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ACS Chemical Biology May 2024Human ornithine aminotransferase (OAT), a pyridoxal 5'-phosphate (PLP)-dependent enzyme, has been shown to play an essential role in the metabolic reprogramming and...
Human ornithine aminotransferase (OAT), a pyridoxal 5'-phosphate (PLP)-dependent enzyme, has been shown to play an essential role in the metabolic reprogramming and progression of hepatocellular carcinoma (HCC). HCC accounts for approximately 75% of primary liver cancers and is within the top three causes of cancer death worldwide. As a result of treatment limitations, the overall 5-year survival rate for all patients with HCC is under 20%. The prevalence of HCC necessitates continued development of novel and effective treatment methods. In recent years, the therapeutic potential of selective inactivation of OAT has been demonstrated for the treatment of HCC. Inspired by previous increased selectivity for OAT by the expansion of the cyclopentene ring scaffold to a cyclohexene, we designed, synthesized, and evaluated a series of novel fluorinated cyclohexene analogues and identified (R)-3-amino-5,5-difluorocyclohex-1-ene-1-carboxylic acid as a time-dependent inhibitor of OAT. Structural and mechanistic studies have elucidated the mechanism of inactivation of OAT by , resulting in a PLP-inactivator adduct tightly bound to the active site of the enzyme. Intact protein mass spectrometry, F NMR spectroscopy, transient state kinetic studies, and X-ray crystallography were used to determine the structure of the final adduct and elucidate the mechanisms of inactivation. Interestingly, despite the highly electrophilic intermediate species conferred by fluorine and structural evidence of solvent accessibility in the OAT active site, Lys292 and water did not participate in nucleophilic addition during the inactivation mechanism of OAT by . Instead, rapid aromatization to yield the final adduct was favored.
Topics: Humans; Ornithine-Oxo-Acid Transaminase; Drug Design; Enzyme Inhibitors; Carboxylic Acids; Cyclohexenes; Carcinoma, Hepatocellular; Crystallography, X-Ray; Models, Molecular
PubMed: 38630468
DOI: 10.1021/acschembio.4c00022 -
Journal of Agricultural and Food... Apr 2024Pyridoxal 5'-phosphate (PLP) is highly valuable in food and medicine. However, achieving the efficient biosynthesis of PLP remains challenging. Here, a salvage pathway...
Pyridoxal 5'-phosphate (PLP) is highly valuable in food and medicine. However, achieving the efficient biosynthesis of PLP remains challenging. Here, a salvage pathway using acid phosphatase from (APase) and pyridoxine oxidase from (PNPO) as pathway enzymes was established for the first time to synthesize PLP from pyridoxine (PN) and pyrophosphate (PPi). APase was identified as a rate-limiting enzyme. Two protein modification strategies were developed based on the PN phosphorylation mechanism: (1) improving the binding of PN into APase and (2) enhancing the hydrophobicity of APase's substrate binding pocket. The / of optimal mutant was 4.9 times higher than that of the wild type. The detailed mechanism of performance improvement was analyzed. Under the catalysis of and PNPO, a PLP high-yielding strain of 14.5 ± 0.55 g/L was engineered with a productivity of 1.0 ± 0.02 g/(L h) (the highest to date). The study suggests a promising method for industrial-scale PLP production.
PubMed: 38602702
DOI: 10.1021/acs.jafc.4c00596 -
Journal of Medicinal Chemistry Apr 2024Weak antigens represented by MUC1 are poorly immunogenic, which greatly constrains the development of relevant vaccines. Herein, we developed a multifunctional lipidated...
Weak antigens represented by MUC1 are poorly immunogenic, which greatly constrains the development of relevant vaccines. Herein, we developed a multifunctional lipidated protein as a carrier, in which the TLR1/2 agonist PamCSK was conjugated to the -terminus of MUC1-loaded carrier protein BSA through pyridoxal 5'-phosphate-mediated transamination reaction. The resulting PamCSK-BSA-MUC1 conjugate was subsequently incorporated into liposomes, which biomimics the membrane structure of tumor cells. The results indicated that this lipidated protein carrier significantly enhanced antigen uptake by APCs and obviously augmented the retention of the vaccine at the injection site. Compared with the BSA-MUC1 and BSA-MUC1 + PamCSK groups, PamCSK-BSA-MUC1 evoked 22- and 11-fold increases in MUC1-specific IgG titers. Importantly, PamCSK-BSA-MUC1 elicited robust cellular immunity and significantly inhibited tumor growth. This is the first time that lipidated protein was constructed to enhance antigen immunogenicity, and this universal carrier platform exhibits promise for utilization in various vaccines, holding the potential for further clinical application.
Topics: Animals; Mucin-1; Mice; Liposomes; Humans; Lipopeptides; Cancer Vaccines; Serum Albumin, Bovine; Adjuvants, Immunologic; Female; Mice, Inbred BALB C; Antigens; Cell Line, Tumor
PubMed: 38588468
DOI: 10.1021/acs.jmedchem.4c00412 -
ELife Apr 2024P2X receptors are extracellular ATP-gated ion channels that form homo- or heterotrimers and consist of seven subtypes. They are expressed in various tissues, including...
P2X receptors are extracellular ATP-gated ion channels that form homo- or heterotrimers and consist of seven subtypes. They are expressed in various tissues, including neuronal and nonneuronal cells, and play critical roles in physiological processes such as neurotransmission, inflammation, pain, and cancer. As a result, P2X receptors have attracted considerable interest as drug targets, and various competitive inhibitors have been developed. However, although several P2X receptor structures from different subtypes have been reported, the limited structural information of P2X receptors in complex with competitive antagonists hampers the understanding of orthosteric inhibition, hindering the further design and optimization of those antagonists for drug discovery. We determined the cryogenic electron microscopy (cryo-EM) structures of the mammalian P2X7 receptor in complex with two classical competitive antagonists of pyridoxal-5'-phosphate derivatives, pyridoxal-5'-phosphate-6-(2'-naphthylazo-6'-nitro-4',8'-disulfonate) (PPNDS) and pyridoxal phosphate-6-azophenyl-2',5'-disulfonic acid (PPADS), and performed structure-based mutational analysis by patch-clamp recording as well as molecular dynamics (MD) simulations. Our structures revealed the orthosteric site for PPADS/PPNDS, and structural comparison with the previously reported apo- and ATP-bound structures showed how PPADS/PPNDS binding inhibits the conformational changes associated with channel activation. In addition, structure-based mutational analysis identified key residues involved in the PPNDS sensitivity of P2X1 and P2X3, which are known to have higher affinity for PPADS/PPNDS than other P2X subtypes.
Topics: Animals; Molecular Dynamics Simulation; Adenosine Triphosphate; Mammals
PubMed: 38578670
DOI: 10.7554/eLife.92829 -
Applied and Environmental Microbiology May 2024Bacteria have two routes for the l-methionine biosynthesis. In one route called the direct sulfuration pathway, acetylated l-homoserine is directly converted into...
UNLABELLED
Bacteria have two routes for the l-methionine biosynthesis. In one route called the direct sulfuration pathway, acetylated l-homoserine is directly converted into l-homocysteine. The reaction using HS as the second substrate is catalyzed by a pyridoxal 5'-phosphate-dependent enzyme, -acetylhomoserine sulfhydrylase (OAHS). In the present study, we determined the enzymatic functions and the structures of OAHS from (OAHS). The OAHS enzyme exhibited the highest catalytic activity under the weak acidic pH condition. In addition, crystallographic analysis revealed that the enzyme takes two distinct structures, open and closed forms. In the closed form, two acidic residues are sterically clustered. The proximity may cause the electrostatic repulsion, inhibiting the formation of the closed form under the neutral to the basic pH conditions. We concluded that the pH-dependent regulation mechanism using the two acidic residues contributes to the acidophilic feature of the enzyme.
IMPORTANCE
In the present study, we can elucidate the pH-dependent regulation mechanism of the acidophilic OAHS. The acidophilic feature of the enzyme is caused by the introduction of an acidic residue to the neighborhood of the key acidic residue acting as a switch for the structural interconversion. The strategy may be useful in the field of protein engineering to change the optimal pH of the enzymes. In addition, this study may be useful for the development of antibacterial drugs because the l-methionine synthesis essential for bacteria is inhibited by the OAHS inhibitors. The compounds that can inhibit the interconversion between the open and closed forms of OAHS may become antibacterial drugs.
Topics: Lactobacillus plantarum; Hydrogen-Ion Concentration; Bacterial Proteins; Carbon-Oxygen Lyases
PubMed: 38568076
DOI: 10.1128/aem.00118-24 -
Journal of Bacteriology Apr 2024In the absence of the RidA deaminase results in the accumulation of the reactive enamine 2-aminoacrylate (2AA). The resulting 2AA stress impacts metabolism and prevents...
In the absence of the RidA deaminase results in the accumulation of the reactive enamine 2-aminoacrylate (2AA). The resulting 2AA stress impacts metabolism and prevents growth in some conditions by inactivating a specific target pyridoxal 5'-phosphate (PLP)-dependent enzyme(s). The detrimental effects of 2AA stress can be overcome by changing the sensitivity of a critical target enzyme or modifying flux in one or more nodes in the metabolic network. The catabolic L-alanine racemase DadX is a target of 2AA, which explains the inability of an strain to use L-alanine as the sole nitrogen source. Spontaneous mutations that suppressed the growth defect of the strain were identified as lesions in which encodes GTP cyclohydrolase and catalyzes the first step of tetrahydrofolate (THF) synthesis. The data here show that THF limitation resulting from a lesion, or inhibition of dihydrofolate reductase (FolA) by trimethoprim, decreases the 2AA generated from endogenous serine. The data are consistent with an increased level of threonine, resulting from low folate levels, decreasing 2AA stress.IMPORTANCERidA is an enamine deaminase that has been characterized as preventing the 2-aminoacrylate (2AA) stress. In the absence of RidA, 2AA accumulates and damages various cellular enzymes. Much of the work describing the 2AA stress system has depended on the exogenous addition of serine to increase the production of the enamine stressor. The work herein focuses on understanding the effect of 2AA stress generated from endogenous serine pools. As such, this work describes the consequences of a subtle level of stress that nonetheless compromises growth in at least two conditions. Describing mechanisms that alter the physiological consequences of 2AA stress increases our understanding of endogenous metabolic stress and how the robustness of the metabolic network allows perturbations to be modulated.
Topics: Sheep; Animals; Salmonella enterica; Scrapie; Acrylates; Bacterial Proteins; Pyridoxal Phosphate; Tetrahydrofolates; Serine
PubMed: 38563759
DOI: 10.1128/jb.00042-24 -
The Biochemical Journal Apr 2024Homocystinuria is a rare disease caused by mutations in the CBS gene that results in a deficiency of cystathionine β-synthase (CBS). CBS is an essential pyridoxal...
Homocystinuria is a rare disease caused by mutations in the CBS gene that results in a deficiency of cystathionine β-synthase (CBS). CBS is an essential pyridoxal 5'-phosphate (PLP)-dependent enzyme in the transsulfuration pathway, responsible for combining serine with homocysteine to produce cystathionine, whose activity is enhanced by the allosteric regulator S-adenosylmethionine (SAM). CBS also plays a role in generating hydrogen sulfide (H2S), a gaseous signaling molecule with diverse regulatory functions within the vascular, nervous, and immune systems. In this study, we present the clinical and biochemical characterization of two novel CBS missense mutations that do not respond to pyridoxine treatment, namely c.689T > A (L230Q) and 215A > T (K72I), identified in a Chinese patient. We observed that the disease-associated K72I genetic variant had no apparent effects on the spectroscopic and catalytic properties of the full-length enzyme. In contrast, the L230Q variant expressed in Escherichia coli did not fully retain heme and when compared with the wild-type enzyme, it exhibited more significant impairments in both the canonical cystathionine-synthesis and the alternative H2S-producing reactions. This reduced activity is consistent with both in vitro and in silico evidence, which indicates that the L230Q mutation significantly decreases the overall protein's stability, which in turn, may represent the underlying cause of its pathogenicity.
Topics: Cystathionine beta-Synthase; Homocystinuria; Humans; Mutation, Missense; Male; Female
PubMed: 38563463
DOI: 10.1042/BCJ20240012 -
International Journal of Molecular... Mar 2024Enzymes reliant on pyridoxal 5'-phosphate (PLP), the metabolically active form of vitamin B, hold significant importance in both biology and medicine. They facilitate... (Review)
Review
Enzymes reliant on pyridoxal 5'-phosphate (PLP), the metabolically active form of vitamin B, hold significant importance in both biology and medicine. They facilitate various biochemical reactions, particularly in amino acid and neurotransmitter metabolisms. Vitamin B is absorbed by organisms in its non-phosphorylated form and phosphorylated within cells via pyridoxal kinase (PLK) and pyridox-(am)-ine 5'-phosphate oxidase (PNPOx). The flavin mononucleotide-dependent PNPOx enzyme converts pyridoxine 5'-phosphate and pyridoxamine 5'-phosphate into PLP. PNPOx is vital for both biosynthesis and salvage pathways in organisms producing B vitamers. However, for those depending on vitamin B as a nutrient, PNPOx participates only in the salvage pathway. Transferring the PLP produced via PNPOx to client apo-enzymes is indispensable for their catalytic function, proper folding and targeting of specific organelles. PNPOx activity deficiencies due to inborn errors lead to severe neurological pathologies, particularly neonatal epileptic encephalopathy. PNPOx maintains PLP homeostasis through highly regulated mechanisms, including structural alterations throughout the catalytic cycle and allosteric PLP binding, influencing substrate transformation at the active site. Elucidation at the molecular level of the mechanisms underlying PNPOx activity deficiencies is a requirement to develop personalized approaches to treat related disorders. Finally, despite shared features, the few PNPOx enzymes molecularly and functionally studied show species-specific regulatory properties that open the possibility of targeting it in pathogenic organisms.
Topics: Humans; Infant, Newborn; Oxidoreductases; Phosphates; Pyridoxaminephosphate Oxidase; Pyridoxal Phosphate; Vitamin B 6; Pyridoxine; Metabolic Diseases; Vitamins
PubMed: 38542149
DOI: 10.3390/ijms25063174 -
Molecular Genetics and Metabolism... Jun 2024A case of an adult with borderline AADC deficiency symptoms is presented here. Genetic analysis revealed that the patient carries two AADC variants (NM_000790.3:...
A case of an adult with borderline AADC deficiency symptoms is presented here. Genetic analysis revealed that the patient carries two AADC variants (NM_000790.3: c.1040G > A and c.679G > C) in compound heterozygosis, resulting in p.Arg347Gln and p.Glu227Gln amino acid alterations. While p.Arg347Gln is a known pathogenic variant, p.Glu227Gln is unknown. Combining clinical features to bioinformatic and molecular characterization of the AADC protein population of the patient (p.Arg347Gln/p.Arg347Gln homodimer, p.Glu227Gln/p.Glu227Gln homodimer, and p.Glu227Gln/p.Arg347Gln heterodimer), we determined that: i) the p.Arg347Gln/p.Arg347Gln homodimer is inactive since the alteration affects a catalytically essential structural element at the active site, ii) the p.Glu227Gln/p.Glu227Gln homodimer is as active as the wild-type AADC since the alteration occurs at the surface and does not change the chemical nature of the amino acid, and iii) the p.Glu227Gln/p.Arg347Gln heterodimer has a catalytic efficiency 75% that of the wild-type since only one of the two active sites is compromised, thus demonstrating a positive complementation. By this approach, the molecular basis for the mild presentation of the disease is provided, and the experience made can also be useful for personalized therapeutic decisions in other mild AADC deficiency patients. Interestingly, in the last few years, many previously undiagnosed or misdiagnosed patients have been identified as mild cases of AADC deficiency, expanding the phenotype of this neurotransmitter disease.
PubMed: 38524666
DOI: 10.1016/j.ymgmr.2024.101071 -
Molecular Neurobiology Mar 2024Subarachnoid hemorrhage (SAH) is a neurological emergency that can lead to fatal outcomes. It occurs when bleeding happens in the subarachnoid space, a small gap between...
Causal Relationships Between Gut Microbiota, Inflammatory Cells/Proteins, and Subarachnoid Hemorrhage: A Multi-omics Bidirectional Mendelian Randomization Study and Meta-analysis.
Subarachnoid hemorrhage (SAH) is a neurological emergency that can lead to fatal outcomes. It occurs when bleeding happens in the subarachnoid space, a small gap between the arachnoid and pia mater. This condition results from the rupture of diseased or damaged blood vessels at the brain's base or surface. This study combined various omics approaches with Mendelian randomization analysis, including MR-IVW, MR Egger, MR weight median, and MR weight mode, to generate preliminary results. It also employed reverse Mendelian randomization, treating SAH as the exposure. Finally, a meta-analysis was conducted to summarize these findings. The study found positive correlations between SAH and both GBPA-Pyridoxal 5 phosphate biosynthesis I (OR=1.48, 95% CI, 1.04-2.12) and GBPA-glucose biosynthesis I (OR=0.68, 95% CI, 0.52-0.90). Increased levels of urokinase-type plasma activator were also associated with SAH (OR=1.17, 95% CI, 1.04-1.32). Associations were observed with SAH for CD80 on CD62L+ plasmacytoid dendritic cells, CD80 on plasmacytoid dendritic cells, CD123 on CD62L+ plasmacytoid dendritic cells, and SSC-A on plasmacytoid dendritic cells. This study, through Mendelian randomization and meta-analysis, established links between SAH and four inflammatory cells, one inflammatory protein, and two gut microbiota-related pathways. These findings suggest potential treatment targets for SAH, highlighting the importance of modulating gut microbiota and utilizing anti-inflammatory drugs in its management.
PubMed: 38523223
DOI: 10.1007/s12035-024-04101-y