Uncoupling histone modification crosstalk by engineering lysine demethylase LSD1.

Biochemical crosstalk between two or more histone modifications is often observed in epigenetic enzyme regulation, but its functional significance in cells has been difficult to discern. Previous enzymatic studies revealed that Lys14 acetylation of histone H3 can inhibit Lys4 demethylation by lysine-specific demethylase 1 (LSD1). In the present study, we engineered a mutant form of LSD1, Y391K, which renders the nucleosome demethylase activity of LSD1 insensitive to Lys14 acetylation. K562 cells with the Y391K LSD1 CRISPR knockin show decreased expression of a set of genes associated with cellular adhesion and myeloid leukocyte activation. Chromatin profiling revealed that the cis-regulatory regions of these silenced genes display a higher level of H3 Lys14 acetylation, and edited K562 cells show diminished H3 mono-methyl Lys4 near these silenced genes, consistent with a role for enhanced LSD1 demethylase activity. These findings illuminate the functional consequences of disconnecting histone modification crosstalk for a key epigenetic enzyme.

Targeting ROS production through inhibition of NADPH oxidases.

NADPH oxidases (NOXs) are transmembrane enzymes that are devoted to the production of reactive oxygen species (ROS). In cancers, dysregulation of NOX enzymes affects ROS production, leading to redox unbalance and tumor progression. Consequently, NOXs are a drug target for cancer therapeutics, although current therapies have off-target effects: there is a need for isoenzyme-selective inhibitors. Here, we describe fully validated human NOX inhibitors, obtained from an in silico screen, targeting the active site of Cylindrospermum stagnale NOX5 (csNOX5). The hits are validated by in vitro and in cellulo enzymatic and binding assays, and their binding modes to the dehydrogenase domain of csNOX5 studied via high-resolution crystal structures. A high-throughput screen in a panel of cancer cells shows activity in selected cancer cell lines and synergistic effects with KRAS modulators. Our work lays the foundation for the development of inhibitor-based methods for controlling the tightly regulated and highly localized ROS sources.

A biosynthetic aspartate N-hydroxylase performs successive oxidations by holding intermediates at a site away from the catalytic center.

Nitrosuccinate is a biosynthetic building block in many microbial pathways. The metabolite is produced by dedicated L-aspartate hydroxylases that use NADPH and molecular oxygen as co-substrates. Here, we investigate the mechanism underlying the unusual ability of these enzymes to perform successive rounds of oxidative modifications. The crystal structure of Streptomyces sp. V2 L-aspartate N-hydroxylase outlines a characteristic helical domain wedged between two dinucleotide-binding domains. Together with NADPH and FAD, a cluster of conserved arginine residues forms the catalytic core at the domain interface. Aspartate is found to bind in an entry chamber that is close to but not in direct contact with the flavin. It is recognized by an extensive H-bond network that explains the enzyme's strict substrate-selectivity. A mutant designed to create steric and electrostatic hindrance to substrate binding disables hydroxylation without perturbing the NADPH oxidase side-activity. Critically, the distance between the FAD and the substrate is far too long to afford N-hydroxylation by the C4a-hydroperoxyflavin intermediate whose formation is confirmed by our work. We conclude that the enzyme functions through a catch-and-release mechanism. L-aspartate slides into the catalytic center only when the hydroxylating apparatus is formed. It is then re-captured by the entry chamber where it waits for the next round of hydroxylation. By iterating these steps, the enzyme minimizes the leakage of incompletely oxygenated products and ensures that the reaction carries on until nitrosuccinate is formed. This unstable product can then be engaged by a successive biosynthetic enzyme or undergoes spontaneous decarboxylation to produce 3-nitropropionate, a mycotoxin.

Characterization of structural, biochemical, pharmacokinetic, and pharmacodynamic properties of the LSD1 inhibitor bomedemstat in preclinical models.

INTRODUCTION: Lysine-specific demethylase 1 (LSD1) is emerging as a critical mediator of tumor progression in metastatic castration-resistant prostate cancer (mCRPC). Neuroendocrine prostate cancer (NEPC) is increasingly recognized as an adaptive mechanism of resistance in mCRPC patients failing androgen receptor axis-targeted therapies. Safe and effective LSD1 inhibitors are necessary to determine antitumor response in prostate cancer models. For this reason, we characterize the LSD1 inhibitor bomedemstat to assess its clinical potential in NEPC as well as other mCRPC pathological subtypes. METHODS: Bomedemstat was characterized via crystallization, flavine adenine dinucleotide spectrophotometry, and enzyme kinetics. On-target effects were assessed in relevant prostate cancer cell models by measuring proliferation and H3K4 methylation using western blot analysis. In vivo, pharmacokinetic (PK) and pharmacodynamic (PD) profiles of bomedemstat are also described. RESULTS: Structural, biochemical, and PK/PD properties of bomedemstat, an irreversible, orally-bioavailable inhibitor of LSD1 are reported. Our data demonstrate bomedemstat has >2500-fold greater specificity for LSD1 over monoamine oxidase (MAO)-A and -B. Bomedemstat also demonstrates activity against several models of advanced CRPC, including NEPC patient-derived xenografts. Significant intra-tumoral accumulation of orally-administered bomedemstat is measured with micromolar levels achieved in vivo (1.2 ± 0.45 µM at the 7.5 mg/kg dose and 3.76 ± 0.43 µM at the 15 mg/kg dose). Daily oral dosing of bomedemstat at 40 mg/kg/day is well-tolerated, with on-target thrombocytopenia observed that is rapidly reversible following treatment cessation. CONCLUSIONS: Bomedemstat provides enhanced specificity against LSD1, as revealed by structural and biochemical data. PK/PD data display an overall safety profile with manageable side effects resulting from LSD1 inhibition using bomedemstat in preclinical models. Altogether, our results support clinical testing of bomedemstat in the setting of mCRPC.

Discovery and biochemical characterization of thermostable glycerol oxidases.

Alditol oxidases are promising tools for the biocatalytic oxidation of glycerol to more valuable chemicals. By integrating in silico bioprospecting with cell-free protein synthesis and activity screening, an effective pipeline was developed to rapidly identify enzymes that are active on glycerol. Three thermostable alditol oxidases from Actinobacteria Bacterium, Streptomyces thermoviolaceus, and Thermostaphylospora chromogena active on glycerol were discovered. The characterization of these three flavoenzymes demonstrated their glycerol oxidation activities, preference for alkaline conditions, and excellent thermostabilities with melting temperatures higher than 75 °C. Structural elucidation of the alditol oxidase from Actinobacteria Bacterium highlighted a constellation of side chains that engage the substrate through several hydrogen bonds, a histidine residue covalently bound to the FAD prosthetic group, and a tunnel leading to the active site. Upon computational simulations of substrate binding, a double mutant targeting a residue pair at the tunnel entrance was created and found to display an improved thermal stability and catalytic efficiency for glycerol oxidation. The hereby described alditol oxidases form a valuable panel of oxidative biocatalysts that can perform regioselective oxidation of glycerol and other polyols. KEY POINTS: • Rapid pipeline designed to identify putative oxidases • Biochemical and structural characterization of alditol oxidases • Glycerol oxidation to more valuable derivatives.

Structural Elucidation and Engineering of a Bacterial Carbohydrate Oxidase.

Flavin-dependent carbohydrate oxidases are valuable tools in biotechnological applications due to their high selectivity in the oxidation of carbohydrates. In this study, we report the biochemical and structural characterization of a recently discovered carbohydrate oxidase from the bacterium Ralstonia solanacearum, which is a member of the vanillyl alcohol oxidase flavoprotein family. Due to its exceptionally high activity toward N-acetyl-d-galactosamine and N-acetyl-d-glucosamine, the enzyme was named N-acetyl-glucosamine oxidase (NagOx). In contrast to most known (fungal) carbohydrate oxidases, NagOx could be overexpressed in a bacterial host, which facilitated detailed biochemical and enzyme engineering studies. Steady state kinetic analyses revealed that non-acetylated hexoses were also accepted as substrates albeit with lower efficiency. Upon determination of the crystal structure, structural insights into NagOx were obtained. A large cavity containing a bicovalently bound FAD, tethered via histidyl and cysteinyl linkages, was observed. Substrate docking highlighted how a single residue (Leu251) plays a key role in the accommodation of N-acetylated sugars in the active site. Upon replacement of Leu251 (L251R mutant), an enzyme variant was generated with a drastically modified substrate acceptance profile, tuned toward non-N-acetylated monosaccharides and disaccharides. Furthermore, the activity toward bulkier substrates such as the trisaccharide maltotriose was introduced by this mutation. Due to its advantage of being overexpressed in a bacterial host, NagOx can be considered a promising alternative engineerable biocatalyst for selective oxidation of monosaccharides and oligosaccharides.

Rationally Guided Improvement of NOV1 Dioxygenase for the Conversion of Lignin-Derived Isoeugenol to Vanillin.

Biocatalysis is a key tool in both green chemistry and biorefinery fields. NOV1 is a dioxygenase that catalyzes the one-step, coenzyme-free oxidation of isoeugenol into vanillin and holds enormous biotechnological potential for the complete valorization of lignin as a sustainable starting material for biobased chemicals, polymers, and materials. This study integrates computational, kinetic, structural, and biophysical approaches to characterize a new NOV1 variant featuring improved activity and stability compared to those of the wild type. The S283F replacement results in a 2-fold increased turnover rate (kcat) for isoeugenol and a 4-fold higher catalytic efficiency (kcat/Km) for molecular oxygen compared to those of the wild type. Furthermore, the variant exhibits a half-life that is 20-fold higher than that of the wild type, which most likely relates to the enhanced stabilization of the iron cofactor in the active site. Molecular dynamics supports this view, revealing that the S283F replacement decreases the optimal pKa and favors conformations of the iron-coordinating histidines compatible with an increased level of binding to iron. Importantly, whole cells containing the S283F variant catalyze the conversion of ≤100 mM isoeugenol to vanillin, yielding >99% molar conversion yields within 24 h. This integrative strategy provided a new enzyme for biotechnological applications and mechanistic insights that will facilitate the future design of robust and efficient biocatalysts.

Efficient Oxidation of 5-Hydroxymethylfurfural Using a Flavoprotein Oxidase from the Honeybee Apis mellifera.

The chemical 5-hydroxymethylfurfural (HMF) can be derived from lignocellulose and is an interesting bio-based platform chemical as it has the potential to be transformed into numerous valuable building blocks such as the polymer-precursor 2,5-diformylfuran (DFF). To date, only a few oxidases acting on HMF are known and by sampling atypical species, we discovered a novel flavin-dependent oxidoreductase from the honeybee Apis mellifera (beeHMFO). The enzyme can perform the chemoselective oxidation of HMF to DFF but can also readily accept other aromatic alcohols as substrates. The function of the enzyme may well be the antimicrobial generation of hydrogen peroxide using HMF, which is very abundant in honey. The discovery of this insect-derived flavoprotein oxidase holds promising potential in the synthesis of renewable products and demonstrates that insects can be an interesting source of novel biocatalysts.

A lonely electron blocks incoming pairs.

Electron bifurcation exploits high energetic states to drive unfavorable single electron reactions and determining the overall mechanism governing these electron transfers represents an arduous task. Using extensive stopped-flow spectroscopy and kinetic simulations, Sucharitakul et al. now explore the bifurcation mechanism of the electron transfer flavoprotein EtfAB from the anaerobic gut bacterium Acidaminococcus fermentans. Strikingly, they illustrated that catalysis is orchestrated by a negatively charged radical, α-FAD, that inhibits further reductions and features an atypical inverted kinetic isotope effect. These results provide additional insight behind electron transfers that are prevalent within multienzyme governed reactions.

Ancestral reconstruction of mammalian FMO1 enables structural determination, revealing unique features that explain its catalytic properties.

Mammals rely on the oxidative flavin-containing monooxygenases (FMOs) to detoxify numerous and potentially deleterious xenobiotics; this activity extends to many drugs, giving FMOs high pharmacological relevance. However, our knowledge regarding these membrane-bound enzymes has been greatly impeded by the lack of structural information. We anticipated that ancestral-sequence reconstruction could help us identify protein sequences that are more amenable to structural analysis. As such, we hereby reconstructed the mammalian ancestral protein sequences of both FMO1 and FMO4, denoted as ancestral flavin-containing monooxygenase (AncFMO)1 and AncFMO4, respectively. AncFMO1, sharing 89.5% sequence identity with human FMO1, was successfully expressed as a functional enzyme. It displayed typical FMO activities as demonstrated by oxygenating benzydamine, tamoxifen, and thioanisole, drug-related compounds known to be also accepted by human FMO1, and both NADH and NADPH cofactors could act as electron donors, a feature only described for the FMO1 paralogs. AncFMO1 crystallized as a dimer and was structurally resolved at 3.0 Å resolution. The structure harbors typical FMO aspects with the flavin adenine dinucleotide and NAD(P)H binding domains and a C-terminal transmembrane helix. Intriguingly, AncFMO1 also contains some unique features, including a significantly porous and exposed active site, and NADPH adopting a new conformation with the 2'-phosphate being pushed inside the NADP+ binding domain instead of being stretched out in the solvent. Overall, the ancestrally reconstructed mammalian AncFMO1 serves as the first structural model to corroborate and rationalize the catalytic properties of FMO1.

Heterocycle-containing tranylcypromine derivatives endowed with high anti-LSD1 activity.

As regioisomers/bioisosteres of 1a, a 4-phenylbenzamide tranylcypromine (TCP) derivative previously disclosed by us, we report here the synthesis and biological evaluation of some (hetero)arylbenzoylamino TCP derivatives 1b-6, in which the 4-phenyl moiety of 1a was shifted at the benzamide C3 position or replaced by 2- or 3-furyl, 2- or 3-thienyl, or 4-pyridyl group, all at the benzamide C4 or C3 position. In anti-LSD1-CoREST assay, all the meta derivatives were more effective than the para analogues, with the meta thienyl analogs 4b and 5b being the most potent (IC50 values = 0.015 and 0.005 µM) and the most selective over MAO-B (selectivity indexes: 24.4 and 164). When tested in U937 AML and prostate cancer LNCaP cells, selected compounds 1a,b, 2b, 3b, 4b, and 5a,b displayed cell growth arrest mainly in LNCaP cells. Western blot analyses showed increased levels of H3K4me2 and/or H3K9me2 confirming the involvement of LSD1 inhibition in these assays.

Flavins as Covalent Catalysts: New Mechanisms Emerge.

With approximately 1% of proteins being flavoproteins, flavins are at the heart of a plethora of redox reactions in all areas of biology. Thanks to a series of fascinating recent discoveries, in addition to redox chemistry, covalent catalysis is now being recognized more frequently as a common strategy in flavoenzymes, with unprecedented mechanisms becoming apparent. Thus, noncanonical covalent reactions by flavins are emerging as a new pervasive concept in basic enzymology and biochemistry. These diverse enzymes are engaged in most biological processes, positioning the knowledge being gained from these new mechanisms to be translated into drugs that function through covalent mechanisms.

Crystallographic snapshots of UDP-glucuronic acid 4-epimerase ligand binding, rotation, and reduction.

UDP-glucuronic acid is converted to UDP-galacturonic acid en route to a variety of sugar-containing metabolites. This reaction is performed by a NAD+-dependent epimerase belonging to the short-chain dehydrogenase/reductase family. We present several high-resolution crystal structures of the UDP-glucuronic acid epimerase from Bacillus cereus The geometry of the substrate-NAD+ interactions is finely arranged to promote hydride transfer. The exquisite complementarity between glucuronic acid and its binding site is highlighted by the observation that the unligated cavity is occupied by a cluster of ordered waters whose positions overlap the polar groups of the sugar substrate. Co-crystallization experiments led to a structure where substrate- and product-bound enzymes coexist within the same crystal. This equilibrium structure reveals the basis for a "swing and flip" rotation of the pro-chiral 4-keto-hexose-uronic acid intermediate that results from glucuronic acid oxidation, placing the C4' atom in position for receiving a hydride ion on the opposite side of the sugar ring. The product-bound active site is almost identical to that of the substrate-bound structure and satisfies all hydrogen-bonding requirements of the ligand. The structure of the apoenzyme together with the kinetic isotope effect and mutagenesis experiments further outlines a few flexible loops that exist in discrete conformations, imparting structural malleability required for ligand rotation while avoiding leakage of the catalytic intermediate and/or side reactions. These data highlight the double nature of the enzymatic mechanism: the active site features a high degree of precision in substrate recognition combined with the flexibility required for intermediate rotation.

Discovery, Biocatalytic Exploration and Structural Analysis of a 4-Ethylphenol Oxidase from Gulosibacter chungangensis.

The vanillyl-alcohol oxidase (VAO) family is a rich source of biocatalysts for the oxidative bioconversion of phenolic compounds. Through genome mining and sequence comparisons, we found that several family members lack a generally conserved catalytic aspartate. This finding led us to study a VAO-homolog featuring a glutamate residue in place of the common aspartate. This 4-ethylphenol oxidase from Gulosibacter chungangensis (Gc4EO) shares 42 % sequence identity with VAO from Penicillium simplicissimum, contains the same 8α-N3 -histidyl-bound FAD and uses oxygen as electron acceptor. However, Gc4EO features a distinct substrate scope and product specificity as it is primarily effective in the dehydrogenation of para-substituted phenols with little generation of hydroxylated products. The three-dimensional structure shows that the characteristic glutamate side chain creates a closely packed environment that may limit water accessibility and thereby protect from hydroxylation. With its high thermal stability, well defined structural properties and high expression yields, Gc4EO may become a catalyst of choice for the specific dehydrogenation of phenolic compounds bearing small substituents.

An Elegant Four-Helical Fold in NOX and STEAP Enzymes Facilitates Electron Transport across Biomembranes-Similar Vehicle, Different Destination.

The ferric reductase superfamily comprises several oxidoreductases that use an intracellular electron source to reduce an extracellular acceptor substrate. NADPH oxidases (NOXs) and six-transmembrane epithelial antigen of the prostate enzymes (STEAPs) are iconic members of the superfamily. NOXs produce extracellular reactive oxygen species that exert potent bactericidal activities and trigger redox-signaling cascades that regulate cell division and differentiation. STEAPs catalyze the reduction of extracellular iron and copper which is necessary for the bioavailability of these essential elements. Both NOXs and STEAPs are present as multiple isozymes with distinct regulatory properties and physiological roles. Despite the important roles of NOXs and STEAPs in human physiology and despite their wide involvement in diseases like cancer, their mode of action at the molecular level remained incompletely understood for a long time, in part due to the absence of high-resolution models of the complete enzymes. Our two laboratories have elucidated the three-dimensional structures of NOXs and STEAPs, providing key insight into their mechanisms and evolution. The enzymes share a conserved transmembrane helical domain with an eye-catching hourglass shape. On the extracellular side, a heme prosthetic group is at the bottom of a pocket where the substrate (O2 in NOX, chelated iron or copper in STEAP) is reduced. On the intracellular side, the inner heme of NOX and the FAD of STEAP are bound to topological equivalent sites. This is a rare case where critical amino acid substitutions and local conformational changes enable a cofactor (heme vs FAD) swap between two structurally and functionally conserved scaffolds. The catalytic core of these enzymes is completed by distinct cytosolic NADPH-binding domains that are topologically unrelated (a ferredoxin reductase-like flavoprotein domain in NOX and a F420H2:NADP+-like domain in STEAP), feature different quaternary structures, and underlie specific regulatory mechanisms. Despite their differences, these domains all establish electron-transfer chains that direct the electrons from NADPH to the transmembrane domain. The multistep nature of the process and the chemical nature of the products pose considerable problems in the enzymatic assays. We learned that great care must be exerted in the validation of a candidate inhibitor. Multiple orthogonal assays are required to rule out off-target effects such as ROS-scavenging activities or nonspecific interference with the enzyme redox chain. The structural analysis of STEAP/NOX enzymes led us to further notice that their transmembrane heme-binding topology is shared by other enzymes. We found that the core domain of the cytochrome b subunits of the mitochondrial complex III and photosynthetic cytochrome b6f are closely related to NOXs and STEAPs and likely arose from the same ancestor protein. This observation expands the substrate portfolio of the superfamily since cytochromes b act on ubiquinone. The rigidly packed helices of the NOX/STEAP/cytochrome b domain contrast with the more malleable membrane proteins like ion channels or amino-acid transporters, which undergo large conformational changes to allow passage of relatively large metabolites. This notion of a rigid hourglass scaffold found an unexpected confirmation in the observation, revealed by structural comparisons, that an helical bundle identical to the NOX/STEAP/cytochrome b enzymes is featured by a de novo designed heme-binding protein, PS1. Apparently, nature and protein designers have independently converged to this fold as a versatile scaffold for heme-mediated reactions. The challenge is now to uncover the molecular mechanisms that implement the isozyme-specific regulation of the enzyme functions and develop much needed inhibitors and modulators for chemical biology and drug design studies.

Touch, act and go: landing and operating on nucleosomes.

Chromatin-associated enzymes are responsible for the installation, removal and reading of precise post-translation modifications on DNA and histone proteins. They are specifically recruited to the target gene by associated factors, and as a result of their activity, they contribute in modulating cell identity and differentiation. Structural and biophysical approaches are broadening our knowledge on these processes, demonstrating that DNA, histone tails and histone surfaces can each function as distinct yet functionally interconnected anchoring points promoting nucleosome binding and modification. The mechanisms underlying nucleosome recognition have been described for many histone modifiers and related readers. Here, we review the recent literature on the structural organization of these nucleosome-associated proteins, the binding properties that drive nucleosome modification and the methodological advances in their analysis. The overarching conclusion is that besides acting on the same substrate (the nucleosome), each system functions through characteristic modes of action, which bring about specific biological functions in gene expression regulation.

Same Substrate, Many Reactions: Oxygen Activation in Flavoenzymes.

Over time, organisms have evolved strategies to cope with the abundance of dioxygen on Earth. Oxygen-utilizing enzymes tightly control the reactions involving O2 mostly by modulating the reactivity of their cofactors. Flavins are extremely versatile cofactors that are capable of undergoing redox reactions by accepting either one electron or two electrons, alternating between the oxidized and the reduced states. The physical and chemical principles of flavin-based chemistry have been investigated widely. In the following pages we summarize the state of the art on a key area of research in flavin enzymology: the molecular basis for the activation of O2 by flavin-dependent oxidases and monooxygenases. In general terms, oxidases use O2 as an electron acceptor to produce H2O2, while monooxygenases activate O2 by forming a flavin intermediate and insert an oxygen atom into the substrate. First, we analyze how O2 reaches the flavin cofactor embedded in the protein matrix through dedicated access pathways. Then we approach O2 activation from the perspective of the monooxygenases, their preferred intermediate, the C(4a)-(hydro)peroxyflavin, and the cases in which other intermediates have been described. Finally, we focus on understanding how the architectures developed in the active sites of oxidases promote O2 activation and which other factors operate in its reactivity.

Crystal structures and atomic model of NADPH oxidase.

NADPH oxidases (NOXs) are the only enzymes exclusively dedicated to reactive oxygen species (ROS) generation. Dysregulation of these polytopic membrane proteins impacts the redox signaling cascades that control cell proliferation and death. We describe the atomic crystal structures of the catalytic flavin adenine dinucleotide (FAD)- and heme-binding domains of Cylindrospermum stagnale NOX5. The two domains form the core subunit that is common to all seven members of the NOX family. The domain structures were then docked in silico to provide a generic model for the NOX family. A linear arrangement of cofactors (NADPH, FAD, and two membrane-embedded heme moieties) injects electrons from the intracellular side across the membrane to a specific oxygen-binding cavity on the extracytoplasmic side. The overall spatial organization of critical interactions is revealed between the intracellular loops on the transmembrane domain and the NADPH-oxidizing dehydrogenase domain. In particular, the C terminus functions as a toggle switch, which affects access of the NADPH substrate to the enzyme. The essence of this mechanistic model is that the regulatory cues conformationally gate NADPH-binding, implicitly providing a handle for activating/deactivating the very first step in the redox chain. Such insight provides a framework to the discovery of much needed drugs that selectively target the distinct members of the NOX family and interfere with ROS signaling.

Impact of Mutations on NPAC Structural Dynamics: Mechanistic Insights from MD Simulations.

NPAC is a cytokine-like nuclear factor involved in chromatin modification and regulation of gene expression. In humans, the C-terminal domain of NPAC has the conserved structure of the ß-hydroxyacid dehydrogenases (ß-HAD) protein superfamily, which forms a stable tetrameric core scaffold for demethylase enzymes and organizes multiple sites for chromatin interactions. In spite of the close structural resemblance to other ß-HAD family members, the human NPAC dehydrogenase domain lacks a highly conserved catalytic lysine, substituted by a methionine. The reintroduction of the catalytic lysine by M437 K mutation results in a significant decrease of stability of the tetramer. Here, we have computationally investigated the molecular determinants of the functional differences between methionine and lysine-containing NPAC proteins. We find that the single mutation can determine strong consequences in terms of dynamics, stability, and ultimately ability to assemble in supramolecular complexes: the higher stability and lower flexibility of the methionine variant structurally preorganizes the monomer for tetramerization, whereas lysine increases flexibility and favors conformations that, while catalytically active, are not optimal for tetrameric assembly. We combine structure-dynamics analysis to an evolutionary study of NPAC sequences, showing that the methionine mutation occurs in a specifically flexible region of the lysine-containing protein, flanked by two domains that concentrate most of the stabilizing interactions. In our model, such separation of stability nuclei and flexible regions appears to favor the functional innovability of the protein.