Role of PLP-Level as a predictive marker for oral health status in adult hypophosphatasia.

AIM: The aim of this study was to investigate the role of pyridoxal-5-phosphate (PLP) level on the oral health status as a predictive marker in patients with hypophosphatasia (HPP). MATERIALS AND METHODS: Throughout a systematic retrospective assessment both bone metabolism and oral health status were analyzed. The oral health status was assessed by the decayed/missing/filled teeth index (DMFT), clinical attachment level (CAL), probing pocket depth (PPD), and the periodontal screening index (PSI). RESULTS: A total of 48 HPP patients (81.3% female) with a mean age of 42.21 years was included in this retrospective study. The study population was divided into two groups using the mean PLP level (87 µg/l) as a cut-off. Patients with a PLP level ≥ 87 µg/l (n = 14) showed a significantly poorer oral health status regarding DMFT index, CAL, PPD and PSI compared to patients with a PLP level < 87 µg/l (n = 34). No significant group differences for tooth loss were found. CONCLUSION: The results of the present study indicate that the PLP level is a suitable diagnostic predictor for the oral health status in HPP patients. HPP patients with PLP levels ≥ 70 µg/l should be included into a regular dental preventive program. CLINICAL RELEVANCE: The oral health status in HPP and its correlation with laboratory parameters (i.e. PLP) has been understudied. For clinical practice, the findings of the present study clearly demonstrated that high PLP levels correlate with a worse oral health status in HPP patients. Therefore, these patients should receive an intensive dental treatment and/or inclusion in a strict maintenance program in a specialized dental practice/university hospital with a PLP level ≥ 70 µg/l.

Increasing 1,4-Diaminobutane Production in Escherichia coli by Optimization of Cofactor PLP and NADPH Synthesis.

1,4-diaminobutane is widely used in the industrial production of polymers, pharmaceuticals, agrochemicals and surfactants. Owing to economic and environmental concerns, there has been a growing interest in using microbes to produce 1,4-diaminobutane. However, there is lack of research on the influence of cofactors pyridoxal phosphate (PLP) and NADPH on the synthesis of 1,4-diaminobutane. PLP serves as a cofactor of ornithine decarboxylase in the synthesis of 1,4-diaminobutane. Additionally, the synthesis of 1 mol 1,4-diaminobutane requires 2 mol NADPH, thus necessitating consideration of NADPH balance in the efficient synthesis of 1,4-diaminobutane by Escherichia coli. The aim of this study was to enhance the synthesis efficiency of 1,4-diaminobutane through increasing production of PLP and NADPH. By optimizing the expression of the genes associated with synthesis of PLP and NADPH in E. coli, cellular PLP and NADPH levels increased, and the yield of 1,4-diaminobutane also increased accordingly. Ultimately, using glucose as the primary carbon source, the yield of 1,4-diaminobutane in the recombinant strain NAP19 reached 272 mg/L·DCW, by increased 79% compared with its chassis strain.

Vitamin B6 and diabetes and its role in counteracting advanced glycation end products.

Naturally occurring forms of vitamin B6 include six interconvertible water-soluble compounds: pyridoxine (PN), pyridoxal (PL), pyridoxamine (PM), and their respective monophosphorylated derivatives (PNP, PLP, and PMP). PLP is the catalytically active form which works as a cofactor in approximately 200 reactions that regulate the metabolism of glucose, lipids, amino acids, DNA, and neurotransmitters. Most of vitamers can counteract the formation of reactive oxygen species and the advanced glycation end-products (AGEs) which are toxic compounds that accumulate in diabetic patients due to prolonged hyperglycemia. Vitamin B6 levels have been inversely associate with diabetes, while vitamin B6 supplementation reduces diabetes onset and its vascular complications. The mechanisms at the basis of the relation between vitamin B6 and diabetes onset are still not completely clarified. In contrast more evidence indicates that vitamin B6 can protect from diabetes complications through its role as scavenger of AGEs. It has been demonstrated that in diabetes AGEs can destroy the functionality of macromolecules such as protein, lipids, and DNA, thus producing tissue damage that result in vascular diseases. AGEs can be in part also responsible for the increased cancer risk associated with diabetes. In this chapter the relationship between vitamin B6, diabetes and AGEs will be discussed by showing the acquired knowledge and questions that are still open.

Variety of genetic defects in GnRH and hypothalamic-pituitary signaling and development in normosmic patients with IHH.

Introduction: Normosmic isolated hypogonadotropic hypogonadism (nIHH) is a clinically and genetically heterogeneous disorder. Deleterious variants in over 50 genes have been implicated in the etiology of IHH, which also indicates a possible role of digenicity and oligogenicity. Both classes of genes controlling GnRH neuron migration/development and hypothalamic/pituitary signaling and development are strongly implicated in nIHH pathogenesis. The study aimed to investigate the genetic background of nIHH and further expand the genotype-phenotype correlation. Methods: A total of 67 patients with nIHH were enrolled in the study. NGS technology and a 38-gene panel were applied. Results: Causative defects regarded as at least one pathogenic/likely pathogenic (P/LP) variant were found in 23 patients (34%). For another 30 individuals, variants of unknown significance (VUS) or benign (B) were evidenced (45%). The most frequently mutated genes presenting P/LP alterations were GNRHR (n = 5), TACR3 (n = 3), and CHD7, FGFR1, NSMF, BMP4, and NROB1 (n = 2 each). Monogenic variants with solid clinical significance (P/LP) were observed in 15% of subjects, whereas oligogenic defects were detected in 19% of patients. Regarding recurrence, 17 novel pathogenic variants affecting 10 genes were identified for 17 patients. The most recurrent pathogenic change was GNRHR:p.Arg139His, detected in four unrelated subjects. Another interesting observation is that P/LP defects were found more often in genes related to hypothalamic-pituitary pathways than those related to GnRH. Conclusions: The growing importance of the neuroendocrine pathway and related genes is drawing increasing attention to nIHH. However, the underestimated potential of VUS variants in IHH etiology, particularly those presenting recurrence, should be further elucidated.

A combined experimental and computational investigation on the OH radical and Cl atom-initiated reaction of 2,3-dichloropropene in troposphere.

Temperature-dependent kinetics of OH radical and Cl atom-initiated reaction of an important halogenated alkene, 2,3-Dichloropropene (23DCP), were investigated using absolute and relative methods over 278-363 K. Pulsed laser photolysis - laser induced fluorescence technique and relative rate method using gas chromatography with flame ionization detector were employed for studying the kinetics of 23DCP with OH radical and Cl atom, respectively. The obtained Arrhenius expressions were kOH(expt)=(4.08 ± 1.63) × 10-13exp{(1043 ± 124)/T} cm3 molecule-1 s-1 and kCl(expt)=(1.54 ± 0.24) × 10-11exp{(705 ± 48)/T} cm3 molecule-1 s-1. Computational calculations were conducted to validate our experimental kinetic results and provide new insights into the importance of a particular pathway among all based on thermodynamic parameters. The addition of OH/Cl to the terminal carbon of the double bond present in 23DCP proved to be the predominant pathway across the selected temperature range for the present study (200-400 K). The degradation mechanism of these reactions was proposed by analyzing the products with the aid of gas chromatography with mass spectrometry. Calculating various atmospheric implication parameters can help to understand how the release of 23DCP may affect the troposphere.

Human-derived fecal microbiota transplantation alleviates social deficits of the BTBR mouse model of autism through a potential mechanism involving vitamin B6 metabolism.

Autism spectrum disorder (ASD) is a heterogeneous neurodevelopmental condition characterized by social communication deficiencies and stereotypic behaviors influenced by hereditary and/or environmental risk factors. There are currently no approved medications for treating the core symptoms of ASD. Human fecal microbiota transplantation (FMT) has emerged as a potential intervention to improve autistic symptoms, but the underlying mechanisms are not fully understood. In this study, we evaluated the effects of human-derived FMT on behavioral and multi-omics profiles of the BTBR mice, an established model for ASD. FMT effectively alleviated the social deficits in the BTBR mice and normalized their distinct plasma metabolic profile, notably reducing the elevated long-chain acylcarnitines. Integrative analysis linked these phenotypic changes to specific Bacteroides species and vitamin B6 metabolism. Indeed, vitamin B6 supplementation improved the social behaviors in BTBR mice. Collectively, these findings shed new light on the interplay between FMT and vitamin B6 metabolism and revealed a potential mechanism underlying the therapeutic role of FMT in ASD.IMPORTANCEAccumulating evidence supports the beneficial effects of human fecal microbiota transplantation (FMT) on symptoms associated with autism spectrum disorder (ASD). However, the precise mechanism by which FMT induces a shift in the microbiota and leads to symptom improvement remains incompletely understood. This study integrated data from colon-content metagenomics, colon-content metabolomics, and plasma metabolomics to investigate the effects of FMT treatment on the BTBR mouse model for ASD. The analysis linked the amelioration of social deficits following FMT treatment to the restoration of mitochondrial function and the modulation of vitamin B6 metabolism. Bacterial species and compounds with beneficial roles in vitamin B6 metabolism and mitochondrial function may further contribute to improving FMT products and designing novel therapies for ASD treatment.

Structure and dynamics of the staphylococcal pyridoxal 5-phosphate synthase complex reveal transient interactions at the enzyme interface.

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.

Carbenoxolone mitigates extensive fibrosis formation in PLP-induced EAE model and multiple sclerosis serum-exposed pericyte culture.

Introduction: Multiple sclerosis (MS) is one of the most common causes of disability in young adults. Nearly, 85% of MS cases start with attacks and remissions, classified as relapsing-remitting multiple sclerosis (RRMS). With repeating attacks, MS causes brain-spinal cord atrophy and enhanced disability as disease progresses. PLP-induced EAE is one of the most established models for pathophysiology and treatment of RRMS. Recent studies demonstrated the possible role of pericytes in perivascular and intra-lesional fibrosis in PLP-induced EAE, whose importance remains elusive. Hence, we have investigated the possible role of pericytes in fibrosis formation and amelioration with a hemichannel blocker, Carbenoxolone (CBX). Methods: PLP-induced experimental autoimmune encephalitis (EAE) model is used and the effect of CBX is investigated. Clinical scores were recorded and followed. Perivascular Collagen 1 and 3 accumulations were demonstrated as markers of fibrosis in the spinal cord. To delineate the role of pericytes, human brain vascular pericytes (HBVP) were incubated with the sera of MS patients to induce in-vitro MS model and the fibrosis formation was investigated. Results: In the PLP induced in-vivo model, both intracerebroventricular and intraperitoneal CBX have significantly mitigated the disease progression followed by clinical scores, demyelination, and fibrosis. Moreover, CBX significantly mitigated MS-serum-induced fibrosis in the HBVP cell culture. Discussion: The study demonstrated two important findings. First, CBX decreases fibrosis formation in both in-vivo and in-vitro MS models. Secondly, it improves neurological scores and decreases demyelination in the EAE model. Therefore, CBX can be potential novel therapeutic option in treating Multiple Sclerosis.

Associations of serum vitamin B6 status with the risks of cardiovascular, cancer, and all-cause mortality in the elderly.

Background: There are few studies investigating the relationship between serum vitamin B6 and mortality risk in the elderly. This study hereby evaluated the associations between biomarkers of serum vitamin B6 status and cardiovascular, cancer, and all-cause mortality risks in the elderly. Methods: Our study included a total of 4,881 participants aged 60 years or older from the National Health and Nutrition Examination Survey (NHANES) 2005-2010. Serum vitamin B6 status was estimated based on levels of pyridoxal 5'-phosphate (PLP), 4-pyridoxic acid (4-PA), and vitamin B6 turnover rate (4-PA/PLP) detected by high-performance liquid chromatography. Survival status and corresponding causes of death were matched through the National Death Index records through December 31, 2019. Multivariate Cox regression model was adopted to assess the relationships between serum vitamin B6 status and the risk of mortality. Results: During a median follow-up period of 10.33 years, 507 cardiovascular deaths, 426 cancer deaths, and 1995 all-cause deaths were recorded, respectively. In the multivariate-adjusted Cox model, the hazard ratios (HRs) and 95% confidence intervals (CIs) for the highest versus the lowest quartiles of PLP, 4-PA, and 4-PA/PLP were 0.70(0.54-0.90), 1.33(0.88-2.02), and 2.01(1.41-2.79) for cardiovascular mortality, 0.73(0.52-1.02), 1.05(0.71-1.57), and 1.95(1.25-3.05) for cancer mortality, and 0.62(0.53-0.74), 1.05(0.82-1.34), and 2.29(1.87-2.79) for all-cause mortality, respectively. Conclusion: Our study found that lower serum PLP levels were associated with increased risks of cardiovascular and all-cause mortality among the elderly population. And higher vitamin B6 turnover rate was associated with increased risks of cardiovascular, cancer, and all-cause mortality.

Whole exome sequencing identified a homozygous novel variant in DOP1A gene in the Pakistan family with neurodevelopmental disabilities: case report and literature review.

Background: Hereditary neurodevelopmental disorders (NDDs) are prevalent in poorly prognostic pediatric diseases, but the pathogenesis of NDDs is still unclear. Irregular myelination could be one of the possible causes of NDDs. Case presentation: Here, whole exome sequencing was carried out for a consanguineous Pakistani family with NDDs to identify disease-associated variants. The co-segregation of candidate variants in the family was validated using Sanger sequencing. The potential impact of the gene on NDDs has been supported by conservation analysis, protein prediction, and expression analysis. A novel homozygous variant DOP1A(NM_001385863.1):c.2561A>G was identified. It was concluded that the missense variant might affect the protein-protein binding sites of the critical MEC interaction region of DOP1A, and DOP1A-MON2 may cause stability deficits in Golgi-endosome protein traffic. Proteolipid protein (PLP) and myelin-associate glycoprotein (MAG) could be targets of the DOP1A-MON2 Golgi-endosome traffic complex, especially during the fetal stage and the early developmental stages. This further supports the perspective that disorganized myelinogenesis due to congenital DOP1A deficiency might cause neurodevelopmental disorders (NDDs). Conclusion: Our case study revealed the potential pathway of myelinogenesis-relevant NDDs and identified DOP1A as a potential NDDs-relevant gene in humans.

Fine-Tuning Pyridoxal 5'-Phosphate Synthesis in Escherichia coli for Cadaverine Production in Minimal Culture Media.

Cadaverine is a critical C5 monomer for the production of polyamides. Pyridoxal 5'-phosphate (PLP), as a crucial cofactor for the key enzyme lysine decarboxylase in the cadaverine biosynthesis pathway, has seen a persistent shortage, leading to limitations in cadaverine production. To address this issue, a dual-pathway strategy was implemented, synergistically enhancing both endogenous and heterologous PLP synthesis modules and resulting in improved PLP synthesis. Subsequently, a growth-stage-dependent molecular switch was introduced to balance the precursor competition between PLP synthesis and cell growth. Additionally, a PLP sensor-based negative feedback circuit was constructed by integrating a newly identified PLP-responsive promoter PygjH and an arabinose-regulated system, dynamically regulating the expression of the PLP synthetic genes and preventing excessive intracellular PLP accumulation. The optimal strain, L18, cultivated in the minimal medium AM1, demonstrated cadaverine production with a titer, yield, and productivity of 64.03 g/L, 0.23 g/g glucose, and 1.33 g/L/h, respectively. This represents the highest titer reported to date in engineered Escherichia coli by fed-batch fermentation in a minimal medium.

Edodin: A New Type of Toxin from Shiitake Mushroom (Lentinula edodes) That Inactivates Mammalian Ribosomes.

Ribosome-inactivating proteins (RIPs) are a group of proteins with rRNA N-glycosylase activity that irreversibly inhibit protein synthesis and consequently cause cell death. Recently, an RIP called ledodin has been found in shiitake; it is cytotoxic, strongly inhibits protein synthesis, and shows rRNA N-glycosylase activity. In this work, we isolated and characterized a 50 kDa cytotoxic protein from shiitake that we named edodin. Edodin inhibits protein synthesis in a mammalian cell-free system, but not in insect-, yeast-, and bacteria-derived systems. It exhibits rRNA N-glycosylase and DNA-nicking activities, which relate it to plant RIPs. It was also shown to be toxic to HeLa and COLO 320 cells. Its structure is not related to other RIPs found in plants, bacteria, or fungi, but, instead, it presents the characteristic structure of the fold type I of pyridoxal phosphate-dependent enzymes. Homologous sequences have been found in other fungi of the class Agaricomycetes; thus, edodin could be a new type of toxin present in many fungi, some of them edible, which makes them of great interest in health, both for their involvement in food safety and for their potential biomedical and biotechnological applications.

Pelizaeus-Merzbacher disease: on the cusp of myelin medicine.

Pelizaeus-Merzbacher disease (PMD) is caused by mutations in the proteolipid protein 1 (PLP1) gene encoding proteolipid protein (PLP). As a major component of myelin, mutated PLP causes progressive neurodegeneration and eventually death due to severe white matter deficits. Medical care has long been limited to symptomatic treatments, but first-in-class PMD therapies with novel mechanisms now stand poised to enter clinical trials. Here, we review PMD disease mechanisms and outline rationale for therapeutic interventions, including PLP1 suppression, cell transplantation, iron chelation, and intracellular stress modulation. We discuss available preclinical data and their implications on clinical development. With several novel treatments on the horizon, PMD is on the precipice of a new era in the diagnosis and treatment of patients suffering from this debilitating disease.

Rational Design of Salmonella typhi Acid Phosphatase for Efficient Production of Pyridoxal 5'-Phosphate.

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 Salmonella typhi (StAPase) and pyridoxine oxidase from Escherichia coli (EcPNPO) as pathway enzymes was established for the first time to synthesize PLP from pyridoxine (PN) and pyrophosphate (PPi). StAPase 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 StAPase and (2) enhancing the hydrophobicity of StAPase's substrate binding pocket. The kcat/Km of optimal mutant M7 was 4.9 times higher than that of the wild type. The detailed mechanism of performance improvement was analyzed. Under the catalysis of M7 and EcPNPO, 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.

Biosynthesis of Strained Amino Acids by a PLP-Dependent Enzyme through Cryptic Halogenation.

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.

Decoding the tumor microenvironment and molecular mechanism: unraveling cervical cancer subpopulations and prognostic signatures through scRNA-Seq and bulk RNA-seq analyses.

Background: Cervical carcinoma (CC) represents a prevalent gynecological neoplasm, with a discernible rise in prevalence among younger cohorts observed in recent years. Nonetheless, the intrinsic cellular heterogeneity of CC remains inadequately investigated. Methods: We utilized single-cell RNA sequencing (scRNA-seq) transcriptomic analysis to scrutinize the tumor epithelial cells derived from four specimens of cervical carcinoma (CC) patients. This method enabled the identification of pivotal subpopulations of tumor epithelial cells and elucidation of their contributions to CC progression. Subsequently, we assessed the influence of associated molecules in bulk RNA sequencing (Bulk RNA-seq) cohorts and performed cellular experiments for validation purposes. Results: Through our analysis, we have discerned C3 PLP2+ Tumor Epithelial Progenitor Cells as a noteworthy subpopulation in cervical carcinoma (CC), exerting a pivotal influence on the differentiation and progression of CC. We have established an independent prognostic indicator-the PLP2+ Tumor EPCs score. By stratifying patients into high and low score groups based on the median score, we have observed that the high-score group exhibits diminished survival rates compared to the low-score group. The correlations observed between these groups and immune infiltration, enriched pathways, single-nucleotide polymorphisms (SNPs), drug sensitivity, among other factors, further underscore their impact on CC prognosis. Cellular experiments have validated the significant impact of ATF6 on the proliferation and migration of CC cell lines. Conclusion: This study enriches our comprehension of the determinants shaping the progression of CC, elevates cognizance of the tumor microenvironment in CC, and offers valuable insights for prospective CC therapies. These discoveries contribute to the refinement of CC diagnostics and the formulation of optimal therapeutic approaches.

Pyridoxal 5'-Phosphate Biosynthesis by Pyridox-(am)-ine 5'-Phosphate Oxidase: Species-Specific Features.

Enzymes reliant on pyridoxal 5'-phosphate (PLP), the metabolically active form of vitamin B6, hold significant importance in both biology and medicine. They facilitate various biochemical reactions, particularly in amino acid and neurotransmitter metabolisms. Vitamin B6 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 B6 vitamers. However, for those depending on vitamin B6 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.

Phosphatase and Tensin Homolog Inhibition in Proteolipid Protein 1-Expressing Cells Stimulates Neurogenesis and Gliogenesis in the Postnatal Enteric Nervous System.

Phosphatase and tensin homolog (Pten) is a key regulator of cell proliferation and a potential target to stimulate postnatal enteric neuro- and/or gliogenesis. To investigate this, we generated two tamoxifen-inducible Cre recombinase murine models in which Pten was conditionally ablated, (1) in glia (Plp1-expressing cells) and (2) in neurons (Calb2-expressing cells). Tamoxifen-treated adult (7-12 weeks of age; n = 4-15) mice were given DSS to induce colitis, EdU to monitor cell proliferation, and were evaluated at two timepoints: (1) early (3-4 days post-DSS) and (2) late (3-4 weeks post-DSS). We investigated gut motility and evaluated the enteric nervous system. Pten inhibition in Plp1-expressing cells elicited gliogenesis at baseline and post-DSS (early and late) in the colon, and neurogenesis post-DSS late in the proximal colon. They also exhibited an increased frequency of colonic migrating motor complexes (CMMC) and slower whole gut transit times. Pten inhibition in Calb2-expressing cells did not induce enteric neuro- or gliogenesis, and no alterations were detected in CMMC or whole gut transit times when compared to the control at baseline or post-DSS (early and late). Our results merit further research into Pten modulation where increased glia and/or slower intestinal transit times are desired (e.g., short-bowel syndrome and rapid-transit disorders).

CLPP-Null Eukaryotes with Excess Heme Biosynthesis Show Reduced L-arginine Levels, Probably via CLPX-Mediated OAT Activation.

The serine peptidase CLPP is conserved among bacteria, chloroplasts, and mitochondria. In humans and mice, its loss causes Perrault syndrome, which presents with growth deficits, infertility, deafness, and ataxia. In the filamentous fungus Podospora anserina, CLPP loss leads to longevity. CLPP substrates are selected by CLPX, an AAA+ unfoldase. CLPX is known to target delta-aminolevulinic acid synthase (ALAS) to promote pyridoxal phosphate (PLP) binding. CLPX may also influence cofactor association with other enzymes. Here, the evaluation of P. anserina metabolomics highlighted a reduction in arginine/histidine levels. In Mus musculus cerebellum, reductions in arginine/histidine and citrulline occurred with a concomitant accumulation of the heme precursor protoporphyrin IX. This suggests that the increased biosynthesis of 5-carbon (C5) chain deltaALA consumes not only C4 succinyl-CoA and C1 glycine but also specific C5 delta amino acids. As enzymes responsible for these effects, the elevated abundance of CLPX and ALAS is paralleled by increased OAT (PLP-dependent, ornithine delta-aminotransferase) levels. Possibly as a consequence of altered C1 metabolism, the proteome profiles of P. anserina CLPP-null cells showed strong accumulation of a methyltransferase and two mitoribosomal large subunit factors. The reduced histidine levels may explain the previously observed metal interaction problems. As the main nitrogen-storing metabolite, a deficiency in arginine would affect the urea cycle and polyamine synthesis. Supplementation of arginine and histidine might rescue the growth deficits of CLPP-mutant patients.

Racemization of the substrate and product by serine palmitoyltransferase from Sphingobacterium multivorum yields two enantiomers of the product from d-serine.

Serine palmitoyltransferase (SPT) catalyzes the pyridoxal-5'-phosphate (PLP)-dependent decarboxylative condensation of l-serine and palmitoyl-CoA to form 3-ketodihydrosphingosine (KDS). Although SPT was shown to synthesize corresponding products from amino acids other than l-serine, it is still arguable whether SPT catalyzes the reaction with d-serine, which is a question of biological importance. Using high substrate and enzyme concentrations, KDS was detected after the incubation of SPT from Sphingobacterium multivorum with d-serine and palmitoyl-CoA. Furthermore, the KDS comprised equal amounts of 2S and 2R isomers. 1H-NMR study showed a slow hydrogen-deuterium exchange at Cα of serine mediated by SPT. We further confirmed that SPT catalyzed the racemization of serine. The rate of the KDS formation from d-serine was comparable to those for the α-hydrogen exchange and the racemization reaction. The structure of the d-serine-soaked crystal (1.65 Å resolution) showed a distinct electron density of the PLP-l-serine aldimine, interpreted as the racemized product trapped in the active site. The structure of the α-methyl-d-serine-soaked crystal (1.70 Å resolution) showed the PLP-α-methyl-d-serine aldimine, mimicking the d-serine-SPT complex prior to racemization. Based on these enzymological and structural analyses, the synthesis of KDS from d-serine was explained as the result of the slow racemization to l-serine, followed by the reaction with palmitoyl-CoA, and SPT would not catalyze the direct condensation between d-serine and palmitoyl-CoA. It was also shown that the S. multivorum SPT catalyzed the racemization of the product KDS, which would explain the presence of (2R)-KDS in the reaction products.