Substrate Promiscuity Research Articles

Correction to “Uncovering Hydroxynitrile Lyase Variants with Promiscuous Diastereoselective Nitroaldolase Activity toward the Highly Stereocontrolled Synthesis of Anti β-Nitroalcohols”

Correction to “Uncovering Hydroxynitrile Lyase Variants with Promiscuous Diastereoselective Nitroaldolase Activity toward the Highly Stereocontrolled Synthesis of <i>Anti</i> β-Nitroalcohols”

Synergistic role of Rubisco inhibitor release and degradation inphotosynthesis.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) exhibits catalytic promiscuity, resulting in error-prone reactions and the formation of inhibitory sugar phosphates. Specifically, Xylulose-1,5-bisphosphate (XuBP) acts as an inhibitor by binding to the active site of Rubisco, thereby impairing its catalytic function. Thermolabile Rubisco activase (Rca) facilitates the release of such inhibitors, including XuBP, by remodelling Rubisco. In Arabidopsis thaliana, the phosphatase pair CbbYA and CbbYB subsequently hydrolyses XuBP to prevent its rebinding to Rubisco. To explore the functional interplay between these components in maintaining photosynthesis, cbbya, cbbyb and cbbyab mutants were crossed with RCA knockdown (rca-2) lines. Additionally, both RCA and CBBYA were overexpressed in wild-type (WT) Arabidopsis thaliana. Phenotypic analyses revealed an exacerbation in decreased growth and photosynthetic efficiency in the cbbyab rca-2 double mutants compared with the control mutants (cbbyab and rca-2), indicating a negative genetic interaction. Furthermore, the co-overexpression of RCA and CBBYA did not improve photosynthesis under short-term heat stress, and light reactions were adversely affected relative to the WT. These findings illustrate the synergistic roles of Rca, CbbYA and CbbYB in maintaining carbon fixation and promoting plant growth in Arabidopsis thaliana. Thus, the coordinated regulation of Rca and CbbY enzymes is crucial for optimizing photosynthetic efficiency.

Structure-guided engineering of 4-coumarate: CoA ligase for efficient production of rosmarinic acid in Saccharomyces cerevisiae

The utilization of genetically modified microbial cells for rosmarinic acid (RA) production is gaining increased attention as a cost-effective and sustainable approach. However, the substrate promiscuity of 4-coumarate: CoA ligase and RA synthase has been considered as a critical factor for low RA yields. In this study, we rationally engineered the substrate preference of 4-coumarate: CoA ligase (OPc4CL2) from Petroselinum crispum, resulting in a significant enhancement in RA production. Particularly, the introduction of the Y240C mutation led to a remarkable 176% increase in RA yield. Subsequent enzymatic analysis of OPc4CL2 variants revealed diminished activity towards p-coumaric acid, resulting in insufficient time for the transformation of p-coumaric acid to 4-coumaroyl CoA to generate byproduct. Furthermore, to minimize the formation of undesired byproducts, the overexpression of 4-hydroxyphenylacetate 3-monooxygenase (OHpaB) and NADPH-flavin oxidoreductase (HpaC) was carried out to facilitate the conversion of p-coumaric acid to caffeic acid and 4-hydroxyphenyllactate to salvianic acid A, thus achieving a significant increase in RA yield of up to 329.9mg/L (16.5mg/g yield on glucose) in shake-flask cultivation. Finally, the engineered strain YRA113-24BHM achieved a notable RA production of 3.6g/L (about 20.2mg/g yield on glucose) by fed-batch fermentation. This study serves as a foundation for the sustainable biosynthesis of RA and other caffeic acid derivatives.

Promiscuous Janus kinase binding to cytokine receptors modulates signaling efficiencies and contributes to cytokine pleiotropy.

Janus kinases (JAKs) bind to class I and II cytokine receptors, activating signaling and regulating gene transcription through signal transducer and activator of transcription (STAT) proteins. Type I interferons (IFNs) require the JAK members TYK2 and JAK1, which bind to the receptor subunits IFNAR1 and IFNAR2, respectively. We investigated the role of JAKs in regulating IFNAR signaling activity. Synthetic IFNARs in which the extracellular domains of IFNAR1 and IFNAR2 are replaced with nanobodies had near-native type I IFN signaling, whereas the homomeric variant of IFNAR2 initiated much weaker signaling, despite harboring docking sites for JAKs and STATs. Cells with JAK1 and TYK2 knockout (KO) showed residual signaling, suggesting partial complementation by the remaining JAKs, particularly when they were overexpressed. Live-cell micropatterning experiments confirmed the promiscuous binding of JAK1, JAK2, and TYK2 to IFNAR1 and IFNAR2, and their recruitment correlated with their relative cellular abundances. However, each JAK had a different efficacy in inducing cross-phosphorylation and downstream signaling. JAK binding was also promiscuous for other cytokine receptors, including IFN-L1, IL-10Rβ, TPOR, and GHR, but not for EPOR, which activated different downstream signaling pathways. These findings suggest that competitive binding of JAKs to cytokine receptors together with the varying absolute and relative abundances of the JAKs in different cell types can account for the cell type-dependent signaling pleiotropy of cytokine receptors.

Development of the Esterase PestE for Amide Bond Synthesis Under Aqueous Conditions: Enzyme Cascades for Converting Waste PET into Tamibarotene.

A growing number of hydrolase enzymes show promiscuous acyltransferase activity, even under aqueous conditions. Here we report, for the first time, the ability of Pyrobaculum calidifontis VA1 esterase (PestE) to catalyse the formation of a wide range of amides in buffer, where the acyl donor forms a significant structural component in the amide product. The reactions occur under mild conditions and can achieve conversions up to 97 % in 6 h for formation of N-benzylfuranamide as the model reaction. We demonstrate PestE's potential in enzyme cascades to make amides from waste PET plastic and the conversion of the terephthalic acid product to tamibarotene, a drug with activity against acute leukemia. Rational mutagenesis led to identification of PestE variants F33L F289A and F33L. F33L F289A increased conversion of N-benzylfuranamide by 1.2-fold, and F33L gave a 4-fold increase in conversion to tamibarotene.

Development of the Esterase PestE for Amide Bond Synthesis Under Aqueous Conditions: Enzyme Cascades for Converting Waste PET into Tamibarotene

AbstractA growing number of hydrolase enzymes show promiscuous acyltransferase activity, even under aqueous conditions. Here we report, for the first time, the ability of Pyrobaculum calidifontis VA1 esterase (PestE) to catalyse the formation of a wide range of amides in buffer, where the acyl donor forms a significant structural component in the amide product. The reactions occur under mild conditions and can achieve conversions up to 97 % in 6 h for formation of N‐benzylfuranamide as the model reaction. We demonstrate PestE's potential in enzyme cascades to make amides from waste PET plastic and the conversion of the terephthalic acid product to tamibarotene, a drug with activity against acute leukemia. Rational mutagenesis led to identification of PestE variants F33L F289A and F33L. F33L F289A increased conversion of N‐benzylfuranamide by 1.2‐fold, and F33L gave a 4‐fold increase in conversion to tamibarotene.

Field-deployable porcine epidemic diarrhea virus diagnostics utilizing CRISPR-Cas13a

ABSTRACT Porcine epidemic diarrhoea virus (PEDV), a pathogenic microorganism that induces epidemic diarrhoea in swine, causes substantial economic damage to swine-farming nations. To prevent and control PEDV infections, the availability of upgraded and rapid virus detection techniques is crucial. The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas)13a system, namely, programmability of CRISPR RNA (crRNA) and “collateral” promiscuous RNase activity of Cas13a after target RNA identification. In this study, we aimed to develop a recombinase polymerase amplification (RPA)-based CRISPR-Cas13a approach for PEDV diagnosis for the first time. The results showed that up to 10 copies of the target PEDV DNA standard/µL were detected after 40 min at 37 °C. PEDV detection exhibited remarkable specificity compared to that of other selected pathogens. Additionally, this RPA-based CRISPR-Cas13a approach could be used to clinical samples, with similar performance to that of reverse transcription-quantitative polymerase chain reaction (RT – qPCR). The results of our proposed approach were visualized using either lateral flow strips or fluorescence for field-deployable viral diagnostics, thereby facilitating its use in endemic regions. Overall, our proposed approach showed good reliability, sensitivity, and specificity, suggesting that it is applicable for detecting other viruses in diagnosing diseases and inspecting food safety.

UGT708S6 from Dendrobium catenatum, catalyzes the formation of flavonoid C-glycosides.

Dendrobium catenatum is a perennial herb of the genus Dendrobium orchidaceae. It has been known as "Golden Grass, Soft Gold" since ancient times with effects of strengthening the body, benefiting the stomach, generating body fluid, nourishing Yin and clearing internal heat. The flowers of D. catenatum have anti-oxidation, immune regulation and other biological activities. The composition analysis of flowers showed that flavonoid glycosides were significantly accumulated in floral tissue. However, in the flowers of D. catenatum, there was only one case of the UDP-glycosyltransferase (UGT) responsible for the glycosylation of flavonoids has been reported. In this study, a new UGT (named UGT708S6) was cloned from D. catenatum flowers rich in O-glycosides and C-glycosides, and its function and biochemical properties were characterized. Through homology comparison and molecular docking, we identified the key amino acid residues affecting the catalytic function of UGT708S6. The glycosyltransferase UGT708S6 was characterized and demonstrated C-glycosyltransferase (CGT) activity in vitro assay using phloretin and 2-hydroxynaringenin as sugar acceptors. The catalytic promiscuity assay revealed that UGT708S6 has a clear sugar donor preference, and displayed O-glycosyltransferase (OGT) activity towards luteolin, naringenin and liquiritigenin. Furthermore, the catalytic characteristics of UGT708S6 were explored, shedding light on the structural basis of substrate promiscuity and the catalytic mechanism involved in the formation of flavonoid C-glycosides. R271 was a key amino acid residue site that sustained the catalytic reaction. The smaller binding pocket resulted in the production of new O-glycosides and the reduction of C-glycosides. This highlighted the importance of the binding pocket in determining whether C-glycosides or O-glycosides were produced. The findings suggest that UGT708S6 holds promise as a new glycosyltransferase for synthesizing flavonoid glycosides and offer valuable insights for further understanding the catalytic mechanisms of flavonoid glycosyltransferases.

Deciphering substrate promiscuity and specificity of indolethylamine N-methyltransferase family enzymes from amphibian toads

N-methylation is a crucial post-modification process in natural product biosynthesis and also contributes to the metabolism of various physiological substances, such as neurotransmitter, hormone, and trace elements. In this study, we identified seven indolethylamine N-methyltransferase (INMT) family enzymes from the amphibian toad Bufo gargarizan with distinct catalytic properties. Among these enzymes, BNMT 1, BNMT 5, BNMT 6 and BNMT 7 exhibited notable promiscuity, demonstrating the ability to methylate multiple derivatives of indolethylamine, phenylethylamine, phenylethanolamine, and nicotinamide. Conversely, BNMT 3 and BNMT 4 exhibited more specific substrate preferences, targeting particular phenylethylamine, phenylethanolamine, and nicotinamide-type substrates. Additionally, one enzyme, BNMT 11, exhibiting high specificity towards phenylethanolamines. By employing molecular docking and mutating key amino acids, we provided a rational explanation for the promiscuity and specificity mechanisms exhibited by these enzymes. This research offers valuable insights into the catalytic mechanisms of INMT family enzymes in B. gargarizans, as well as other organisms. Moreover, the identification of these broadly substrate-specific enzymes holds promise for leveraging synthetic biology in the production of a wide variety of naturally occurring N-methylated compounds.

Expanding the Substrate Selectivity of the Fimsbactin Biosynthetic Adenylation Domain, FbsH.

Nonribosomal peptide synthetases (NRPSs) produce diverse natural products including siderophores, chelating agents that many pathogenic bacteria produce to survive in low iron conditions. Engineering NRPSs to produce diverse siderophore analogs could lead to the generation of novel antibiotics and imaging agents that take advantage of this unique iron uptake system in bacteria. The highly pathogenic and antibiotic-resistant bacteria Acinetobacter baumannii produces fimsbactin, an unusual branched siderophore with iron-binding catechol groups bound to a serine or threonine side chain. To explore the substrate promiscuity of the assembly line enzymes, we report a structure-guided investigation of the stand-alone aryl adenylation enzyme FbsH. We report structures bound to its native substrate 2,3-dihydroxybenzoic acid (DHB) as well as an inhibitor that mimics the adenylate intermediate. We produced enzyme variants with an expanded binding pocket that are more tolerant for analogs containing a DHB C4 modification. Wild-type and mutant enzymes were then used in an in vitro reconstitution analysis to assess the production of analogs of the final product as well as several early stage intermediates. This analysis shows that some altered substrates progress down the fimsbactin assembly line to the downstream domains. However, analogs from alternate building blocks are produced at lower levels, indicating that selectivity exists in the downstream catalytic domains. These findings expand the substrate scope of producing condensation products between serine and aryl acids and identify the bottlenecks for chemoenzymatic production of fimsbactin analogs.

Biosynthetic Origin of the Methoxy Group in Quinine and Related Alkaloids.

Quinine is a historically important natural product containing a methoxy group that has been assumed to be incorporated at a late pathway stage. Here we show that the methoxy group in quinine and related alkaloids is introduced onto the starting substrate tryptamine. Feeding studies definitively show that 5-methoxytryptamine is utilized as a quinine biosynthetic intermediate in planta. We discover the biosynthetic genes that encode the responsible oxidase and methyltransferase, and we use these genes to reconstitute the early steps of the alkaloid biosynthetic pathway in Nicotiana benthamiana to produce a mixture of methoxylated and non-methoxylated alkaloid intermediates. Importantly, we show that the co-occurrence of both tryptamine and 5-methoxytryptamine substrates, along with the substrate promiscuity of downstream pathway enzymes, enable parallel formation of both methoxylated and non-methoxylated alkaloids.

Repurposing a Fully Reducing Polyketide Synthase toward 2-Methyl Guerbet-like Lipids.

In nature, thousands of diverse and bioactive polyketides are assembled by a family of multifunctional, "assembly line" enzyme complexes called polyketide synthases (PKS). Since the late 20th century, there have been several attempts to decode, rearrange, and "reprogram" the PKS assembly line to generate valuable materials such as biofuels and platform chemicals. Here, the first module from Mycobacterium tuberculosis (Mt) PKS12, an unorthodox, "modularly iterative" PKS, was modified and repurposed toward the formation of 2-methyl Guerbet lipids, which have wide applications in industry. We established a robust method for the recombinant expression and purification of this modified module (named [M1*]), and we demonstrated its ability to catalyze the formation of several 2-methyl Guerbet-like lipids (C13-C21). Furthermore, we studied and applied the promiscuous thioesterase activity of a neighboring β-ketoacyl synthase (KS) to release [M1*]-bound condensation products in a one-pot biosynthetic cascade. Finally, starting from lauric acid, we could generate our primary target compound (2-methyltetradecanoic acid) by coupling the Escherichia coli fatty acyl-CoA synthetase FadD to [M1*]. This work supports the biosynthetic utility of engineered PKS modules such as [M1*] and their ability to derive valuable Guerbet-like lipids from inexpensive fatty acids.

Doubly Homologated Tyrosine-Containing Peptides from the Cyanobacterium Microcystis aeruginosa NIES-4285 and Their Biosynthesis.

Chemical investigation of the cyanobacterium Microcystis aeruginosa NIES-4285 led to the isolation of six new natural products, microginins 705 (1), 719 (2), 733A (3), 733B (4), and 733C (5), and anabaenopeptin 885 (7), and three known compounds, anabaenopeptins 871 (6), B (8), and F (9). Planar structures and absolute configurations for 1-7 were determined by 2D NMR, HRMS, and Marfey's analyses. Microginin 733C (5), and anabaenopeptins 871 (6) and 885 (7) contained a unique residue of 2-amino-5-(4-hydroxyphenyl)pentanoic acid (Ahppa): doubly homologated tyrosine (di-hTyr). The biosynthetic origin of this nonproteinogenic amino acid di-hTyr was investigated, and it was found that MaHphABCDE are involved in the production of di-hTyr. In addition, biochemical characterization of aminotransferase MaHphE showed that it is a promiscuous enzyme. This result expanded the biocatalytic toolbox for amino acid homologation.

Adverse effects of CXCR2 deficiency in mice reared under non-gnotobiotic conditions

The family of pro-inflammatory and pro-angiogenic chemokines including Interleukin-8 (IL-8, aka CXCL8) and its homologues (CXCL1,2,3,5,6, and 7) exhibit promiscuous binding and activation of several G-protein-coupled receptors (i.e., CXCR2, CXCR1, and the atypical chemokine receptor (ACKR1)). A high proportion of their biological activity is attributed to CXCR2 activation, thus many CXCR2 inhibitors are in clinical trials for several chronic diseases. However, CXCR2 inhibition is often only investigated acutely in these trials or in Cxcr2−/− mice grown in gnotobiotic conditions. Since humans do not live in germ-free environments, our first goal is to highlight novel retinal and systemic observations in Cxcr2−/− mice grown in non-gnotobiotic conditions that suggest potential harmful consequences of long-term CXCR2 deficiency or blockade. Beyond confirmation of circulating blood/immune cell-related phenotypes, we report novel findings in Cxcr2−/− mice including: (1) delayed dye transit to the retinal vasculature, (2) alterations in the density and distribution of retinal vessels, astrocytes and microglia, (3) decreased electroretinogram a- and b-wave amplitudes, (4) reduced visual acuity, and (5) increased polymorphonuclear cell accumulation in vascular lumina abutting venular walls in the retina and in vital non-ocular tissues (lung and liver). Furthermore, PheWAS of CXCR2 CXCR1, and ACKR1 gene variants using data from UK Biobank participants suggest clinical associations with both retinal and vascular disease phenotypes. We conclude that chronic CXCR2 deficiency in mice contributes to functional damage to the retina and that the long-term safety of CXCR1/2 inhibitors designed for chronic use in humans should be explored before clinical adoption to safeguard sight and overall vascular health.

Illuminating Substrate Preferences of Promiscuous F420H2-Dependent Dehydroamino Acid Reductases with 4-Track mRNA Display.

Stereoselective reduction of dehydroamino acids is a common biosynthetic strategy to introduce d-amino acids into peptidic natural products. The reduction, often observed during the biosynthesis of lanthipeptides, is performed by dedicated dehydroamino acid reductases (dhAARs). Enzymes from the three known dhAAR families utilize nicotinamide, flavin, or F420H2 coenzymes as hydride donors, and little is known about the catalysis performed by the latter family proteins. Here, we perform a bioinformatics-guided identification and large-scale in vitro characterization of five F420H2-dependent dhAARs. We construct an mRNA display-based pipeline for ultrahigh throughput substrate specificity profiling of the enzymes. The pipeline relies on a 4-track selection strategy to deliver large quantities of clean data, which were leveraged to build accurate substrate fitness models. Our results identify a remarkably promiscuous enzyme, referred to as MaeJC, that is capable of installing d-Ala residues into arbitrary substrates with minimal recognition requirements. We integrate MaeJC into a thiopeptide biosynthetic pathway to produce d-amino acids-containing thiopeptides, demonstrating the utility of MaeJC for the programmable installation of d-amino acids in ribosomal peptides.

Selective regulation of aspartyl intramembrane protease activity by calnexin

Signal peptide peptidase-like 2c (SPPL2c) is a testis-specific aspartyl intramembrane protease that contributes to male gamete function both by catalytic and non-proteolytic mechanisms. Here, we provide an unbiased characterisation of the in vivo interactome of SPPL2c identifying the ER chaperone calnexin as novel binding partner of this enzyme. Recruitment of calnexin specifically required the N-glycosylation within the N-terminal protease-associated domain of SPPL2c. Importantly, mutation of the single glycosylation site of SPPL2c or loss of calnexin expression completely prevented SPPL2c-mediated intramembrane proteolysis of all tested substrates. By contrast and despite rather promiscuous binding of calnexin to other SPP/SPPL proteases, expression of the chaperone was exclusively required for SPPL2c-mediated proteolysis. Despite some impact on the stability of SPPL2c most presumably due to assistance in folding of the luminal domain of the protease, calnexin appeared to be recruited rather constitutively to the protease thereby boosting its catalytic activity. In summary, we describe a novel, highly specific mode of intramembrane protease regulation, highlighting the need to systematically approach control mechanisms governing the proteolytic activity of other members of the aspartyl intramembrane protease family.

Frameshift Mutations in Genes Encoding PBP3 and PBP4 Trigger an Unusual, Extreme β-Lactam Resistance Phenotype in Burkholderia multivorans.

In our curated panel of Burkholderia cepacia complex isolates, Burkholderia multivorans strain AU28442 was unusually highly β-lactam resistant. To explore the molecular mechanisms leading to this phenotype, we performed whole genome sequencing (WGS) and microbiological and biochemical assays. WGS analysis revealed that strain AU28442 produced two β-lactamases, AmpC22 and a novel PenA-like β-lactamase denominated PenA39. Additionally, the strain presented frame-shift mutations in the genes encoding penicillin binding proteins 3 (PBP3) and 4 (PBP4). The antibiotic susceptibilities of the parent AU28442 strain carrying blaPenA39 vs the isogenic E. colistrain producing blaPenA39 were discrepant with ceftazidime MICs of >512 and 1 μg/mL, respectively. Accordingly, PenA39 was found to poorly hydrolyze β-lactams with kcat values of ≤8.8 s-1. An overlay of the crystal structure of PenA39 with PenA1 revealed a shift in the SDN loop in the variant, which may affect the catalytic efficiency of PenA39 toward substrates and inhibitors. Moreover, microscopic examination of AU28442 revealed shortened rod-shaped cells compared to B. multivoransATCC 17616, which carries a full complement of intact PBPs. Further complementation assays confirmed that the loss of PBP3 and PBP4 was the main factor contributing to the high-level β-lactam resistance observed in B. multivoransAU28442. This information allowed us to revert susceptibility by pairing a potent β-lactamase inhibitor with a β-lactam with promiscuous PBP binding. This detailed characterization of B. multivoransprovides an illustration of the myriad ways in which bacteria under antibiotic selection can develop resistance and demonstrates a mechanism to overcome it.

Exploring the Substrate Scope and Catalytic Promiscuity of Nitroreductase‐Like Enzymes

AbstractFlavin‐dependent nitroreductases are gaining attention as biocatalysts for the synthesis of pharmaceutically active compounds and their precursors. Here, we examined a panel of nitroreductase‐like flavoenzymes for their reductase activity towards a wide variety of aromatic and aliphatic nitro compounds, nitroolefins, and α,β‐unsaturated carbonyl compounds. Several of these flavoenzymes displayed high reductase activity and achieved excellent conversion of diverse nitroarenes, nitroolefins and α,β‐unsaturated carbonyl compounds, accomplishing good product yields in semi‐preparative scale reactions (up to 97%). In addition to the catalytic promiscuity of several of these flavoenzymes, being able to perform the reduction of nitro groups (nitroreductase activity) as well as C=C groups (ene‐reductase activity), this study also revealed that some flavoenzymes exhibit high chemo‐, regio‐ and/or enantioselectivity, making them attractive enzymes for use in organic synthesis.

How to drug a cloud? Targeting intrinsically disordered proteins.

Biologically active proteins/regions without stable structure (i.e., intrinsically disordered proteins and regions (IDPs and IDRs)) are commonly found in all proteomes. They have a unique functional repertoire that complements the functionalities of ordered proteins and domains. IDPs/IDRs are multifunctional promiscuous binders capable of folding at interaction with specific binding partners on a template- or context-dependent manner, many of which undergo liquid-liquid phase separation, leading to the formation of membrane-less organelles and biomolecular condensates. Many of them are frequently related to the pathogenesis of various human diseases. All this defines IDPs/IDRs as attractive targets for the development of novel drugs. However, their lack of unique structures, multifunctionality, binding promiscuity, and involvement in unusual modes of action preclude direct use of traditional structure-based drug design approaches for targeting IDPs/IDRs, and make disorder-based drug discovery for these "protein clouds" challenging. Despite all these complexities there is continuing progress in the design of small molecules affecting IDPs/IDRs. This article describes the major structural features of IDPs/IDRs and the peculiarities of the disorder-based functionality. It also discusses the roles of IDPs/IDRs in various pathologies, and shows why the approaches elaborated for finding drugs targeting ordered proteins cannot be directly used for the intrinsic disorder-based drug design, and introduces some novel methodologies suitable for these purposes. Finally, it emphasizes that regardless of their multifunctionality, binding promiscuity, lack of unique structures, and highly dynamic nature, "protein clouds" are principally druggable. Significance Statement Intrinsically disordered proteins and regions are highly abundant in nature, have multiple important biological functions, are commonly involved in the pathogenesis of a multitude of human diseases, and are therefore considered as very attractive drug targets. Although dealing with these unstructured multifunctional protein/regions is a challenging task, multiple innovative approaches have been designed to target them by small molecules.

Adaptive laboratory evolution recruits the promiscuity of succinate semialdehyde dehydrogenase to repair different metabolic deficiencies

Promiscuous enzymes often serve as the starting point for the evolution of novel functions. Yet, the extent to which the promiscuity of an individual enzyme can be harnessed several times independently for different purposes during evolution is poorly reported. Here, we present a case study illustrating how NAD(P)+-dependent succinate semialdehyde dehydrogenase of Escherichia coli (Sad) is independently recruited through various evolutionary mechanisms for distinct metabolic demands, in particular vitamin biosynthesis and central carbon metabolism. Using adaptive laboratory evolution (ALE), we show that Sad can substitute for the roles of erythrose 4-phosphate dehydrogenase in pyridoxal 5’-phosphate (PLP) biosynthesis and glyceraldehyde 3-phosphate dehydrogenase in glycolysis. To recruit Sad for PLP biosynthesis and glycolysis, ALE employs various mechanisms, including active site mutation, copy number amplification, and (de)regulation of gene expression. Our study traces down these different evolutionary trajectories, reports on the surprising active site plasticity of Sad, identifies regulatory links in amino acid metabolism, and highlights the potential of an ordinary enzyme as innovation reservoir for evolution.