their dosing schedule and clinical effects.

A Clinically Relevant Variant of the Human Hydrogen Sulfide-Synthesizing Enzyme Cystathionine ß-Synthase: Increased CO Reactivity as a Novel Molecular Mechanism of Pathogenicity?

The human disease classical homocystinuria results from mutations in the gene encoding the pyridoxal 5'-phosphate- (PLP-) dependent cystathionine ß-synthase (CBS), a key enzyme in the transsulfuration pathway that controls homocysteine levels, and is a major source of the signaling molecule hydrogen sulfide (H2S). CBS activity, contributing to cellular redox homeostasis, is positively regulated by S-adenosyl-L-methionine (AdoMet) but fully inhibited upon CO or NO• binding to a noncatalytic heme moiety. Despite extensive studies, the molecular basis of several pathogenic CBS mutations is not yet fully understood. Here we found that the ferrous heme of the reportedly mild p.P49L CBS variant has altered spectral properties and markedly increased affinity for CO, making the protein much more prone than wild type (WT) CBS to inactivation at physiological CO levels. The higher CO affinity could result from the slightly higher flexibility in the heme surroundings revealed by solving at 2.80-Å resolution the crystallographic structure of a truncated p.P49L. Additionally, we report that p.P49L displays impaired H2S-generating activity, fully rescued by PLP supplementation along the purification, despite a minor responsiveness to AdoMet. Altogether, the results highlight how increased propensity to CO inactivation of an otherwise WT-like variant may represent a novel pathogenic mechanism in classical homocystinuria.

PKU mutation p.G46S prevents the stereospecific binding of l-phenylalanine to the dimer of human phenylalanine hydroxylase regulatory domain.

Mammalian phenylalanine hydroxylase (PAH) has a potential allosteric regulatory binding site for l-phenylalanine (l-Phe), in addition to its catalytic site. This arrangement is supported by a crystal structure of a homodimeric truncated form of the regulatory domain of human PAH (hPAH-RD 1-118/19-118) [Patel D et al. (2016) Sci Rep doi: 10.1038/srep23748]. In this study, a fusion protein of the domain (MBP-(pepXa)-hPAH-RD 1-120) was overexpressed and recovered in a metastable and soluble state, which allowed the isolation of a dimeric and a monomeric fusion protein. When cleaved from MBP, hPAH-RD forms aggregates which are stereospecifically inhibited by l-Phe (> 95%) at low physiological concentrations. Aggregation of the cleaved dimer of the mutant form hPAH-G46S-RD was not inhibited by l-Phe, which is compatible with structurally/conformationally changed ßαßßαß ACT domain folds in the mutant.

Unveiling the Pathogenic Molecular Mechanisms of the Most Common Variant (p.K329E) in Medium-Chain Acyl-CoA Dehydrogenase Deficiency by in Vitro and in Silico Approaches.

Medium-chain acyl-CoA dehydrogenase deficiency (MCADD) is the most common genetic disorder affecting the mitochondrial fatty acid ß-oxidation pathway. The mature and functional form of human MCAD (hMCAD) is a homotetramer assembled as a dimer of dimers (monomers A/B and C/D). Each monomer binds a FAD cofactor, necessary for the enzyme's activity. The most frequent mutation in MCADD results from the substitution of a lysine with a glutamate in position 304 of mature hMCAD (p.K329E in the precursor protein). Here, we combined in vitro and in silico approaches to assess the impact of the p.K329E mutation on the protein's structure and function. Our in silico results demonstrated for the first time that the p.K329E mutation, despite lying at the dimer-dimer interface and being deeply buried inside the tetrameric core, seems to affect the tetramer surface, especially the ß-domain that forms part of the catalytic pocket wall. Additionally, the molecular dynamics data indicate a stronger impact of the mutation on the protein's motions in dimer A/B, while dimer C/D remains similar to the wild type. For dimer A/B, severe disruptions in the architecture of the pockets and in the FAD and octanoyl-CoA binding affinities were also observed. The presence of unaffected pockets (C/D) in the in silico studies may explain the decreased enzymatic activity determined for the variant protein (46% residual activity). Moreover, the in silico structural changes observed for the p.K329E variant protein provide an explanation for the structural instability observed experimentally, namely, the disturbed oligomeric profile, thermal stability, and conformational flexibility, with respect to the wild-type.

Insights into Medium-chain Acyl-CoA Dehydrogenase Structure by Molecular Dynamics Simulations.

The medium-chain acyl-CoA dehydrogenase (MCAD) is a mitochondrial enzyme that catalyzes the first step of mitochondrial fatty acid ß-oxidation (mFAO) pathway. Its deficiency is the most common genetic disorder of mFAO. Many of the MCAD disease-causing variants, including the most common p.K304E variant, show loss of function due to protein misfolding. Herein, we used molecular dynamics simulations to provide insights into the structural stability and dynamic behavior of MCAD wild-type (MCADwt) and validate a structure that would allow reliable new studies on its variants. Our results revealed that in both proteins the flavin adenine dinucleotide (FAD) has an important structural role on the tetramer stability and also in maintaining the volume of the enzyme catalytic pockets. We confirmed that the presence of substrate changes the dynamics of the catalytic pockets and increases FAD affinity. A comparison between the porcine MCADwt (pMCADwt) and human MCADwt (hMCADwt) structures revealed that both proteins are essentially similar and that the reversion of the double mutant E376G/T255E of hMCAD enzyme does not affect the structure of the protein neither its behavior in simulation. Our validated hMCADwt structure is crucial for complementing and accelerating the experimental studies aiming for the discovery and development of potential stabilizers of MCAD variants as candidates for the treatment of MCAD deficiency (MCADD).