Nicotinamide Moiety Research Articles

Chemo-enzymatic synthesis of NPN cofactor taking advantage of ADP-ribosyl cyclase and LarC cyclometallase promiscuous activities

The nickel-pincer nucleotide cofactor (NPN) is a widespread organometallic cofactor required for lactate racemase (LarA) and for α-hydroxy acid racemases and epimerases of the LarA superfamily. Its biosynthesis, which starts with nicotinic acid adenine dinucleotide (NaAD), requires three enzymes: LarB, LarC, and LarE, and can be performed in vitro with purified enzymes. Nevertheless, as LarE and LarC are single turnover enzymes, the in vitro NPN biosynthesis requires huge amounts of enzymes (particularly 2 equivalents of LarE), which hampers the study of NPN and of NPN-dependent enzymes. By using adenosine diphosphate (ADP)-ribosyl cyclase (ARC), we exchanged the nicotinamide moiety in NAD+ with synthetic pyridine-3,5-bisthiocarboxylic acid in order to synthesize the novel intermediate pyridinium-3,5-bisthiocarboxylic acid adenine dinucleotide (P2TAD). The latter could be produced at a multimilligram scale allowing its characterization by Nuclear Magnetic Resonance (NMR) and mass spectrometry. Interestingly, P2TAD could directly be used by LarC in order to generate the NPN cofactor, bypassing both LarB and LarE. Globally, a new chemoenzymatic route towards NPN was developed via the intermediate P2TAD, which should facilitate the biochemical and biotechnological investigations on NPN binding enzymes.

Nicotinamide Riboside: What It Takes to Incorporate It into RNA.

Nicotinamide is an important functional compound and, in the form of nicotinamide adenine dinucleotide (NAD), is used as a co-factor by protein-based enzymes to catalyze redox reactions. In the context of the RNA world hypothesis, it is therefore reasonable to assume that ancestral ribozymes could have used co-factors such as NAD or its simpler analog nicotinamide riboside (NAR) to catalyze redox reactions. The only described example of such an engineered ribozyme uses a nicotinamide moiety bound to the ribozyme through non-covalent interactions. Covalent attachment of NAR to RNA could be advantageous, but the demonstration of such scenarios to date has suffered from the chemical instability of both NAR and its reduced form, NARH, making their use in oligonucleotide synthesis less straightforward. Here, we review the literature describing the chemical properties of the oxidized and reduced species of NAR, their synthesis, and previous attempts to incorporate either species into RNA. We discuss how to overcome the stability problem and succeed in generating RNA structures incorporating NAR.

A desthiobiotin labelled NAD+ analogue to uncover Poly(ADP-ribose) polymerase 1 protein targets.

ADP-ribosylation is a post-translational modification catalyzed by the enzyme family of polyadenosine diphosphate (ADP)-ribose) polymerases (PARPs). This enzymatic process involves the transfer of single or multiple ADP-ribose molecules onto proteins, utilizing nicotinamide adenine dinucleotide (NAD+ ) as a substrate. It, thus, plays a pivotal role in regulating various biological processes. Unveiling PARP-selective protein targets is crucial for a better understanding of their biological functions. Nonetheless, this task proves challenging due to overlapping targets shared among PARP family members. Therefore, we applied the "bump-and-hole" strategy to modify the nicotinamide binding site of PARP1 by introducing a hydrophobic pocket ("hole"). This PARP1-mutant binds an orthogonal NAD+ (Et-DTB-NAD+ ) containing an ethyl group ("bump") at the nicotinamide moiety. Furthermore, we added a desthiobiotin (DTB) tag directly to the adenosine moiety, enabling affinity enrichment of ADP-ribosylated proteins. Employing this approach, we successfully identified protein targets modified by PARP1 in cell lysate. This strategy expands the arsenal of chemically modified NAD+ analogs available for studying ADP-ribosylation, providing a powerful tool to study these critical post-translational modifications.

Supramolecular Block Copolymers from Tricarboxamides. Biasing Co-assembly by the Incorporation of Pyridine Rings.

The synthesis of a series of triangular-shape tricarboxamides endowed with three picolin or nicotin (compounds2and3) or just one nicotine unit (compound4) is reported and their self-assembling features investigated. The pyridine rings make compounds2-4electronically complementary with our previously reported OPETA1to form supramolecular copolymers.C3-symmetric tricarboxamide2forms highly stable intramolecular five-membered pseudocycles that impedes its supramolecular polymerization intopoly-2and the co-assembly with1to yield copolymerpoly-1-co-2. On the other hand,C3-symmetric tricarboxamide3readily formpoly-3with great stability but unable to form helical supramolecular polymers despite the presence of the peripheral chiral side chains. The copolymerpoly-1-co-3can only be obtained by a previous complete disassembly of the constitutive homopolymers in CHCl3. Helicalpoly-1-co-3arises in a process involving the transfer of the helicity from racemicpoly-1topoly-3, and the amplification of asymmetry from chiralpoly-3topoly-1. Importantly,C2v-symmetric4, endowed with only one nicotinamide moiety and three chiral side chains, self-assembles intoP-type helical supramolecular polymer (poly-4) in a thermodynamically controlled cooperative process. The combination ofpoly-1andpoly-4generates chiral supramolecular copolymerpoly-1-co-4, whose blocky microstructure has been investigated by applying the supramolecular copolymerization model.

Supramolecular Block Copolymers from Tricarboxamides. Biasing Co‐assembly by the Incorporation of Pyridine Rings

AbstractThe synthesis of a series of triangular‐shaped tricarboxamides endowed with three picoline or nicotine units (compounds 2 and 3, respectively) or just one nicotine unit (compound 4) is reported, and their self‐assembling features investigated. The pyridine rings make compounds 2–4 electronically complementary with our previously reported oligo(phenylene ethynylene)tricarboxamides (OPE‐TA) 1 to form supramolecular copolymers. C3‐symmetric tricarboxamide 2 forms highly stable intramolecular five‐membered pseudocycles that impede its supramolecular polymerization into poly‐2 and the co‐assembly with 1 to yield copolymer poly‐1‐co‐2. On the other hand, C3‐symmetric tricarboxamide 3 readily forms poly‐3 with great stability but unable to form helical supramolecular polymers despite the presence of the peripheral chiral side chains. The copolymer poly‐1‐co‐3 can only be obtained by a previous complete disassembly of the constitutive homopolymers in CHCl3. Helical poly‐1‐co‐3 arises in a process involving the transfer of the helicity from racemic poly‐1 to poly‐3, and the amplification of asymmetry from chiral poly‐3 to poly‐1. Importantly, C2v‐symmetric 4, endowed with only one nicotinamide moiety and three chiral side chains, self‐assembles into a P‐type helical supramolecular polymer (poly‐4) in a thermodynamically controlled cooperative process. The combination of poly‐1 and poly‐4 generates chiral supramolecular copolymer poly‐1‐co‐4, whose blocky microstructure has been investigated by applying the previously reported supramolecular copolymerization model.

Competitive inhibition of a non-natural cofactor dependent formaldehyde dehydrogenase by imidazole.

To better understand the unique inhibitory behavior of a non-natural cofactor preferred formaldehyde dehydrogenase (FalDH) mutant 9B2. We described our serendipitous observation that 9B2 was reversibly inhibited by residual imidazole introduced during protein preparation, while the wild-type enzyme was not sensitive to imidazole. Kinetic analysis showed that imidazole was a competitive inhibitor of formaldehyde with a Ki of 16 μM and an uncompetitive inhibitor of Nicotinamide Cytosine Dinucleotide for 9B2, indicating that formaldehyde and imidazole were combined in the same position. Molecular docking results of 9B2 showed that imidazole could favorably bind very close to the nicotinamide moiety of the cofactor, where formaldehyde was expected to reside for catalysis, which was in line with a competitive inhibition. The mutant 9B2 can be competitively inhibited by imidazole, suggesting that cautions should be taken to evaluate activities as protein mutants might attain unexpected sensitivity to a component in buffers for purification or activity assays.

Crystal structures of the NAD+-II riboswitch reveal two distinct ligand-binding pockets.

We present crystal structures of a new NAD+-binding riboswitch termed NAD+-II, bound to nicotinamide mononucleotide (NMN), nicotinamide adenine dinucleotide (NAD+) and nicotinamide riboside (NR). The RNA structure comprises a number of structural features including three helices, one of which forms a triple helix by interacting with an A5 strand in its minor-groove, and another formed from a long-range pseudoknot. The core of the structure (centrally located and coaxial with the triplex and the pseudoknot) includes two consecutive quadruple base interactions. Unusually the riboswitch binds two molecules of ligand, bound at distinct, non-overlapping sites in the RNA. Binding occurs primarily through the nicotinamide moiety of each ligand, held by specific hydrogen bonding and stacking interactions with the pyridyl ring. The mode of binding is the same for NMN, NR and the nicotinamide moiety of NAD+. In addition, when NAD+ is bound into one site it adopts an elongated conformation such that its diphosphate linker occupies a groove on the surface of the RNA, following which the adenine portion inserts into a pocket and makes specific hydrogen bonding interactions. Thus the NAD+-II riboswitch is distinct from the NAD+-I riboswitch in that it binds two molecules of ligand at separate sites, and that binding occurs principally through the nicotinamide moiety.

Structural analysis of carboxyspermidine dehydrogenase from Helicobacter pylori

Spermidine is a cationic polyamine that plays key roles in diverse biological processes, including biofilm formation and cell viability in bacteria. In some human gastrointestinal bacteria, such as Helicobacter pylori and Campylobacter jejuni, spermidine is biosynthesized using carboxyspermidine dehydrogenase (CASDH) and carboxyspermidine decarboxylase through an alternative pathway rather than the classical pathway found in most bacteria and eukaryotes. CASDH condenses putrescine and aspartate β-semialdehyde into carboxyspermidine in an NADPH-dependent manner. Because structural information on CASDH is not available, the exact enzymatic mechanism of CASDH has not been elucidated. To reveal the structural features of CASDH required for cofactor and substrate recruitment, we determined the crystal structures of the H. pylori CASDH protein alone and in complex with NADP. CASDH consists of three domains (D1, D2, and D3) and assembles into a homodimer exclusively using the D3 domain. The CASDH structure harbors a dent between the D1 and D3 domains. The NADP cofactor is inserted into the interdomain dent and induces structural rearrangements in CASDH, including dent closure and local structural changes in the D1 and D3 domains. A comparative analysis suggests that the substrate of CASDH binds in a cavity near the nicotinamide moiety of NADPH for the condensation reaction.

Structure-based rational design of a short-chain dehydrogenase/reductase for improving activity toward mycotoxin patulin

Patulin is a fatal mycotoxin that is widely detected in drinking water and fruit-derived products contaminated by diverse filamentous fungi. CgSDR from Candida guilliermondii represents the first NADPH-dependent short-chain dehydrogenase/reductase that catalyzes the reduction of patulin to the nontoxic E-ascladiol. To elucidate the catalytic mechanism of CgSDR, we solved its crystal structure in complex with cofactor and substrate. Structural analyses indicate that patulin is situated in a hydrophobic pocket adjacent to the cofactor, with the hemiacetal ring orienting toward the nicotinamide moiety of NADPH. In addition, we conducted structure-guided engineering to modify substrate-binding residue V187 and obtained variant V187F, V187K and V187W, whose catalytic activity was elevated by 3.9-, 2.2- and 1.7-fold, respectively. The crystal structures of CgSDR variants suggest that introducing additional aromatic stacking or hydrogen-bonding interactions to bind the lactone ring of patulin might account for the observed enhanced activity. These results illustrate the catalytic mechanism of SDR-mediated patulin detoxification for the first time and provide the upgraded variants that exhibit tremendous potentials in industrial applications.

Structures of lactaldehyde reductase, FucO, link enzyme activity to hydrogen bond networks and conformational dynamics.

A group-III iron containing 1,2-propanediol oxidoreductase, FucO, (also known as lactaldehyde reductase) from Escherichia coli was examined regarding its structure-dynamics-function relationships in the catalysis of the NADH-dependent reduction of (2S)-lactaldehyde. Crystal structures of FucO variants in the presence or absence of cofactors have been determined, illustrating large domain movements between the apo and holo enzyme structures. Different structures of FucO variants co-crystallized with NAD+ or NADH together with substrate further suggest dynamic properties of the nicotinamide moiety of the coenzyme that are important for the reaction mechanism. Modelling of the native substrate (2S)-lactaldehyde into the active site can explain the stereoselectivity exhibited by the enzyme, with a critical hydrogen bond interaction between the (2S)-hydroxyl and the side-chain of N151, as well as the previously experimentally demonstrated pro-(R) selectivity in hydride transfer from NADH to the aldehydic carbon. Furthermore, the deuterium kinetic isotope effect of hydride transfer suggests that reduction chemistry is the main rate-limiting step for turnover which is not the case in FucO catalysed alcohol oxidation. We further propose that a water molecule in the active site - hydrogen bonded to a conserved histidine (H267) and the 2'-hydroxyl of the coenzyme ribose - functions as a catalytic proton donor in the protonation of the product alcohol. A hydrogen bond network of water molecules and the side-chains of amino acid residues D360 and H267 links bulk solvent to this proposed catalytic water molecule.

Structural basis of SARM1 activation, substrate recognition, and inhibition by small molecules

The NADase SARM1 (sterile alpha and TIR motif containing 1) is a key executioner of axon degeneration and a therapeutic target for several neurodegenerative conditions. We show that a potent SARM1 inhibitor undergoes base exchange with the nicotinamide moiety of nicotinamide adenine dinucleotide (NAD+) to produce the bona fide inhibitor 1AD. We report structures of SARM1 in complex with 1AD, NAD+ mimetics and the allosteric activator nicotinamide mononucleotide (NMN). NMN binding triggers reorientation of the armadillo repeat (ARM) domains, which disrupts ARM:TIR interactions and leads to formation of a two-stranded TIR domain assembly. The active site spans two molecules in these assemblies, explaining the requirement of TIR domain self-association for NADase activity and axon degeneration. Our results reveal the mechanisms of SARM1 activation and substrate binding, providing rational avenues for the design of new therapeutics targeting SARM1.

Nanomechanical Study of Enzyme: Coenzyme Complexes: Bipartite Sites in Plastidic Ferredoxin-NADP+ Reductase for the Interaction with NADP.

Plastidic ferredoxin-NADP+ reductase (FNR) transfers two electrons from two ferredoxin or flavodoxin molecules to NADP+, generating NADPH. The forces holding the Anabaena FNR:NADP+ complex were analyzed by dynamic force spectroscopy, using WT FNR and three C-terminal Y303 variants, Y303S, Y303F, and Y303W. FNR was covalently immobilized on mica and NADP+ attached to AFM tips. Force–distance curves were collected for different loading rates and specific unbinding forces were analyzed under the Bell–Evans model to obtain the mechanostability parameters associated with the dissociation processes. The WT FNR:NADP+ complex presented a higher mechanical stability than that reported for the complexes with protein partners, corroborating the stronger affinity of FNR for NADP+. The Y303 mutation induced changes in the FNR:NADP+ interaction mechanical stability. NADP+ dissociated from WT and Y303W in a single event related to the release of the adenine moiety of the coenzyme. However, two events described the Y303S:NADP+ dissociation that was also a more durable complex due to the strong binding of the nicotinamide moiety of NADP+ to the catalytic site. Finally, Y303F shows intermediate behavior. Therefore, Y303, reported as crucial for achieving catalytically competent active site geometry, also regulates the concerted dissociation of the bipartite nucleotide moieties of the coenzyme.

Synthesis, characterization, X-ray structural analysis, DFT and BSA binding study of a Zn(II) complex, [Zn(II)Cl2(nia)2].2nia

A Zn(II) complex with two nicotinamide (nia) ligands, two chloride ions, and two co-crystallized nicotinamide molecules, [ZnCl2(nia)2].2nia, was synthesized and characterized on the basis of elemental analysis, spectroscopic techniques (UV-vis, FT-IR, 1H and 13C NMR), conductivity measurements, DFT and single-crystal X-ray crystallography. The complex crystallized in orthorhombic crystal system with pccn space group and cell dimensions: a = 27.1852(2), b = 7.6705(4), c = 12.7502(8) Å and α = β = γ = 90°. Single-crystal structural analysis revealed the presence of a neutral complex having tetrahedral geometry. In this complex, Zn(II) is coordinated to two chloride and two nicotinamide ligands in the coordination sphere and two nicotinamide molecules are co-crystallized. Hydrogen bonding interactions (N-H…O, C-H…O, N-H…N, C-H…Cl) along with π…π bonding interactions (between the coordinated nicotinamide and free nicotinamide moiety) stabilize the crystal lattice. The DFT studies consolidated the experimental findings and provided deeper insight into vibrational spectra, excitation spectra and hydrogen bonding interactions between nicotinamide molecules. The interactions and binding parameters of this complex with bovine serum albumin (BSA) have been probed through absorption and fluorescence spectroscopy which revealed the presence of static quenching mechanism and good binding propensity of the Zn complex.

Potent Inhibition of Nicotinamide N-Methyltransferase by Alkene-Linked Bisubstrate Mimics Bearing Electron Deficient Aromatics.

Nicotinamide N-methyltransferase (NNMT) methylates nicotinamide (vitamin B3) to generate 1-methylnicotinamide (MNA). NNMT overexpression has been linked to a variety of diseases, most prominently human cancers, indicating its potential as a therapeutic target. The development of small-molecule NNMT inhibitors has gained interest in recent years, with the most potent inhibitors sharing structural features based on elements of the nicotinamide substrate and the S-adenosyl-l-methionine (SAM) cofactor. We here report the development of new bisubstrate inhibitors that include electron-deficient aromatic groups to mimic the nicotinamide moiety. In addition, a trans-alkene linker was found to be optimal for connecting the substrate and cofactor mimics in these inhibitors. The most potent NNMT inhibitor identified exhibits an IC50 value of 3.7 nM, placing it among the most active NNMT inhibitors reported to date. Complementary analytical techniques, modeling studies, and cell-based assays provide insights into the binding mode, affinity, and selectivity of these inhibitors.

Enzymatic and Chemical Syntheses of Vacor Analogs of Nicotinamide Riboside, NMN and NAD.

It has recently been demonstrated that the rat poison vacor interferes with mammalian NAD metabolism, because it acts as a nicotinamide analog and is converted by enzymes of the NAD salvage pathway. Thereby, vacor is transformed into the NAD analog vacor adenine dinucleotide (VAD), a molecule that causes cell toxicity. Therefore, vacor may potentially be exploited to kill cancer cells. In this study, we have developed efficient enzymatic and chemical procedures to produce vacor analogs of NAD and nicotinamide riboside (NR). VAD was readily generated by a base-exchange reaction, replacing the nicotinamide moiety of NAD by vacor, catalyzed by Aplysia californica ADP ribosyl cyclase. Additionally, we present the chemical synthesis of the nucleoside version of vacor, vacor riboside (VR). Similar to the physiological NAD precursor, NR, VR was converted to the corresponding mononucleotide (VMN) by nicotinamide riboside kinases (NRKs). This conversion is quantitative and very efficient. Consequently, phosphorylation of VR by NRKs represents a valuable alternative to produce the vacor analog of NMN, compared to its generation from vacor by nicotinamide phosphoribosyltransferase (NamPT).

PH-Dependent Flavin Adenine Dinucleotide and Nicotinamide Adenine Dinucleotide Ultraviolet Resonance Raman (UVRR) Spectra at Intracellular Concentration.

The ultraviolet resonance Raman spectra of the adenine-containing enzymatic redox cofactors nicotinamide adenine dinucleotide and flavin adenine dinucleotide in aqueous solution of physiological concentration are compared with the aim of distinguishing between them and their building block adenine in potential co-occurrence in biological materials. At an excitation wavelength of 266 nm, the spectra are dominated by the strong resonant contribution from adenine; nevertheless, bands assigned to vibrational modes of the nicotinamide and the flavin unit are found to appear at similar signal strength. Comparison of spectra measured at pH 7 with data obtained pH 10 and pH 3 shows characteristic changes when pH is increased or lowered, mainly due to deprotonation of the flavin and nicotinamide moieties, and protonation of the adenine, respectively.

Crystal structure of the ferredoxin reductase component of carbazole 1,9a-dioxygenase from Janthinobacterium sp. J3.

Carbazole 1,9a-dioxygenase (CARDO), which consists of an oxygenase component and the electron-transport components ferredoxin (CARDO-F) and ferredoxin reductase (CARDO-R), is a Rieske nonheme iron oxygenase (RO). ROs are classified into five subclasses (IA, IB, IIA, IIB and III) based on their number of constituents and the nature of their redox centres. In this study, two types of crystal structure (type I and type II) were resolved of the class III CARDO-R from Janthinobacterium sp. J3 (CARDO-RJ3). Superimposition of the type I and type II structures revealed the absence of flavin adenine dinucleotide (FAD) in the type II structure along with significant conformational changes to the FAD-binding domain and the C-terminus, including movements to fill the space in which FAD had been located. Docking simulation of NADH into the FAD-bound form of CARDO-RJ3 suggested that shifts of the residues at the C-terminus caused the nicotinamide moiety to approach the N5 atom of FAD, which might facilitate electron transfer between the redox centres. Differences in domain arrangement were found compared with RO reductases from the ferredoxin-NADP reductase family, suggesting that these differences correspond to differences in the structures of their redox partners ferredoxin and terminal oxygenase. The results of docking simulations with the redox partner class III CARDO-F from Pseudomonas resinovorans CA10 suggested that complex formation suitable for efficient electron transfer is stabilized by electrostatic attraction and complementary shapes of the interacting regions.

Stereospecificity of hydride transfer and molecular docking in FMN-dependent NADH-indigo reductase of Bacillussmithii.

In this study, we investigated the stereospecificity of hydride transfer from NADH to flavin mononucleotide (FMN) in reactions catalyzed by the FMN‐dependent NADH‐indigo reductase expressed by thermophilic Bacillus smithii. We performed 1H‐NMR spectroscopy using deuterium‐labeled NADH (4R‐2H‐NADH) and molecular docking simulations to reveal that the pro‐S hydrogen at the C4 position of the nicotinamide moiety in NADH was specifically transferred to the flavin‐N5 atom of FNM. Altogether, our findings may aid in the improvement of the indigo dyeing (Aizome) process.

Crystal structure and magnetic properties of Cu(II) dinuclear complex with equatorial-axial bridging thiocyanate ligand: Showing ferromagnetic coupling

A new Cu (II)-dinuclear, [Cu2-(µ-NCS)2 (NCS)2(Nicotinamide)4] complex was synthesized, and its single crystal X-ray structure was determined. The complex was further characterized by IR spectra, TGA and EPR spectroscopy and its magnetic behaviour was studied in detail. The complex consists of a binuclear unit in which two copper atoms are connected through two thiocyanate end to end (equatorial N-axial S) bridges. The coordination geometry around each of the metal centre in the dinuclear is square pyramidal, where basal plane is occupied by two nitrogen atoms from two nicotinamide moieties (Pyridine ring-N), one nitrogen atom from bridging thiocyanate and one nitrogen of non-bridging thiocyanate anion. The axial positions in the square pyramid of each copper centre of the dinuclear is occupied by sulphur atom of the two bridging thiocyanate moieties which are equatorially bonded to neighbouring copper atom of same dinuclear unit through nitrogen end. The dinuclear chain is extended by hydrogen bonding resulting into a 1D and 2D structure. The temperature dependent magnetic susceptibility of compound was carried out at 1000 Oe in the temperature range of 2–300 K. This behaviour indicates that the intra dinuclear magnetic interactions between the paramagnetic Cu(II) atoms is of ferromagnetic nature.

Characterization of Enzymes Catalyzing the Formation of the Nonproteinogenic Amino Acid l-Dap in Capreomycin Biosynthesis.

Capreomycin (CMN) and viomycin (VIO) are nonribosomal peptide antituberculosis antibiotics, the structures of which contain four nonproteinogenic amino acids, including l-2,3-diaminopropionic acid (l-Dap), β-ureidodehydroalanine, l-capreomycidine, and β-lysine. Previous bioinformatics analysis suggested that CmnB/VioB and CmnK/VioK participate in the formation of l-Dap; however, the real substrates of these enzymes are yet to be confirmed. We herein show that starting from O-phospho-l-Ser (OPS) and l-Glu precursors, CmnB catalyzes the condensation reaction to generate a metabolite intermediate N-(1-amino-1-carboxyl-2-ethyl)glutamic acid (ACEGA), which undergoes NAD+-dependent oxidative hydrolysis by CmnK to generate l-Dap. Furthermore, the binding site of ACEGA and the catalytic mechanism of CmnK were elucidated with the assistance of three crystal structures, including those of apo-CmnK, the NAD+-CmnK complex, and CmnK in an alternative conformation. The CmnK-ACEGA docking model revealed that the glutamate α-hydrogen points toward the nicotinamide moiety. It provides evidence that the reaction is dependent on hydride transfer to form an imine intermediate, which is subsequently hydrolyzed by a water molecule to produce l-Dap. These findings modify the original proposed pathway and provide insights into l-Dap formation in the biosynthesis of other related natural products.