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1.
J Enzyme Inhib Med Chem ; 39(1): 2372734, 2024 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-39149761

RESUMEN

The current therapies against gastric pathogen Helicobacter pylori are ineffective in over 20% of patients. Enzymes belonging to the purine salvage pathway are considered as novel drug targets in this pathogen. Therefore, the main aim of the current study was to determine the antibacterial activity of pyridoxal 5'-phosphate (PLP), an active form of vitamin B6, against reference and clinical strains of H. pylori. Using a broad set of microbiological, physicochemical (UV absorption, LC-MS, X-ray analysis) and in silico experiments, we were able to prove that PLP inhibits adenylosuccinate synthetase (AdSS) from H. pylori by the competition with GTP (IC50eq ∼30 nM). This behaviour was attributed to formation of a Schiff base with a lysine residue (a covalent bond with Lys322 in the GTP binding site of AdSS) and was potentiated by the presence of vitamin C. This antibacterial activity of PLP gives hope for its future use against H. pylori.


Asunto(s)
Adenilosuccinato Sintasa , Antibacterianos , Relación Dosis-Respuesta a Droga , Helicobacter pylori , Pruebas de Sensibilidad Microbiana , Vitamina B 6 , Helicobacter pylori/efectos de los fármacos , Helicobacter pylori/enzimología , Antibacterianos/farmacología , Antibacterianos/química , Antibacterianos/síntesis química , Vitamina B 6/farmacología , Vitamina B 6/química , Vitamina B 6/síntesis química , Relación Estructura-Actividad , Adenilosuccinato Sintasa/metabolismo , Adenilosuccinato Sintasa/química , Adenilosuccinato Sintasa/antagonistas & inhibidores , Adenilosuccinato Sintasa/farmacología , Estructura Molecular , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/síntesis química , Farmacorresistencia Bacteriana/efectos de los fármacos , Fosfato de Piridoxal/farmacología , Fosfato de Piridoxal/química , Modelos Moleculares
2.
J Am Chem Soc ; 146(29): 20263-20269, 2024 Jul 24.
Artículo en Inglés | MEDLINE | ID: mdl-39001849

RESUMEN

α,ß-Diamino acids are important structural motifs and building blocks for numerous bioactive natural products, peptidomimetics, and pharmaceuticals, yet efficient asymmetric synthesis to access these stereoarrays remains a challenge. Herein, we report the development of a pyridoxal 5'-phosphate (PLP)-dependent enzyme that is engineered to catalyze stereoselective Mannich-type reactions between free α-amino acids and enolizable cyclic imines. This biocatalyst enabled one-step asymmetric enzymatic synthesis of the unusual pyrrolidine-containing amino acid L-tambroline at gram-scale with high enantio- and diastereocontrol. Furthermore, this enzymatic platform is capable of utilizing a diverse range of α-amino acids as the Mannich donor and various cyclic imines as the acceptor. By coupling with different imine-generating enzymes, we established versatile biocatalytic cascades and demonstrated a general, concise, versatile, and atom-economic approach to access unprotected α,ß-diamino acids, including structurally complex α,α-disubstituted α,ß-diamino acids with contiguous stereocenters.


Asunto(s)
Aminoácidos , Iminas , Iminas/química , Iminas/metabolismo , Estereoisomerismo , Aminoácidos/química , Aminoácidos/síntesis química , Aminoácidos/metabolismo , Biocatálisis , Fosfato de Piridoxal/química , Fosfato de Piridoxal/metabolismo , Estructura Molecular
3.
Phys Chem Chem Phys ; 26(23): 16579-16588, 2024 Jun 12.
Artículo en Inglés | MEDLINE | ID: mdl-38832404

RESUMEN

The transsulfuration pathway plays a key role in mammals for maintaining the balance between cysteine and homocysteine, whose concentrations are critical in several biochemical processes. Human cystathionine ß-synthase is a heme-containing, pyridoxal 5'-phosphate (PLP)-dependent enzyme found in this pathway. The heme group does not participate directly in catalysis, but has a regulatory function, whereby CO or NO binding inhibits the PLP-dependent reactions. In this study, we explore the detailed structural changes responsible for inhibition using quantum chemical calculations to validate the experimentally observed bonding patterns associated with heme CO and NO binding and molecular dynamics simulations to explore the medium-range structural changes triggered by gas binding and propagating to the PLP active site, which is more than 20 Å distant from the heme group. Our results support a previously proposed mechanical signaling model, whereby the cysteine decoordination associated with gas ligand binding leads to breaking of a hydrogen bond with an arginine residue on a neighbouring helix. In turn, this leads to a shift in position of the helix, and hence also of the PLP cofactor, ultimately disrupting a key hydrogen bond that stabilizes the PLP in its catalytically active form.


Asunto(s)
Cistationina betasintasa , Simulación de Dinámica Molecular , Fosfato de Piridoxal , Cistationina betasintasa/metabolismo , Cistationina betasintasa/química , Humanos , Fosfato de Piridoxal/metabolismo , Fosfato de Piridoxal/química , Gases/química , Gases/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico/química , Enlace de Hidrógeno , Monóxido de Carbono/química , Monóxido de Carbono/metabolismo , Hemo/química , Hemo/metabolismo , Dominio Catalítico , Teoría Cuántica , Cisteína/química , Cisteína/metabolismo
4.
J Biol Chem ; 300(6): 107404, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38782204

RESUMEN

Infectious diseases are a significant cause of death, and recent studies estimate that common bacterial infectious diseases were responsible for 13.6% of all global deaths in 2019. Among the most significant bacterial pathogens is Staphylococcus aureus, accounting for more than 1.1 million deaths worldwide in 2019. Vitamin biosynthesis has been proposed as a promising target for antibacterial therapy. Here, we investigated the biochemical, structural, and dynamic properties of the enzyme complex responsible for vitamin B6 (pyridoxal 5-phosphate, PLP) biosynthesis in S. aureus, which comprises enzymes SaPdx1 and SaPdx2. The crystal structure of the 24-mer complex of SaPdx1-SaPdx2 enzymes indicated that the S. aureus PLP synthase complex forms a highly dynamic assembly with transient interaction between the enzymes. Solution scattering data indicated that SaPdx2 typically binds to SaPdx1 at a substoichiometric ratio. We propose a structure-based view of the PLP synthesis mechanism initiated with the assembly of SaPLP synthase complex that proceeds in a highly dynamic interaction between Pdx1 and Pdx2. This interface interaction can be further explored as a potentially druggable site for the design of new antibiotics.


Asunto(s)
Proteínas Bacterianas , Fosfato de Piridoxal , Staphylococcus aureus , Staphylococcus aureus/enzimología , Staphylococcus aureus/metabolismo , Fosfato de Piridoxal/metabolismo , Fosfato de Piridoxal/química , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Cristalografía por Rayos X , Conformación Proteica , Unión Proteica
5.
J Am Chem Soc ; 146(21): 14672-14684, 2024 May 29.
Artículo en Inglés | MEDLINE | ID: mdl-38743881

RESUMEN

Pyridoxal 5'-phosphate (PLP)-dependent enzymes are the most versatile biocatalysts for synthesizing nonproteinogenic amino acids. α,α-Disubstituted quaternary amino acids, such as 1-aminocyclopentane-1-carboxylic acid (cycloleucine), are useful building blocks for pharmaceuticals. In this study, starting with the biosynthesis of fusarilin A, we discovered a family of PLP-dependent enzymes that can facilitate tandem carbon-carbon forming steps to catalyze an overall [3 + 2]-annulation. In the first step, the cycloleucine synthases use SAM as the latent electrophile and an in situ-generated enamine as the nucleophile for γ-substitution. Whereas previously characterized γ-replacement enzymes protonate the resulting α-carbon and release the acyclic amino acid, cycloleucine synthases can catalyze an additional, intramolecular aldol or Mannich reaction with the nucleophilic α-carbon to form the substituted cyclopentane. Overall, the net [3 + 2]-annulation reaction can lead to 2-hydroxy or 2-aminocycloleucine products. These studies further expand the biocatalytic scope of PLP-dependent enzymes.


Asunto(s)
Fosfato de Piridoxal , Fosfato de Piridoxal/metabolismo , Fosfato de Piridoxal/química , Biocatálisis , Estructura Molecular , Ciclopentanos/química , Ciclopentanos/metabolismo
6.
Int J Mol Sci ; 25(9)2024 May 06.
Artículo en Inglés | MEDLINE | ID: mdl-38732264

RESUMEN

Pyridoxal and pyridoxal 5'-phosphate are aldehyde forms of B6 vitamin that can easily be transformed into each other in the living organism. The presence of a phosphate group, however, provides the related compounds (e.g., hydrazones) with better solubility in water. In addition, the phosphate group may sometimes act as a binding center for metal ions. In particular, a phosphate group can be a strong ligand for a gold(III) ion, which is of interest for researchers for the anti-tumor and antimicrobial potential of gold(III). This paper aims to answer whether the phosphate group is involved in the complex formation between gold(III) and hydrazones derived from pyridoxal 5'-phosphate. The answer is negative, since the comparison of the stability constants determined for the gold(III) complexes with pyridoxal- and pyridoxal 5'-phosphate-derived hydrazones showed a negligible difference. In addition, quantum chemical calculations confirmed that the preferential coordination of two series of phosphorylated and non-phosphorylated hydrazones to gold(III) ion is similar. The preferential protonation modes for the gold(III) complexes were also determined using experimental and calculated data.


Asunto(s)
Oro , Hidrazonas , Piridoxal , Hidrazonas/química , Oro/química , Piridoxal/química , Fosfato de Piridoxal/química , Complejos de Coordinación/química , Espectrofotometría Ultravioleta , Estructura Molecular
7.
Curr Opin Chem Biol ; 81: 102472, 2024 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-38815536

RESUMEN

Pyridoxal phosphate (PLP) is a cofactor that is widely employed in enzymology. This pyridine-containing cofactor can be used for reactions ranging from transaminations to oxidations. The catalytic versatility can be understood by considering the chemical features of this cofactor. In recent years, exciting new reactions involving PLP have been discovered in natural products biosynthesis, upending our understanding of what this cofactor is capable of. Here we review some of the most exciting PLP-dependent reactions from the last five years.


Asunto(s)
Fosfato de Piridoxal , Fosfato de Piridoxal/metabolismo , Fosfato de Piridoxal/química , Enzimas/metabolismo , Enzimas/química , Humanos , Oxidación-Reducción , Animales
8.
Int J Biol Macromol ; 268(Pt 1): 131696, 2024 May.
Artículo en Inglés | MEDLINE | ID: mdl-38642679

RESUMEN

Carbon­carbon (C-C) bonds serve as the fundamental structural backbone of organic molecules. As a critical CC bond forming enzyme, α-oxoamine synthase is responsible for the synthesis of α-amino ketones by performing the condensation reaction between amino acids and acyl-CoAs. We previously identified an α-oxoamine synthase (AOS), named as Alb29, involved in albogrisin biosynthesis in Streptomyces albogriseolus MGR072. This enzyme belongs to the α-oxoamine synthase family, a subfamily under the pyridoxal 5'-phosphate (PLP) dependent enzyme superfamily. In this study, we report the crystal structures of Alb29 bound to PLP and L-Glu, which provide the atomic-level structural insights into the substrate recognition by Alb29. We discover that Alb29 can catalyze the amino transformation from L-Gln to L-Glu, besides the condensation of L-Glu with ß-methylcrotonyl coenzyme A. Subsequent structural analysis has revealed that one flexible loop in Alb29 plays an important role in both amino transformation and condensation. Based on the crystal structure of the S87G mutant in the loop region, we capture two distinct conformations of the flexible loop in the active site, compared with the wild-type Alb29. Our study offers valuable insights into the catalytic mechanism underlying substrate recognition of Alb29.


Asunto(s)
Ácido Glutámico , Especificidad por Sustrato , Ácido Glutámico/química , Modelos Moleculares , Streptomyces/enzimología , Cristalografía por Rayos X , Dominio Catalítico , Conformación Proteica , Fosfato de Piridoxal/metabolismo , Fosfato de Piridoxal/química , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Relación Estructura-Actividad
9.
Arch Biochem Biophys ; 756: 110011, 2024 06.
Artículo en Inglés | MEDLINE | ID: mdl-38649133

RESUMEN

Structure-function relationships are key to understanding enzyme mechanisms, controlling enzyme activities, and designing biocatalysts. Here, we investigate the functions of arginine residues in the active sites of pyridoxal-5'-phosphate (PLP)-dependent non-canonical d-amino acid transaminases, focusing on the analysis of a transaminase from Haliscomenobacter hydrossis. Our results show that the tandem of arginine residues R28* and R90, which form the conserved R-[RK] motif in non-canonical d-amino acid transaminases, not only facilitates effective substrate binding but also regulates the catalytic properties of PLP. Non-covalent interactions between residues R28*, R90, and Y147 strengthen the hydrogen bond between Y147 and PLP, thereby maintaining the reactivity of the cofactor. Next, the R90 residue contributes to the stability of the holoenzyme. Finally, the R90I substitution induces structural changes that lead to substrate promiscuity, as evidenced by the effective binding of substrates with and without the α-carboxylate group. This study sheds light on the structural determinants of the activity of non-canonical d-amino acid transaminases. Understanding the structural basis of the active site plasticity in the non-canonical transaminase from H. hydrossis, which is characterized by effective conversion of d-amino acids and α-keto acids, may help to tailor it for industrial applications.


Asunto(s)
Arginina , Dominio Catalítico , Fosfato de Piridoxal , Transaminasas , Transaminasas/metabolismo , Transaminasas/química , Arginina/química , Arginina/metabolismo , Fosfato de Piridoxal/metabolismo , Fosfato de Piridoxal/química , Especificidad por Sustrato , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Modelos Moleculares
10.
Angew Chem Int Ed Engl ; 63(31): e202319344, 2024 07 29.
Artículo en Inglés | MEDLINE | ID: mdl-38519422

RESUMEN

Amino acids (AAs) are modular building blocks which nature uses to synthesize both macromolecules, such as proteins, and small molecule natural products, such as alkaloids and non-ribosomal peptides. While the 20 main proteinogenic AAs display relatively limited side chain diversity, a wide range of non-canonical amino acids (ncAAs) exist that are not used by the ribosome for protein synthesis, but contain a broad array of structural features and functional groups. In this communication, we report the discovery of the biosynthetic pathway for a new ncAA, pazamine, which contains a cyclopropane ring formed in two steps. In the first step, a chlorine is added onto the C4 position of lysine by a radical halogenase, PazA. The cyclopropane ring is then formed in the next step by a pyridoxal-5'-phosphate-dependent enzyme, PazB, via an SN2-like attack at C4 to eliminate chloride. Genetic studies of this pathway in the native host, Pseudomonas azotoformans, show that pazamine potentially inhibits ethylene biosynthesis in growing plants based on alterations in the root phenotype of Arabidopsis thaliana seedlings. We further show that PazB can be utilized to make an alternative cyclobutane-containing AA. These discoveries may lead to advances in biocatalytic production of specialty chemicals and agricultural biotechnology.


Asunto(s)
Aminoácidos , Halogenación , Aminoácidos/metabolismo , Aminoácidos/química , Aminoácidos/biosíntesis , Fosfato de Piridoxal/metabolismo , Fosfato de Piridoxal/química , Arabidopsis/metabolismo , Arabidopsis/enzimología , Pseudomonas/metabolismo , Pseudomonas/enzimología , Ciclopropanos/química , Ciclopropanos/metabolismo
11.
Angew Chem Int Ed Engl ; 63(13): e202317161, 2024 03 22.
Artículo en Inglés | MEDLINE | ID: mdl-38308582

RESUMEN

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.


Asunto(s)
Aminoácidos , Fosfato de Piridoxal , Fosfato de Piridoxal/química , Fosfato de Piridoxal/metabolismo , Cristalografía por Rayos X , Especificidad por Sustrato , Carbono , Catálisis
12.
J Phys Chem B ; 128(1): 77-85, 2024 01 11.
Artículo en Inglés | MEDLINE | ID: mdl-38131279

RESUMEN

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.


Asunto(s)
Iminas , Nitrilos , Protones , Bases de Schiff , Transaminasas , Bases de Schiff/química , Fosfatos , Piridoxal , Fosfato de Piridoxal/química
13.
J Phys Chem B ; 127(38): 8139-8149, 2023 09 28.
Artículo en Inglés | MEDLINE | ID: mdl-37721415

RESUMEN

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.


Asunto(s)
Ornitina Descarboxilasa , Bases de Schiff , Dominio Catalítico , Ornitina Descarboxilasa/metabolismo , Bases de Schiff/química , Protones , Fosfato de Piridoxal/química , Fosfatos
14.
Science ; 381(6656): 444-451, 2023 07 28.
Artículo en Inglés | MEDLINE | ID: mdl-37499030

RESUMEN

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.


Asunto(s)
Aminoácidos , Fosfato de Piridoxal , Aminoácidos/biosíntesis , Aminoácidos/química , Biocatálisis , Fosfato de Piridoxal/química , Estereoisomerismo
15.
Biochemistry (Mosc) ; 88(5): 600-609, 2023 May.
Artículo en Inglés | MEDLINE | ID: mdl-37331706

RESUMEN

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.


Asunto(s)
Clostridioides difficile , Clostridioides difficile/metabolismo , Cisteína Sintasa/química , Cisteína Sintasa/metabolismo , Dominio Catalítico , Clostridioides/metabolismo , Tirosina , Fosfato de Piridoxal/química , Fosfato de Piridoxal/metabolismo , Metionina , Cinética
16.
ACS Chem Biol ; 18(4): 794-802, 2023 04 21.
Artículo en Inglés | MEDLINE | ID: mdl-37005433

RESUMEN

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.


Asunto(s)
Nucleósidos , Seudouridina , Vías Biosintéticas , ARN Polimerasas Dirigidas por ADN/metabolismo , Nucleósidos/metabolismo , Seudouridina/biosíntesis , Seudouridina/metabolismo , Fosfato de Piridoxal/química , Streptomyces/química , Streptomyces/metabolismo
17.
Protein Sci ; 32(4): e4600, 2023 04.
Artículo en Inglés | MEDLINE | ID: mdl-36807942

RESUMEN

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.


Asunto(s)
5-Aminolevulinato Sintetasa , Saccharomyces cerevisiae , Humanos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , 5-Aminolevulinato Sintetasa/química , Fosfato de Piridoxal/química , Dominio Catalítico , Hemo/química
18.
J Chem Inf Model ; 63(4): 1313-1322, 2023 02 27.
Artículo en Inglés | MEDLINE | ID: mdl-36745546

RESUMEN

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.


Asunto(s)
Oxo-Ácido-Liasas , Protones , Fosfato de Piridoxal/química , Oxo-Ácido-Liasas/química , Fosfatos
19.
Methods Enzymol ; 680: 35-83, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-36710018

RESUMEN

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.


Asunto(s)
Fosfato de Piridoxal , Transaminasas , Transaminasas/química , Transaminasas/metabolismo , Fosfato de Piridoxal/química , Fosfato de Piridoxal/metabolismo , Cetoácidos , Aminoácidos Aromáticos , Especificidad por Sustrato
20.
Chembiochem ; 24(7): e202200669, 2023 04 03.
Artículo en Inglés | MEDLINE | ID: mdl-36652345

RESUMEN

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.


Asunto(s)
Fosfato de Piridoxal , Fosfato de Piridoxal/química , Modelos Moleculares , Espectrometría de Masas
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