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.

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.

The pathway for coenzyme M biosynthesis in bacteria.

Mercaptoethane sulfonate or coenzyme M (CoM) is the smallest known organic cofactor and is most commonly associated with the methane-forming step in all methanogenic archaea but is also associated with the anaerobic oxidation of methane to CO2 in anaerobic methanotrophic archaea and the oxidation of short-chain alkanes in Syntrophoarchaeum species. It has also been found in a small number of bacteria capable of the metabolism of small organics. Although many of the steps for CoM biosynthesis in methanogenic archaea have been elucidated, a complete pathway for the biosynthesis of CoM in archaea or bacteria has not been reported. Here, we present the complete CoM biosynthesis pathway in bacteria, revealing distinct chemical steps relative to CoM biosynthesis in methanogenic archaea. The existence of different pathways represents a profound instance of convergent evolution. The five-step pathway involves the addition of sulfite, the elimination of phosphate, decarboxylation, thiolation, and the reduction to affect the sequential conversion of phosphoenolpyruvate to CoM. The salient features of the pathway demonstrate reactivities for members of large aspartase/fumarase and pyridoxal 5'-phosphate-dependent enzyme families.

Atomic-resolution chemical characterization of (2x)72-kDa tryptophan synthase via four- and five-dimensional 1H-detected solid-state NMR.

NMR chemical shifts provide detailed information on the chemical properties of molecules, thereby complementing structural data from techniques like X-ray crystallography and electron microscopy. Detailed analysis of protein NMR data, however, often hinges on comprehensive, site-specific assignment of backbone resonances, which becomes a bottleneck for molecular weights beyond 40 to 45 kDa. Here, we show that assignments for the (2x)72-kDa protein tryptophan synthase (665 amino acids per asymmetric unit) can be achieved via higher-dimensional, proton-detected, solid-state NMR using a single, 1-mg, uniformly labeled, microcrystalline sample. This framework grants access to atom-specific characterization of chemical properties and relaxation for the backbone and side chains, including those residues important for the catalytic turnover. Combined with first-principles calculations, the chemical shifts in the ß-subunit active site suggest a connection between active-site chemistry, the electrostatic environment, and catalytically important dynamics of the portal to the ß-subunit from solution.

Structural insights into the substrate recognition of serine palmitoyltransferase from Sphingobacterium multivorum.

Serine palmitoyltransferase (SPT) is a key enzyme of sphingolipid biosynthesis, which catalyzes the pyridoxal-5'-phosphate-dependent decarboxylative condensation reaction of l-serine (l-Ser) and palmitoyl-CoA (PalCoA) to form 3-ketodihydrosphingosine called long chain base (LCB). SPT is also able to metabolize l-alanine (l-Ala) and glycine (Gly), albeit with much lower efficiency. Human SPT is a membrane-bound large protein complex containing SPTLC1/SPTLC2 heterodimer as the core subunits, and it is known that mutations of the SPTLC1/SPTLC2 genes increase the formation of deoxy-type of LCBs derived from l-Ala and Gly to cause some neurodegenerative diseases. In order to study the substrate recognition of SPT, we examined the reactivity of Sphingobacterium multivorum SPT on various amino acids in the presence of PalCoA. The S. multivorum SPT could convert not only l-Ala and Gly but also l-homoserine, in addition to l-Ser, into the corresponding LCBs. Furthermore, we obtained high-quality crystals of the ligand-free form and the binary complexes with a series of amino acids, including a nonproductive amino acid, l-threonine, and determined the structures at 1.40 to 1.55 Å resolutions. The S. multivorum SPT accommodated various amino acid substrates through subtle rearrangements of the active-site amino acid residues and water molecules. It was also suggested that non-active-site residues mutated in the human SPT genes might indirectly influence the substrate specificity by affecting the hydrogen-bonding networks involving the bound substrate, water molecules, and amino acid residues in the active site of this enzyme. Collectively, our results highlight SPT structural features affecting substrate specificity for this stage of sphingolipid biosynthesis.

One-pot chemoenzymatic syntheses of non-canonical amino acids.

Despite their prevalent use in drug discovery and protein biochemistry, non-canonical amino acids are still challenging to synthesize through purely chemical means. In recent years, biocatalysis has emerged as a transformative paradigm for small-molecule synthesis. One strategy to further empower biocatalysis is to use it in combination with modern chemical reactions and take advantage of the strengths of each method to enable access to challenging structural motifs that were previously unattainable using each method alone. In this Mini-Review, we highlight several recent case studies that feature the synergistic use of chemical and enzymatic transformations in one pot to synthesize novel non-canonical amino acids. ONE-SENTENCE SUMMARY: This Mini-Review highlights several recent case studies that feature the synergistic use of chemical and enzymatic transformations in one pot to synthesize novel non-canonical amino acids.

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.

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.

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.

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.

Evolutionary origin and functional diversification of aminotransferases.

Aminotransferases (ATs) are pyridoxal 5'-phosphate-dependent enzymes that catalyze the transamination reactions between amino acid donor and keto acid acceptor substrates. Modern AT enzymes constitute ∼2% of all classified enzymatic activities, play central roles in nitrogen metabolism, and generate multitude of primary and secondary metabolites. ATs likely diverged into four distinct AT classes before the appearance of the last universal common ancestor and further expanded to a large and diverse enzyme family. Although the AT family underwent an extensive functional specialization, many AT enzymes retained considerable substrate promiscuity and multifunctionality because of their inherent mechanistic, structural, and functional constraints. This review summarizes the evolutionary history, diverse metabolic roles, reaction mechanisms, and structure-function relationships of the AT family enzymes, with a special emphasis on their substrate promiscuity and multifunctionality. Comprehensive characterization of AT substrate specificity is still needed to reveal their true metabolic functions in interconnecting various branches of the nitrogen metabolic network in different organisms.

Clinical and biochemical impact of vitamin B6 deficiency in primary sclerosing cholangitis before and after liver transplantation.

BACKGROUND AND AIMS: We previously demonstrated that people with primary sclerosing cholangitis (PSC) had reduced gut microbial capacity to produce active vitamin B6 (pyridoxal 5'-phosphate [PLP]), which corresponded to lower circulating PLP levels and poor outcomes. Here, we define the extent and biochemical and clinical impact of vitamin B6 deficiency in people with PSC from several centers before and after liver transplantation (LT). METHODS: We used targeted liquid chromatography-tandem mass spectrometry to measure B6 vitamers and B6-related metabolic changes in blood from geographically distinct cross-sectional cohorts totaling 373 people with PSC and 100 healthy controls to expand on our earlier findings. Furthermore, we included a longitudinal PSC cohort (n = 158) sampled prior to and serially after LT, and cohorts of people with inflammatory bowel disease (IBD) without PSC (n = 51) or with primary biliary cholangitis (PBC) (n = 100), as disease controls. We used Cox regression to measure the added value of PLP to predict outcomes before and after LT. RESULTS: In different cohorts, 17-38% of people with PSC had PLP levels below the biochemical definition of a vitamin B6 deficiency. The deficiency was more pronounced in PSC than in IBD without PSC and PBC. Reduced PLP was associated with dysregulation of PLP-dependent pathways. The low B6 status largely persisted after LT. Low PLP independently predicted reduced LT-free survival in both non-transplanted people with PSC and in transplant recipients with recurrent disease. CONCLUSIONS: Low vitamin B6 status with associated metabolic dysregulation is a persistent feature of PSC. PLP was a strong prognostic biomarker for LT-free survival both in PSC and recurrent disease. Our findings suggest that vitamin B6 deficiency modifies the disease and provides a rationale for assessing B6 status and testing supplementation. IMPACT AND IMPLICATIONS: We previously found that people with PSC had reduced gut microbial potential to produce essential nutrients. Across several cohorts, we find that the majority of people with PSC are either vitamin B6 deficient or have a marginal deficiency, which remains prevalent even after liver transplantation. Low vitamin B6 levels strongly associate with reduced liver transplantation-free survival as well as deficits in biochemical pathways dependent on vitamin B6, suggesting that the deficiency has a clinical impact on the disease. The results provide a rationale for measuring vitamin B6 and to investigate whether vitamin B6 supplementation or modification of the gut microbial community can help improve outcomes for people with PSC.

PLP2 drives collective cell migration via ZO-1-mediated cytoskeletal remodeling at the leading edge in human colorectal cancer cells.

Collective cell migration (CCM), in which cell-cell integrity remains preserved during movement, plays an important role in the progression of cancer. However, studies describing CCM in cancer progression are majorly focused on the effects of extracellular tissue components on moving cell plasticity. The molecular and cellular mechanisms of CCM during cancer progression remain poorly explored. Here, we report that proteolipid protein 2 (PLP2), a colonic epithelium-enriched transmembrane protein, plays a vital role in the CCM of invasive human colorectal cancer (CRC) epithelium by modulating leading-edge cell dynamics in 2D. The extracellular pool of PLP2, secreted via exosomes, was also found to contribute to the event. During CCM, the protein was found to exist in association with ZO-1 (also known as TJP1) and to be involved in the positioning of the latter at the migrating edge. PLP2-mediated positioning of ZO-1 at the leading edge further alters actin cytoskeletal organization that involves Rac1 activation. Taken together, our findings demonstrate that PLP2, via its association with ZO-1, drives CCM in CRC epithelium by modulating the leading-edge actin cytoskeleton, thereby opening up new avenues of cancer research. This article has an associated First Person interview with the first author of the paper.

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.

Cronobacter sakazakii Pyridoxal Kinase PdxY Mediated by TreR and pESA3 Is Essential for Vitamin B6 (PLP) Maintenance and Virulence.

Cronobacter sakazakii is an opportunistic pathogen capable of causing severe infections, particularly in neonates. Despite the bacterium's strong pathogenicity, the pathogenicity of C. sakazakii is not yet well understood. Using a comparative proteomic profiling approach, we successfully identified pdxY, encoding a pyridoxal kinase involved in the recycling of pyridoxal 5'-phosphate (PLP), as a gene essential for the successful pathogenesis of C. sakazakii. Knocking out the pdxY gene resulted in slower growth and reduced virulence. Our study sheds light on the fundamental importance of pyridoxal kinase for the survival and virulence of C. sakazakii. The identification of pdxY as gene essential for successful pathogenesis provides a potential target for the development of new antibiotic treatments. IMPORTANCE The opportunistic pathogen Cronobacter sakazakii is known to cause severe infections, particularly in neonates, and can result in high mortality rates. In this study, we used a comparative proteomic profiling approach to identify genes essential for the successful pathogenesis of C. sakazakii. We successfully identified pdxY, encoding a pyridoxal kinase involved in the salvage pathway of pyridoxal 5'-phosphate (PLP), as a gene essential for the successful pathogenesis of C. sakazakii. Knocking out the pdxY gene resulted in impaired growth and reduced virulence. This study sheds light on the fundamental importance of pyridoxal kinase for the survival and virulence of C. sakazakii, which can be a potential target for the development of new antibiotic treatments. This study highlights the importance of comparative proteomic profiling in identifying virulence factors that can be targeted for the development of new antibiotics.

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.

Impact of lifestyle factors on dietary vitamin B6 intake and plasma pyridoxal 5'-phosphate level in UK adults: National Diet and Nutrition Survey Rolling Programme (NDNS) (2008-2017).

Reduction in dietary vitamin B6 intake is associated with an increased relative risk of diseases such as cancer, atherosclerosis and cognitive dysfunction. The current research has assessed vitamin B6 intakes and PLP concentrations as a marker of vitamin B6 status among the UK adult (≥ 19 years) population. This study was carried out using a cross-sectional analysis of the National Diet and Nutrition Survey Rolling Programme (NDNS) (2008-2017). The impacts of lifestyle factors, including type of diet, smoking, alcohol consumption, and commonly used medications grouped by therapeutic usage, were determined, and data were analysed using IBM SPSS®. Results are expressed as medians (25th-75th percentiles), with P values ≤ 0·05 considered statistically significant. Among UK adults, the median intakes of total population of dietary vitamin B6 met the reference nutrient intake and median plasma PLP concentrations were above the cut-off of vitamin B6 deficiency; however, we found an association between reduction in vitamin B6 intake and plasma PLP concentration and age group (P < 0·001). Smokers had significantly lower plasma PLP concentrations than non-smokers (P < 0·001). Moreover, regression analysis showed some commonly used medications were associated with plasma PLP levels reduction (P < 0·05). Taken together, we report on a tendency for dietary vitamin B6 intake and plasma PLP concentrations to decrease with age and lifestyle factors such as smoking and medication usage. This information could have important implications for smokers and in the elderly population using multiple medications (polypharmacy).

Defining the scope of extended NIPS in Western China: evidence from a large cohort of fetuses with normal ultrasound scans.

BACKGROUND: Standard noninvasive prenatal screening(NIPS) is an accurate and reliable method to screen for common chromosome aneuploidies, such as trisomy 21, 18 and 13. Extended NIPS has been used in clinic for not only aneuploidies but also copy number variants(CNVs). Here we aim to define the range of chromosomal abnormalities that should be able to identify by NIPS in order to be an efficient extended screening test for chromosomal abnormalities. METHODS: A prospective study was conducted, involving pregnant women without fetal sonographic structural abnormalities who underwent amniocentesis. Prenatal samples were analyzed using copy number variation sequencing(CNV-seq) to identify fetal chromosomal abnormalities. RESULTS: Of 28,469 pregnancies included 1,022 (3.59%) were identified with clinically significant fetal chromosome abnormalities, including 587 aneuploidies (2.06%) and 435 (1.53%) pathogenic (P) / likely pathogenic (LP) CNVs. P/LP CNVs were found in all chromosomes, but the distribution was not uniform. Among them, P/LP CNVs in chromosomes 16, 22, and X exhibited the highest frequencies. In addition, P/LP CNVs were most common on distal ends of the chromosomes and in low copy repeat regions. Recurrent microdeletion/microduplication syndromes (MMS) accounted for 40.69% of total P/LP CNVs. The size of most P/LP CNVs (77.47%) was < 3 Mb. CONCLUSIONS: In addition to aneuploidies, the scope of extended NIPS should include the currently known P/LP CNVs, especially the regions with recurrent MMS loci, distal ends of the chromosomes, and low copy repeat regions. To be effective detection should include CNVs of < 3 Mb. Meanwhile, sufficient preclinical validation is still needed to ensure the clinical effect of extended NIPS.

Pyridoxal kinase inhibition by artemisinins down-regulates inhibitory neurotransmission.

The antimalarial artemisinins have also been implicated in the regulation of various cellular pathways including immunomodulation of cancers and regulation of pancreatic cell signaling in mammals. Despite their widespread application, the cellular specificities and molecular mechanisms of target recognition by artemisinins remain poorly characterized. We recently demonstrated how these drugs modulate inhibitory postsynaptic signaling by direct binding to the postsynaptic scaffolding protein gephyrin. Here, we report the crystal structure of the central metabolic enzyme pyridoxal kinase (PDXK), which catalyzes the production of the active form of vitamin B6 (also known as pyridoxal 5'-phosphate [PLP]), in complex with artesunate at 2.4-Šresolution. Partially overlapping binding of artemisinins with the substrate pyridoxal inhibits PLP biosynthesis as demonstrated by kinetic measurements. Electrophysiological recordings from hippocampal slices and activity measurements of glutamic acid decarboxylase (GAD), a PLP-dependent enzyme synthesizing the neurotransmitter γ-aminobutyric acid (GABA), define how artemisinins also interfere presynaptically with GABAergic signaling. Our data provide a comprehensive picture of artemisinin-induced effects on inhibitory signaling in the brain.

Pyridoxal-5'-phosphate-dependent bifunctional enzyme catalyzed biosynthesis of indolizidine alkaloids in fungi.

Indolizidine alkaloids such as anticancer drugs vinblastine and vincristine are exceptionally attractive due to their widespread occurrence, prominent bioactivity, complex structure, and sophisticated involvement in the chemical defense for the producing organisms. However, the versatility of the indolizidine alkaloid biosynthesis remains incompletely addressed since the knowledge about such biosynthetic machineries is only limited to several representatives. Herein, we describe the biosynthetic gene cluster (BGC) for the biosynthesis of curvulamine, a skeletally unprecedented antibacterial indolizidine alkaloid from Curvularia sp. IFB-Z10. The molecular architecture of curvulamine results from the functional collaboration of a highly reducing polyketide synthase (CuaA), a pyridoxal-5'-phosphate (PLP)-dependent aminotransferase (CuaB), an NADPH-dependent dehydrogenase (CuaC), and a FAD-dependent monooxygenase (CuaD), with its transportation and abundance regulated by a major facilitator superfamily permease (CuaE) and a Zn(II)Cys6 transcription factor (CuaF), respectively. In contrast to expectations, CuaB is bifunctional and capable of catalyzing the Claisen condensation to form a new C-C bond and the α-hydroxylation of the alanine moiety in exposure to dioxygen. Inspired and guided by the distinct function of CuaB, our genome mining effort discovers bipolamines A-I (bipolamine G is more antibacterial than curvulamine), which represent a collection of previously undescribed polyketide alkaloids from a silent BGC in Bipolaris maydis ATCC48331. The work provides insight into nature's arsenal for the indolizidine-coined skeletal formation and adds evidence in support of the functional versatility of PLP-dependent enzymes in fungi.