Molecular and Structural Basis for Cγ-C Bond Formation by PLP-Dependent Enzyme Fub7.

Pyridoxal 5'-phosphate (PLP)-dependent enzymes that catalyze γ-replacement reactions are prevalent, yet their utilization of carbon nucleophile substrates is rare. The recent discovery of two PLP-dependent enzymes, CndF and Fub7, has unveiled unique C-C bond forming capabilities, enabling the biocatalytic synthesis of alkyl- substituted pipecolic acids from O-acetyl-L-homoserine and ß-keto acid or aldehyde derived enolates. This breakthrough presents fresh avenues for the biosynthesis of pipecolic acid derivatives. However, the catalytic mechanisms of these enzymes remain elusive, and a dearth of structural information hampers their extensive application. Here, we have broadened the catalytic scope of Fub7 by employing ketone-derived enolates as carbon nucleophiles, revealing Fub7's capacity for substrate-dependent regioselective α-alkylation of unsymmetrical ketones. Through an integrated approach combining X-ray crystallography, spectroscopy, mutagenesis, and computational docking studies, we offer a detailed mechanistic insight into Fub7 catalysis. Our findings elucidate the structural basis for its substrate specificity, stereoselectivity, and regioselectivity. Our work sets the stage ready for subsequent protein engineering effort aimed at expanding the synthetic utility of Fub7, potentially unlocking novel methods to access a broader array of noncanonical amino acids.

1,3-Proton Transfer of Pyridoxal 5'-Phosphate Schiff Base in the Branched-Chain Aminotransferase: Concerted or Stepwise Mechanism?

The branched-chain aminotransferase from Mycobacterium tuberculosis (MtIlvE) is a pyridoxal 5'-phosphate (PLP) dependent enzyme, and it is essential for the synthesis of the branched-chain amino acids. Ketimine is an important intermediate in the catalytic process. We have investigated the mechanism of ketimine formation and the energy landscape using the multiple computational methods. It is found that the 1,3-proton transfer involved in ketimine formation occurs through a stepwise process rather than a one-step process. Lys204 is identified as a key residue for ligand binding and as a base that abstracts the Cα proton from the PLP-Glu Schiff base, yielding a carbanionic intermediate. The first proton transfer is the rate-limiting step with an energy barrier of 17.8 kcal mol-1. Our study disclosed the detailed pathway of the proton transfer from external aldimine to ketimine, providing novel insights into the catalytic mechanism of other PLP-dependent enzymes.

Free Energy Landscapes of the Tautomeric Interconversion of Pyridoxal 5'-Phosphate Aldimines at the Active Site of Ornithine Decarboxylase in Aqueous Media.

The pyridoxal 5'-phosphate (PLP) acts as a coenzyme for a large number of biochemical reactions. It exists in mainly two bound forms at the active site of the concerned enzyme: the internal aldimine, in which the PLP is bound with the ϵ-amino group of lysine at the active site, and the external aldimine, where the PLP is bound to the substrate amino acid. Both the internal and external aldimines have Schiff base linkage (N-H-O) and can exist in two tautomeric structures of ketoenamine and enolimine forms. In this work, we have investigated the free energy landscape for the tautomeric proton transfer in the internal and external aldimines at the active site of the ornithine decarboxylase enzyme in an aqueous medium. We performed hybrid quantum-classical metadynamics and force field-based molecular dynamics simulations, which revealed that the ketoenamine tautomer is more stable than the enolimine form. The QM/MM metadynamics calculations show that the free energy difference between the ketoenamine and enolimine forms for the internal aldimine is 3.9 kcal/mol, and it is found to be 5.8 kcal/mol for the external aldimine, with the ketoenamine form being more stable in both cases. The results are further supported by calculations of the binding free energies from classical simulations and static quantum chemical calculations in different environments. We have also analyzed the configurational structure of the microenvironment at the active site in order to have better insights into the interactions of the active site residues with the PLP in its two tautomeric forms.

Stereoselective amino acid synthesis by synergistic photoredox-pyridoxal radical biocatalysis.

Developing synthetically useful enzymatic reactions that are not known in biochemistry and organic chemistry is an important challenge in biocatalysis. Through the synergistic merger of photoredox catalysis and pyridoxal 5'-phosphate (PLP) biocatalysis, we developed a pyridoxal radical biocatalysis approach to prepare valuable noncanonical amino acids, including those bearing a stereochemical dyad or triad, without the need for protecting groups. Using engineered PLP enzymes, either enantiomeric product could be produced in a biocatalyst-controlled fashion. Synergistic photoredox-pyridoxal radical biocatalysis represents a powerful platform with which to discover previously unknown catalytic reactions and to tame radical intermediates for asymmetric catalysis.

O-Acetylhomoserine Sulfhydrylase from Clostridioides difficile: Role of Tyrosine Residues in the Active Site.

O-acetylhomoserine sulfhydrylase is one of the key enzymes in biosynthesis of methionine in Clostridioides difficile. The mechanism of γ-substitution reaction of O-acetyl-L-homoserine catalyzed by this enzyme is the least studied among the pyridoxal-5'-phosphate-dependent enzymes involved in metabolism of cysteine and methionine. To clarify the role of active site residues Tyr52 and Tyr107, four mutant forms of the enzyme with replacements of these residues with phenylalanine and alanine were generated. Catalytic and spectral properties of the mutant forms were investigated. The rate of γ-substitution reaction catalyzed by the mutant forms with replaced Tyr52 residue decreased by more than three orders of magnitude compared to the wild-type enzyme. The Tyr107Phe and Tyr107Ala mutant forms practically did not catalyze this reaction. Replacements of the Tyr52 and Tyr107 residues led to the decrease in affinity of apoenzyme to coenzyme by three orders of magnitude and changes in the ionic state of the internal aldimine of the enzyme. The obtained results allowed us to assume that Tyr52 is involved in ensuring optimal position of the catalytic coenzyme-binding lysine residue at the stages of C-α-proton elimination and elimination of the side group of the substrate. Tyr107 could act as a general acid catalyst at the stage of acetate elimination.

Pseudouridine-Modifying Enzymes SapB and SapH Control Entry into the Pseudouridimycin Biosynthetic Pathway.

Pseudouridimycin is a microbial C-nucleoside natural product that specifically inhibits bacterial RNA polymerases by binding to the active site and competing with uridine triphosphate for the nucleoside triphosphate (NTP) addition site. Pseudouridimycin consists of 5'-aminopseudouridine and formamidinylated, N-hydroxylated Gly-Gln dipeptide moieties to allow Watson-Crick base pairing and to mimic protein-ligand interactions of the triphosphates of NTP, respectively. The metabolic pathway of pseudouridimycin has been studied in Streptomyces species, but no biosynthetic steps have been characterized biochemically. Here, we show that the flavin-dependent oxidase SapB functions as a gate-keeper enzyme selecting pseudouridine (KM = 34 µM) over uridine (KM = 901 µM) in the formation of pseudouridine aldehyde. The pyridoxal phosphate (PLP)-dependent SapH catalyzes transamination, resulting in 5'-aminopseudouridine with a preference for arginine, methionine, or phenylalanine as cosubstrates as amino group donors. The binary structure of SapH in complex with pyridoxamine-5'-phosphate and site-directed mutagenesis identified Lys289 and Trp32 as key residues for catalysis and substrate binding, respectively. The related C-nucleoside oxazinomycin was accepted as a substrate by SapB with moderate affinity (KM = 181 µM) and was further converted by SapH, which opens possibilities for metabolic engineering to generate hybrid C-nucleoside pseudouridimycin analogues in Streptomyces.

Catalytic Mechanism of Pyridoxal 5'-Phosphate-Dependent Aminodeoxychorismate Lyase: A Computational QM/MM Study.

Aminodeoxychorismate lyase (ADCL) is a kind of pyridoxal-5'-phosphate (PLP)-dependent enzyme that catalyzes the conversion of 4-amino-4-deoxychorismate (ADC) to p-aminobenzoate (PABA), which is a key step for the biosynthesis of folate. To illuminate the reaction details at the atomistic level, an enzyme-substrate reactant model has been constructed, and QM/MM calculations have been performed. Our calculation results reveal that the overall catalytic cycle contains 11 elementary steps, which can be described by three stages, including the transamination reaction of PLP, the release of pyruvate and aromatization of ADC, and the recovery to the initial aldimine. During the reaction, a series of intramolecular proton transfer are involved, which are the key for the C-N bond formation and cleavage as well as the aromatization of the ADC ring. In addition to forming the Schiff base with the pocket residue Lys251 and substrate in the internal aldimine and the external aldimine, respectively, the coenzyme PLP also plays a critical role in the intramolecular proton transfer by employing its hydroxyl oxygen anion and phosphate group. These findings may provide useful information for further understanding the catalytic mechanism of other PLP-dependent enzymes.

The yeast ALA synthase C-terminus positively controls enzyme structure and function.

5-Aminolevulinic acid synthase (ALAS) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that catalyzes the first and rate-limiting step of heme biosynthesis in α-proteobacteria and several non-plant eukaryotes. All ALAS homologs contain a highly conserved catalytic core, but eukaryotes also have a unique C-terminal extension that plays a role in enzyme regulation. Several mutations in this region are implicated in multiple blood disorders in humans. In Saccharomyces cerevisiae ALAS (Hem1), the C-terminal extension wraps around the homodimer core to contact conserved ALAS motifs proximal to the opposite active site. To determine the importance of these Hem1 C-terminal interactions, we determined the crystal structure of S. cerevisiae Hem1 lacking the terminal 14 amino acids (Hem1 ΔCT). With truncation of the C-terminal extension, we show structurally and biochemically that multiple catalytic motifs become flexible, including an antiparallel ß-sheet important to Fold-Type I PLP-dependent enzymes. The changes in protein conformation result in an altered cofactor microenvironment, decreased enzyme activity and catalytic efficiency, and ablation of subunit cooperativity. These findings suggest that the eukaryotic ALAS C-terminus has a homolog-specific role in mediating heme biosynthesis, indicating a mechanism for autoregulation that can be exploited to allosterically modulate heme biosynthesis in different organisms.

A Nucleophilic Activity-Based Probe Enables Profiling of PLP-Dependent Enzymes.

PLP-dependent enzymes represent an important class of highly "druggable" enzymes that perform a wide array of critical reactions to support all organisms. Inhibition of individual members of this family of enzymes has been validated as a therapeutic target for pathologies ranging from infection with Mycobacterium tuberculosis to epilepsy. Given the broad nature of the activities within this family of enzymes, we envisioned a universally acting probe to characterize existing and putative members of the family that also includes the necessary chemical moieties to enable activity-based protein profiling experiments. Hence, we developed a probe that contains an N-hydroxyalanine warhead that acts as a covalent inhibitor of PLP-dependent enzymes, a linear diazirine for UV crosslinking, and an alkyne moiety to enable enrichment of crosslinked proteins. Our molecule was used to study PLP-dependent enzymes in vitro as well as look at whole-cell lysates of M. tuberculosis and assess inhibitory activity. The probe was able to enrich and identify LysA, a PLP-dependent enzyme crucial for lysine biosynthesis, through mass spectrometry. Overall, our study shows the utility of this trifunctional first-generation probe. We anticipate further optimization of probes for PLP-dependent enzymes will enable the characterization of rationally designed covalent inhibitors of PLP-dependent enzymes, which will expedite the preclinical characterization of these important therapeutic targets.

Biochemical characterization of plant aromatic aminotransferases.

Aromatic aminotransferases (Aro ATs) are pyridoxal-5-phosphate (PLP)-dependent enzymes that catalyze the transamination reactions of an aromatic amino acid (AAA) or a keto acid. Aro ATs are involved in biosynthesis or degradation of AAAs and play important functions in controlling the production of plant hormones and secondary metabolites, such as auxin, tocopherols, flavonoids, and lignin. Most Aro ATs show substrate promiscuity and can accept multiple aromatic and non-aromatic amino and keto acid substrates, which complicates and limits our understanding of their in planta functions. Considering the critical roles Aro ATs play in plant primary and secondary metabolism, it is important to accurately determine substrate specificity and kinetic properties of Aro ATs. This chapter describes various methodologies of protein expression, purification and enzymatic assays, which can be used for biochemical characterization of Aro ATs.

A direct enzymatic evaluation platform (DEEP) to fine-tuning pyridoxal 5'-phosphate-dependent proteins for cadaverine production.

Pyridoxal 5'-phosphate (pyridoxal phosphate, PLP) is an essential cofactor for multiple enzymatic reactions in industry. However, cofactor engineering based on PLP regeneration and related to the performance of enzymes in chemical production has rarely been discussed. First, we found that MG1655 strain was sensitive to nitrogen source and relied on different amino acids, thus the biomass was significantly reduced when PLP excess in the medium. Then, the six KEIO collection strains were applied to find out the prominent gene in deoxyxylulose-5-phosphate (DXP) pathway, where pdxB was superior in controlling cell growth. Therefore, the clustered regularly interspaced short palindromic repeats interference (CRISPRi) targeted on pdxB in MG1655 was employed to establish a novel direct enzymatic evaluation platform (DEEP) as a high-throughput tool and obtained the optimal modules for incorporating of PLP to enhance the biomass and activity of PLP-dependent enzymes simultaneously. As a result, the biomass has increased by 55% using PlacI promoter driven pyridoxine 5'-phosphate oxidase (PdxH) with a trace amount of precursor. When the strains incorporated DEEP and lysine decarboxylase (CadA), the cadaverine productivity was increased 32% due to the higher expression of CadA. DEEP is not only feasible for high-throughput screening of the best chassis for PLP engineering but also practical in fine-tuning the quantity and quality of enzymes.

Mechanistic aspects of the transamination reactions catalyzed by D-amino acid transaminase from Haliscomenobacter hydrossis.

Pyridoxal-5'-phosphate-(PLP-) dependent D-amino acid transaminases (DAATs) catalyze stereoselective reversible transfer of the amino group between D-amino acids and keto acids. In vivo DAATs are commonly known to synthesize D-glutamate for cell wall peptidoglycans. Today DAATs meet increasing attention for application in the synthesis of D-amino acids, whereas little is known about the mechanism of substrate recognition and catalytic steps of the D-amino acids conversion by DAATs. In this work, the pre-steady-state kinetics of the half-reactions of DAAT from Haliscomenobacter hydrossis with D-glutamate, D-alanine, D-leucine, and D-phenylalanine was examined at two wavelengths, 416 and 330 nm, using a stopped-flow technique. Monophasic kinetics was observed with specific substrates D-glutamate and D-alanine, whereas half-reactions with D-leucine and D-phenylalanine exhibited biphasic kinetics. All half-reactions proceeded until the complete conversion of PLP due to the release of the pyridoxamine-5'-phosphate form of cofactor from the holoenzyme . Comparison of kinetic parameters of half-reactions and the overall transamination reactions for D-leucine, D-phenylalanine revealed the increase in the rates of deamination of these substrates in the overall reaction with α-ketoglutarate. In the overall transamination reaction, the catalytic turnover rates for D-leucine and D-phenylalanine increased by 260 and 60 times, correspondingly, comparing with the slowest step rate constants in the half-reactions. We suggested the activating effect by a specific substrate α-ketoglutarate in the overall transamination reaction. The study of half-reactions helped to quantify the specificity of DAAT from H. hydrossis for D-amino acids with different properties. The results obtained are the first detailed analysis of half-reactions catalyzed by DAAT.

Complexation of Gold(III) with Pyridoxal 5'-Phosphate-Derived Hydrazones in Aqueous Solution.

Today, complexes of gold(I) and gold(III) are recognized as promising drugs for the treatment of bacterial infectious diseases and oncological diseases, respectively. It is of interest to broaden the area of potential use of gold(III) compounds to the pathogenic microorganism as well. The first step towards the development of new antibacterial drugs based on Au3+ complexes is the study of their stability in an aqueous solution. The present contribution reports on the investigation of gold(III) complexation with five hydrazones derived from a well-known biologically active compound, pyridoxal 5'-phosphate (one of the aldehyde forms of the B6 vitamin). The complex formation in aqueous solutions was confirmed by mass spectrometry and fluorescent spectroscopy. The stoichiometric composition of the complexes formed and their stability constants were determined using a UV-Vis titration method. The complexes are quite stable at physiological values of pH, as the speciation diagrams show. The results of the paper are helpful for further studies of gold(III) complexes interaction with biomacromolecules.

The Y430F mutant of Salmonella d-ornithine/d-lysine decarboxylase has altered stereospecificity and a putrescine allosteric activation site.

Tyrosine-430 of d-ornithine/d-lysine decarboxylase (DOKDC) is located in the active site, and was suggested to be responsible for the D-stereospecificity of the enzyme. We have prepared the Y430F mutant form of Salmonella enterica serovar typhimurium DOKDC and evaluated its catalytic activity with D- and l-lysine and ornithine. The kinetic results show that the Y430F mutant has measurable decarboxylase activity with both D- and l-lysine and ornithine, which wild type DOKDC does not. Spectroscopic experiments show that these amino acids bind to form external aldimine complexes with the pyridoxal-5'-phosphate with λmax = 425 nm. In addition, we have obtained crystal structures of Y430F DOKDC bound to HEPES, putrescine, d-ornithine, d-lysine, and d-arginine. The d-amino acids bind in the crystals to form equilibrium mixtures of gem-diamine and external aldimine complexes. Furthermore, the crystal structures reveal an unexpected allosteric product activator site for putrescine located on the 2-fold axis between the two active sites. Putrescine binds by donating hydrogen bonds from the ammonium groups to Asp-361 and Gln-358 in the specificity helix of both chains. Addition of 0.1-1 mM putrescine eliminates the lag in steady state kinetics and abolishes the sigmoid kinetics. The catalytic loop was modeled with AlphaFold2, and the model shows that Glu-181 can form additional hydrogen bonds with the bound putrescine, likely stabilizing the catalytic closed conformation.

Conformational change of organic cofactor PLP is essential for catalysis in PLP-dependent enzymes.

Pyridoxal 5'-phosphate (PLP)-dependent enzymes are ubiquitous, catalyzing various biochemical reactions of approximately 4% of all classified enzymatic activities. They transform amines and amino acids into important metabolites or signaling molecules and are important drug targets in many diseases. In the crystal structures of PLP-dependent enzymes, organic cofactor PLP showed diverse conformations depending on the catalytic step. The conformational change of PLP is essential in the catalytic mechanism. In the study, we review the sophisticated catalytic mechanism of PLP, especially in transaldimination reactions. Most drugs targeting PLP-dependent enzymes make a covalent bond to PLP with the transaldimination reaction. A detailed understanding of organic cofactor PLP will help develop a new drug against PLP-dependent enzymes. [BMB Reports 2022; 55(9): 439-446].

Fluorescent turn-on sensing of albumin proteins (BSA and ovalbumin) using vitamin B6 cofactor derived Schiff base.

The detection of albumin proteins with high accuracy by facile analytical approaches is important for the diagnosis of various diseases. This manuscript introduced an easy-to-prepare Schiff base L by condensing vitamin B6 cofactor pyridoxal 5'-phosphate (PLP) with 2-aminothiophenol for the fluorescence turn-on sensing of bovine serum albumin (BSA) and ovalbumin (OVA). The weakly emissive L showed a significant fluorescence enhancement at 485 and 490 nm in the presence of OVA and BSA with an estimated sensitivity limit of 1.7 µM and 0.3 µM, respectively. The formation of protein-ligand complex restricted the free intramolecular rotation of L is expected to show the selective fluorescence enhancement. The molecular docking and molecular dynamics simulations were performed to examine the binding affinity and modes between BSA/OVA and L. The practical utility of L as a fluorescent turn-on sensor was validated by quantifying BSA and OVA in various real biological samples of milk, serum, egg white and urine with good recovery percentages.

α-Helix in Cystathionine ß-Synthase Enzyme Acts as an Electron Reservoir.

The modulation of electron density at the Pyridoxal 5'-phosphate (PLP) catalytic center, because of charge transfer across the α-helix/PLP interface, is the determining factor for the enzymatic activities in the human Cystathionine ß-Synthase (hCBS) enzyme. Applying density functional theory calculations, in conjunction with the real space density analysis, we investigated the charge density delocalization across the entire heme-α-helix-PLP electron communication channels. The electron delocalization due to hydrogen bonds at the heme/α-helix and α-helix/PLP interfaces are found to be extended over a very long range, as a result of redistribution of electron densities of the cofactors. Moreover, the internal hydrogen bonds of α-helix that are crucial for its secondary structure also participate in the electron redistribution through the structured hydrogen-bond network. α-Helix is found to accumulate the electron density at the ground state from both of the cofactors and behaves as an electron reservoir for catalytic reaction at the electrophilic center of PLP.

Molecular basis and functional development of enzymes related to amino acid metabolism.

Enzymology, the study of enzyme structures and reaction mechanisms can be considered a classical discipline. However, enzymes cannot be freely designed to catalyze desired reactions yet, and enzymology is by no means a complete science. I have long studied the reaction mechanisms of enzymes related to amino acid metabolism, such as aminotransferases and racemases, which depend on pyridoxal 5'-phosphate, a coenzyme form of vitamin B6. During these studies, I have often been reminded that enzymatic reactions are extremely sophisticated processes based on chemical principles and enzyme structures, and have often been amazed at the evolutionary mechanisms that bestowed them with such structures. In this review, I described the reaction mechanism of various pyridoxal enzymes especially related to d-amino acids metabolism, whose roles in mammals have recently attracted attention. I hope to convey some of the significance and interest in enzymology through this review.

Cryptic Oxidative Transamination of Hydroxynaphthoquinone in Natural Product Biosynthesis.

Pyridoxal 5'-phosphate (PLP)-dependent enzymes are a group of versatile enzymes that catalyze various reactions, but only a small number of them react with O2. Here, we report an unprecedented PLP-dependent enzyme, NphE, that catalyzes both transamination and two-electron oxidation using O2 as an oxidant. Our intensive analysis reveals that NphE transfers the l-glutamate-derived amine to 1,3,6,8-tetrahydroxynaphthalene-derived mompain to form 8-amino-flaviolin (8-AF) via a highly conjugated quinonoid intermediate that is reactive with O2. During the NphE reaction, O2 is reduced to yield H2O2. An integrated technique involving NphE structure prediction by AlphaFold v2.0 and molecular dynamics simulation suggested the O2-accessible cavity. Our in vivo results demonstrated that 8-AF is a genuine biosynthetic intermediate for the 1,3,6,8-tetrahydroxynaphthalene-derived meroterpenoid naphterpin without an amino group, which was supported by site-directed mutagenesis. This study clearly establishes the NphE reaction product 8-AF as a common intermediate with a cryptic amino group for the biosynthesis of terpenoid-polyketide hybrid natural products.

Rational Design, Synthesis, and Mechanism of (3S,4R)-3-Amino-4-(difluoromethyl)cyclopent-1-ene-1-carboxylic Acid: Employing a Second-Deprotonation Strategy for Selectivity of Human Ornithine Aminotransferase over GABA Aminotransferase.

Human ornithine aminotransferase (hOAT) is a pyridoxal 5'-phosphate (PLP)-dependent enzyme that contains a similar active site to that of γ-aminobutyric acid aminotransferase (GABA-AT). Recently, pharmacological inhibition of hOAT was recognized as a potential therapeutic approach for hepatocellular carcinoma. In this work, we first studied the inactivation mechanisms of hOAT by two well-known GABA-AT inactivators (CPP-115 and OV329). Inspired by the inactivation mechanistic difference between these two aminotransferases, a series of analogues were designed and synthesized, leading to the discovery of analogue 10b as a highly selective and potent hOAT inhibitor. Intact protein mass spectrometry, protein crystallography, and dialysis experiments indicated that 10b was converted to an irreversible tight-binding adduct (34) in the active site of hOAT, as was the unsaturated analogue (11). The comparison of kinetic studies between 10b and 11 suggested that the active intermediate (17b) was only generated in hOAT and not in GABA-AT. Molecular docking studies and pKa computational calculations highlighted the importance of chirality and the endocyclic double bond for inhibitory activity. The turnover mechanism of 10b was supported by mass spectrometric analysis of dissociable products and fluoride ion release experiments. Notably, the stopped-flow experiments were highly consistent with the proposed mechanism, suggesting a relatively slow hydrolysis rate for hOAT. The novel second-deprotonation mechanism of 10b contributes to its high potency and significantly enhanced selectivity for hOAT inhibition.