Biochemical and structural impact of two novel missense mutations in cystathionine ß-synthase gene associated with homocystinuria.

Homocystinuria is a rare disease caused by mutations in the CBS gene that results in a deficiency of cystathionine ß-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.

Imaging active site chemistry and protonation states: NMR crystallography of the tryptophan synthase α-aminoacrylate intermediate.

NMR-assisted crystallography-the integrated application of solid-state NMR, X-ray crystallography, and first-principles computational chemistry-holds significant promise for mechanistic enzymology: by providing atomic-resolution characterization of stable intermediates in enzyme active sites, including hydrogen atom locations and tautomeric equilibria, NMR crystallography offers insight into both structure and chemical dynamics. Here, this integrated approach is used to characterize the tryptophan synthase α-aminoacrylate intermediate, a defining species for pyridoxal-5'-phosphate-dependent enzymes that catalyze ß-elimination and replacement reactions. For this intermediate, NMR-assisted crystallography is able to identify the protonation states of the ionizable sites on the cofactor, substrate, and catalytic side chains as well as the location and orientation of crystallographic waters within the active site. Most notable is the water molecule immediately adjacent to the substrate ß-carbon, which serves as a hydrogen bond donor to the ε-amino group of the acid-base catalytic residue ßLys87. From this analysis, a detailed three-dimensional picture of structure and reactivity emerges, highlighting the fate of the L-serine hydroxyl leaving group and the reaction pathway back to the preceding transition state. Reaction of the α-aminoacrylate intermediate with benzimidazole, an isostere of the natural substrate indole, shows benzimidazole bound in the active site and poised for, but unable to initiate, the subsequent bond formation step. When modeled into the benzimidazole position, indole is positioned with C3 in contact with the α-aminoacrylate Cß and aligned for nucleophilic attack. Here, the chemically detailed, three-dimensional structure from NMR-assisted crystallography is key to understanding why benzimidazole does not react, while indole does.

The ß-latch structural element of the SufS cysteine desulfurase mediates active site accessibility and SufE transpersulfurase positioning.

Under oxidative stress and iron starvation conditions, Escherichia coli uses the Suf pathway to assemble iron-sulfur clusters. The Suf pathway mobilizes sulfur via SufS, a type II cysteine desulfurase. SufS is a pyridoxal-5'-phosphate-dependent enzyme that uses cysteine to generate alanine and an active-site persulfide (C364-S-S-). The SufS persulfide is protected from external oxidants/reductants and requires the transpersulfurase, SufE, to accept the persulfide to complete the SufS catalytic cycle. Recent reports on SufS identified a conserved "ß-latch" structural element that includes the α6 helix, a glycine-rich loop, a ß-hairpin, and a cis-proline residue. To identify a functional role for the ß-latch, we used site-directed mutagenesis to obtain the N99D and N99A SufS variants. N99 is a conserved residue that connects the α6 helix to the backbone of the glycine-rich loop via hydrogen bonds. Our x-ray crystal structures for N99A and N99D SufS show a distorted beta-hairpin and glycine-rich loop, respectively, along with changes in the dimer geometry. The structural disruption of the N99 variants allowed the external reductant TCEP to react with the active-site C364-persulfide intermediate to complete the SufS catalytic cycle in the absence of SufE. The substitutions also appear to disrupt formation of a high-affinity, close approach SufS-SufE complex as measured with fluorescence polarization. Collectively, these findings demonstrate that the ß-latch does not affect the chemistry of persulfide formation but does protect it from undesired reductants. The data also indicate the ß-latch plays an unexpected role in forming a close approach SufS-SufE complex to promote persulfide transfer.

Maintenance of cellular vitamin B6 levels and mitochondrial oxidative function depend on pyridoxal 5'-phosphate homeostasis protein.

Recently, biallelic variants in PLPBP coding for pyridoxal 5'-phosphate homeostasis protein (PLPHP) were identified as a novel cause of early-onset vitamin B6-dependent epilepsy. The molecular function and precise role of PLPHP in vitamin B6 metabolism are not well understood. To address these questions, we used PLPHP-deficient patient skin fibroblasts and HEK293 cells and YBL036C (PLPHP ortholog)-deficient yeast. We showed that independent of extracellular B6 vitamer type (pyridoxine, pyridoxamine, or pyridoxal), intracellular pyridoxal 5'-phosphate (PLP) was lower in PLPHP-deficient fibroblasts and HEK293 cells than controls. Culturing cells with pyridoxine or pyridoxamine led to the concentration-dependent accumulation of pyridoxine 5'-phosphate and pyridoxamine 5'-phosphate (PMP), respectively, suggesting insufficient pyridox(am)ine 5'-phosphate oxidase activity. Experiments utilizing 13C4-pyridoxine confirmed lower pyridox(am)ine 5'-phosphate oxidase activity and revealed increased fractional turnovers of PLP and pyridoxal, indicating increased PLP hydrolysis to pyridoxal in PLPHP-deficient cells. This effect could be partly counteracted by inactivation of pyridoxal phosphatase. PLPHP deficiency had a distinct effect on mitochondrial PLP and PMP, suggesting impaired activity of mitochondrial transaminases. Moreover, in YBL036C-deficient yeast, PLP was depleted and PMP accumulated only with carbon sources requiring mitochondrial metabolism. Lactate and pyruvate accumulation along with the decrease of tricarboxylic acid cycle intermediates downstream of α-ketoglutarate suggested impaired mitochondrial oxidative metabolism in PLPHP-deficient HEK293 cells. We hypothesize that impaired activity of mitochondrial transaminases may contribute to this depletion. Taken together, our study provides new insights into the pathomechanisms of PLPBP deficiency and reinforces the link between PLPHP function, vitamin B6 metabolism, and mitochondrial oxidative metabolism.

Mass spectrometry imaging-based assays for aminotransferase activity reveal a broad substrate spectrum for a previously uncharacterized enzyme.

Aminotransferases (ATs) catalyze pyridoxal 5'-phosphate-dependent transamination reactions between amino donor and keto acceptor substrates and play central roles in nitrogen metabolism of all organisms. ATs are involved in the biosynthesis and degradation of both proteinogenic and nonproteinogenic amino acids and also carry out a wide variety of functions in photorespiration, detoxification, and secondary metabolism. Despite the importance of ATs, their functionality is poorly understood as only a small fraction of putative ATs, predicted from DNA sequences, are associated with experimental data. Even for characterized ATs, the full spectrum of substrate specificity, among many potential substrates, has not been explored in most cases. This is largely due to the lack of suitable high-throughput assays that can screen for AT activity and specificity at scale. Here we present a new high-throughput platform for screening AT activity using bioconjugate chemistry and mass spectrometry imaging-based analysis. Detection of AT reaction products is achieved by forming an oxime linkage between the ketone groups of transaminated amino donors and a probe molecule that facilitates mass spectrometry-based analysis using nanostructure-initiator mass spectrometry or MALDI-mass spectrometry. As a proof-of-principle, we applied the newly established method and found that a previously uncharacterized Arabidopsis thaliana tryptophan AT-related protein 1 is a highly promiscuous enzyme that can utilize 13 amino acid donors and three keto acid acceptors. These results demonstrate that this oxime-mass spectrometry imaging AT assay enables high-throughput discovery and comprehensive characterization of AT enzymes, leading to an accurate understanding of the nitrogen metabolic network.

Structural analysis of the CJ0600 protein from Campylobacter jejuni.

The Campylobacter jejuni bacterium, which causes foodborne enteritis in humans, expresses the uncharacterized protein CJ0600. Based on sequence analysis, CJ0600 has been proposed to function as a 1-aminocyclopropane-1-carboxylate (ACC) deaminase (AccDA) or cysteine desulfhydrase (CysDS). However, it has never been investigated whether CJ0600 exerts AccDA or CysDS activity or how CJ0600 mediates its enzymatic activity. To reveal the structural features necessary for the function of CJ0600, we determined the crystal structure of CJ0600 and characterized its enzymatic activity. CJ0600 contains two domains and features an interdomain pocket, which accommodates a pyridoxal 5'-phosphate (PLP) molecule as a Schiff base with its lysine residue (K35), as observed in its structural homologs, including AccDA, CysDS, and serine deaminase (SerDA). However, unlike its structural homologs, CJ0600 exists as a monomer and exhibits unique structural features throughout its structure. Moreover, CJ0600 contains unique active site residues that are not observed in AccDA, CysDS, or SerDA. Consistently, phylogenetic analysis indicates that CJ0600 and its orthologs are evolutionarily distinct from AccDA, CysDS, and SerDA. Indeed, CJ0600 showed no CysDS or SerDA activity and extremely weak AccDA activity. These observations suggest that CJ0600 functions as a unique PLP-dependent enzyme that has not been reported.

Pyridoxal-5'-phosphate: A cost-effective treatment candidate for hypertensive patients?

OBJECTIVES: Because angiotensin (Ang) II is an essential vasoconstrictive peptide, we analyzed the impact of its post-translational modification to pyruvamide-Ang II (Ang P) by pyridoxal-5'-phosphate (PLP) on blood pressure. PLP is a less expensive vitamin B6 derivative and, therefore, could be a cost-effective drug against hypertension. METHODS: Effect of Ang P on calcium ion entry into vascular smooth muscle cells (VSMCs) was analyzed. Binding affinity of Ang P to angiotensin II type 1 receptor (AT1R) was measured. Vasoconstrictive effect of Ang P was investigated using the bioassay of isolated perfused rat kidneys. Spontaneously hypertensive rats (SHR) were administered PLP. Additionally, Wistar Kyoto rats (WKY) received Ang II and PLP. Blood pressure was measured time-dependently. RESULTS: Ang II, incubated with PLP, was post-translationally modified to Ang P. Calcium ion entry in VSMCs was significantly lower with Ang P compared to Ang II. Binding affinity of Ang P to AT1R was lower compared to Ang II. Perfusion pressure of isolated perfused rat kidneys increased less by Ang P than by Ang II. Blood pressure of SHR treated with PLP decreased significantly. Blood pressure of WKY rats treated with Ang II was increased to hypertensive values, whereas blood pressure of WKY rats cotreated with Ang II and PLP was not. CONCLUSION: PLP induces a post-translational modification of Ang II decreasing blood pressure in rats. Assuming that increased PLP intake in the form of vitamin B6 might reduce blood pressure in hypertensive patients, PLP might be a cost-effective drug against hypertension.

Extending diagnostic practices in gyrate atrophy: Enzymatic characterization and the development of an in vitro pyridoxine responsiveness assay.

Gyrate atrophy of the choroid and retina (GACR) is caused by pathogenic biallelic variants in the gene encoding ornithine-δ-aminotransferase (OAT), and is characterized by progressive vision loss leading to blindness. OAT is a pyridoxal-5'-phosphate (PLP) dependent enzyme that is mainly involved in ornithine catabolism, and patients with a deficiency develop profound hyperornithinemia. Therapy is aimed at lowering ornithine levels through dietary arginine restriction and, in some cases, through enhancement of OAT activity via supraphysiological dosages of pyridoxine. In this study, we aimed to extend diagnostic practices in GACR by extensively characterizing the consequences of pathogenic variants on the enzymatic function of OAT, both at the level of the enzyme itself as well as the flux through the ornithine degradative pathway. In addition, we developed an in vitro pyridoxine responsiveness assay. We identified 14 different pathogenic variants, of which one variant was present in all patients of Dutch ancestry (p.(Gly353Asp)). In most patients the enzymatic activity of OAT as well as the rate of [14C]-ornithine flux was below the limit of quantification (LOQ). Apart from our positive control, only one patient cell line showed responsiveness to pyridoxine in vitro, which is in line with the reported in vivo pyridoxine responsiveness in this patient. None of the patients harboring the p.(Gly353Asp) substitution were responsive to pyridoxine in vivo or in vitro. In silico analysis and small-scale expression experiments showed that this variant causes a folding defect, leading to increased aggregation properties that could not be rescued by PLP. Using these results, we developed a diagnostic pipeline for new patients suspected of having GACR. Adding OAT enzymatic analyses and in vitro pyridoxine responsiveness to diagnostic practices will not only increase knowledge on the consequences of pathogenic variants in OAT, but will also enable expectation management for therapeutic modalities, thus eventually improving clinical care.

Vitamin B6 Cofactor Pyridoxal 5'-phosphate Conjugated Papain-Stabilized Fluorescent Gold Nanoclusters for Switch-on Detection of Zinc(II).

In this study, fluorescent gold nanoclusters (AuNCs) conjugated with pyridoxal-5-phosphate (PLP) were synthesized, characterized, and used for Zn2+ fluorescence turn-on sensing. PLP was conjugated over the surface of papain-stabilized fluorescent gold nanoclusters (pap-AuNCs; λex = 380 nm, λem = 670 nm) by forming imine linkage. Due to this modification, the red color emitting pap-AuNCs changed to orange color emitting nanoclusters PLP_pap-AuNCs. The nano-assembly PLP_pap-AuNCs detect Zn2+ selectively by showing a notable fluorescence enhancement at 477 nm. Zn2+ detection with PLP_pap-AuNCs was quick and easy, with an estimated detection limit of 0.14 µM. Further, paper strips and cotton buds coated with PLP_pap-AuNCs were developed for affordable on-site visual detection of Zn2+. Finally, the detection of Zn2+ in actual environmental water samples served as validation of the usefulness of PLP_pap-AuNCs.

Establishing reference intervals for thiamine pyrophosphate and pyridoxal 5'-phosphate in whole blood in a Danish cohort using liquid chromatography tandem-mass spectrometry (LC-ms/ms).

Vitamin B1 (thiamine pyrophosphate (TPP)) and B6 (pyridoxal 5'- phosphate (PLP)) deficiencies pose significant health risks. The current measurement method employs High-Performance Liquid Chromatography (HPLC), though, Liquid Chromatography with tandem Mass Spectrometry (LC-MS/MS) is considered a more sensitive and selective analytical method. However, there is a lack of LC-MS/MS-based reference intervals. Moreover, none of the existing reference intervals are established in Danish populations. Therefore, the aim of this study was to establish a reference interval for whole blood concentrations of TPP and PLP in Danish blood donors using LC-MS/MS. Blood samples were collected from healthy Danish blood donors and analysed using the reagent kit, MassChrom® Vitamins B1 and B6 in whole blood (Chromsystems Instruments & Chemicals GmbH, Munich, Germany) for quantitative determination of both TPP and PLP concentration in whole blood, using LC-MS/MS. Reference intervals were determined with non-parametric methods as the 2.5th and 97.5th percentile and presented with 90% confidence intervals (CI). In total 120 blood donors were included. The concentrations of TTP or PLP were not statistically different between sexes just as age did not affect the concentrations, hence, combined reference intervals were employed. The resulting reference intervals are: TPP, nmol/L: 101.0 (90% CI: 96.4-108.5) - 189.0 (90% CI: 184.7-192.0) and PLP, nmol/L: 64.0 (90% CI: 60.9-66.7) - 211.8 (90% CI: 168.3-231.0). In conclusion, reference intervals for whole blood TTP and PLP in a healthy Danish population were established based on a LC-MS/MS method. Furthermore, the reference intervals were not affected by age or sex.

Does Vitamin B6 Act as an Exercise Mimetic in Skeletal Muscle?

Marginal vitamin B6 (B6) deficiency is common in various segments worldwide. In a super-aged society, sarcopenia is a major concern and has gained significant research attention focused on healthy aging. To date, the primary interventions for sarcopenia have been physical exercise therapy. Recent evidence suggests that inadequate B6 status is associated with an increased risk of sarcopenia and mortality among older adults. Our previous study showed that B6 supplementation to a marginal B6-deficient diet up-regulated the expression of various exercise-induced genes in the skeletal muscle of rodents. Notably, a supplemental B6-to-B6-deficient diet stimulates satellite cell-mediated myogenesis in rodents, mirroring the effects of physical exercise. These findings suggest the potential role of B6 as an exercise-mimetic nutrient in skeletal muscle. To test this hypothesis, we reviewed relevant literature and compared the roles of B6 and exercise in muscles. Here, we provide several pieces of evidence supporting this hypothesis and discuss the potential mechanisms behind the similarities between the effects of B6 and exercise on muscle. This research, for the first time, provides insight into the exercise-mimetic roles of B6 in skeletal muscle.

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.

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

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

Ichthyosis linked to sphingosine 1-phosphate lyase insufficiency is due to aberrant sphingolipid and calcium regulation.

Sphingosine 1-phosphate lyase (SGPL1) insufficiency (SPLIS) is a syndrome which presents with adrenal insufficiency, steroid-resistant nephrotic syndrome, hypothyroidism, neurological disease, and ichthyosis. Where a skin phenotype is reported, 94% had abnormalities such as ichthyosis, acanthosis, and hyperpigmentation. To elucidate the disease mechanism and the role SGPL1 plays in the skin barrier we established clustered regularly interspaced short palindromic repeats-Cas9 SGPL1 KO and a lentiviral-induced SGPL1 overexpression (OE) in telomerase reverse-transcriptase immortalised human keratinocytes (N/TERT-1) and thereafter organotypic skin equivalents. Loss of SGPL1 caused an accumulation of S1P, sphingosine, and ceramides, while its overexpression caused a reduction of these species. RNAseq analysis showed perturbations in sphingolipid pathway genes, particularly in SGPL1_KO, and our gene set enrichment analysis revealed polar opposite differential gene expression between SGPL1_KO and _OE in keratinocyte differentiation and Ca2+ signaling genesets. SGPL1_KO upregulated differentiation markers, while SGPL1_OE upregulated basal and proliferative markers. The advanced differentiation of SGPL1_KO was confirmed by 3D organotypic models that also presented with a thickened and retained stratum corneum and a breakdown of E-cadherin junctions. We conclude that SPLIS associated ichthyosis is a multifaceted disease caused possibly by sphingolipid imbalance and excessive S1P signaling, leading to increased differentiation and an imbalance of the lipid lamellae throughout the epidermis.

2-Aminoacrylate stress damages diverse PLP-dependent enzymes in vivo.

Pyridoxal 5'-phosphate (PLP) is an essential cofactor for a class of enzymes that catalyze diverse reactions in central metabolism. The catalytic mechanism of some PLP-dependent enzymes involves the generation of reactive enamine intermediates like 2-aminoacrylate (2AA). 2AA can covalently modify PLP in the active site of some PLP-dependent enzymes and subsequently inactivate the enzyme through the formation of a PLP-pyruvate adduct. In the absence of the enamine/imine deaminase RidA, Salmonella enterica experiences 2AA-mediated metabolic stress. Surprisingly, PLP-dependent enzymes that generate endogenous 2AA appear to be immune to its attack, while other PLP-dependent enzymes accumulate damage in the presence of 2AA stress; however, structural determinants of 2AA sensitivity are unclear. In this study, we refined a molecular method to query proteins from diverse systems for their sensitivity to 2AA in vivo. This method was then used to examine active site residues of Alr, a 2AA-sensitive PLP-dependent enzyme, that affect its sensitivity to 2AA in vivo. Unexpectedly, our data also showed that a low level of 2AA stress can persist even in the presence of a functional RidA. In summary, this study expands our understanding of 2AA metabolism and takes an initial step toward characterizing the structural determinants influencing enzyme susceptibility to damage by free 2AA.

4'-Deoxypyridoxine disrupts vitamin B6 homeostasis in Escherichia coli K12 through combined inhibition of cumulative B6 uptake and PLP-dependent enzyme activity.

Pyridoxal 5'-phosphate (PLP) is the active form of vitamin B6 and a cofactor for many essential metabolic processes such as amino acid biosynthesis and one carbon metabolism. 4'-deoxypyridoxine (4dPN) is a long known B6 antimetabolite but its mechanism of action was not totally clear. By exploring different conditions in which PLP metabolism is affected in the model organism Escherichia coli K12, we showed that 4dPN cannot be used as a source of vitamin B6 as previously claimed and that it is toxic in several conditions where vitamin B6 homeostasis is affected, such as in a B6 auxotroph or in a mutant lacking the recently discovered PLP homeostasis gene, yggS. In addition, we found that 4dPN sensitivity is likely the result of multiple modes of toxicity, including inhibition of PLP-dependent enzyme activity by 4'-deoxypyridoxine phosphate (4dPNP) and inhibition of cumulative pyridoxine (PN) uptake. These toxicities are largely dependent on the phosphorylation of 4dPN by pyridoxal kinase (PdxK).

Active site serine-193 modulates activity of human aromatic amino acid decarboxylase.

Aromatic amino acid decarboxylase is a pyridoxal 5'-phosphate-dependent enzyme responsible for the synthesis of the neurotransmitters, dopamine and serotonin. Here, by a combination of bioinformatic predictions and analyses, phosphorylation assays, spectroscopic investigations and activity measurements, we determined that Ser-193, a conserved residue located at the active site, can be phosphorylated, increasing catalytic efficiency. In order to determine the molecular basis for this functional improvement, we determined the structural and kinetic properties of the site-directed variants S193A, S193D and S193E. While S193A retains 27% of the catalytic efficiency of wild-type, the two acidic side chain variants are impaired in catalysis with efficiencies of about 0.15% with respect to the wild-type. Thus, even if located at the active site, Ser-193 is not essential for enzyme activity. We advance the idea that this residue is fundamental for the correct architecture of the active site in terms of network of interactions triggering catalysis. This role has been compared with the properties of the Ser-194 of the highly homologous enzyme histidine decarboxylase whose catalytic loop is visible in the spatial structure, allowing us to propose the validation for the effect of the phosphorylation. The effect could be interesting for AADC deficiency, a rare monogenic disease, whose broad clinical phenotype could be also related to post translational AADC modifications.

Stereoselective Biocatalytic α-Deuteration of L-Amino Acids by a Pyridoxal 5'-Phosphate-Dependent Mannich Cyclase.

α-Deuterated amino acids are valuable building blocks for developing deuterated drugs, and are important tools for studying biological systems. Biocatalytic deuteration represents an attractive strategy to directly access enantiopure α-deuterated amino acids. Here, we show that a PLP-dependent Mannich cyclase, LolT, involved in the biosynthesis of loline alkaloids, is capable of deuterating a diverse range of L-amino acids, including basic and acidic, nonpolar and polar, aliphatic and aromatic amino acids. Furthermore, complete deuteration of many amino acids can be achieved within minutes with exquisite control on the site- and stereoselectivity. During the course of this investigation, we also unexpectedly discovered that LolT exhibits ß-elimination activity with L-cystine and O-acetyl-L-serine, confirming our previous hypothesis based on structural and phylogenetic analysis that LolT, a Cα-C bond forming enzyme, is evolved from a primordial Cß-S lyase family. Overall, our study demonstrates that LolT is an extremely versatile biocatalyst, and can be used for not only heterocyclic quaternary amino acid biosynthesis, but also biocatalytic amino acid deuteration.

Mangiferin is a new potential antimalarial and anticancer drug for targeting serine hydroxymethyltransferase.

Mangiferin, a polyphenolic xanthone glycoside found in various botanical sources, including mango (Mangifera indica L.) leaves, can exhibit a variety of bioactivities. Although mangiferin has been reported to inhibit many targets, none of the studies have investigated the inhibition of serine hydroxymethyltransferase (SHMT), an attractive target for antimalarial and anticancer drugs. SHMT, one of the key enzymes in the deoxythymidylate synthesis cycle, catalyzes the reversible conversion of l-serine and (6S)-tetrahydrofolate (THF) into glycine and 5,10-methylene THF. Here, in vitro and in silico studies were used to probe how mangiferin isolated from mango leaves inhibits Plasmodium falciparum and human cytosolic SHMTs. The inhibition kinetics at pH 7.5 revealed that mangiferin is a competitive inhibitor against THF for enzymes from both organisms. Molecular docking and molecular dynamic (MD) simulations demonstrated the inhibitory effects of the deprotonated forms of mangiferin, specifically the C6-O- species and its resonance C9-O- species appearing at pH 7.5, combined with two docked poses, either a xanthone or glucose moiety, placed inside the THF-binding pocket. The MD analysis revealed that both C6-O- and its resonance-stabilized C9-O- species can favorably bind to SHMT in a similar fashion to THF, supporting the THF competitive inhibition of mangiferin. In addition, characterization of the proton dissociation equilibria of isolated mangiferin revealed that only three hydroxy groups of the xanthone moiety, C6-OH, C3-OH, and C7-OH, underwent varying degrees of deprotonation with pKa values of 6.38 ± 0.11, 8.21 ± 0.35, and 12.37 ± 0.30, respectively, while C1-OH remained protonated. Altogether, our findings demonstrate a new bioactivity of mangiferin and provide the basis for the future development of mangiferin as a potent antimalarial and anticancer drug.

Expanding the ß-substitution reactions of serine synthase through mutagenesis of aromatic active site residues.

The Gram-negative bacterium, Fusobacterium nucleatum, possesses a fold II type pyridoxal 5'-phosphate-dependent enzyme that catalyzes the reversible ß-replacement of l-cysteine and l-serine, generating H2S and H2O, respectively. This enzyme, termed serine synthase (FN1055), contains an active site Asp232 that serves as a general base in the activation of a water molecule for nucleophilic attack of the ⍺-aminoacrylate intermediate. A network of hydrophobic residues surrounding Asp232 are key to catalysis as they increase the basicity of the side chain. However, these residues severely restrict the range of nucleophilic substrates that can react with the ⍺-aminoacrylate, making the enzyme an ineffective biocatalyst for noncanonical amino acid biosynthesis. Herein, we systematically substituted four aromatic active residues (Trp99, Phe125, Phe148 and Phe234) to an alanine to determine their catalytic importance in serine/cysteine synthase reactions and if their substitution could broaden the scope of nucleophiles that could react with the ⍺-aminoacrylate intermediate. All four single site mutants W99A, F125A, F148A, and F234A could form the ⍺-aminoacrylate intermediate upon reaction with either l-cysteine or l-serine; however, the rate constant associated with the elimination of the ß-hydroxyl group from l-serine was 150 to 200-fold lower in the F125A and F148A variants. Substitution of Phe125 and Phe148, situated ∼3-4 Å from the general base, also abolished the serine synthase reaction due to their inability to activate a water molecule for nucleophilic attack of the ⍺-aminoacrylate. Overall, the mutational studies indicate that the clustering of aromatic residues disproportionately benefits the serine synthase reaction as they increase the binding affinity for l-cysteine, decrease the binding of the product, l-serine, and promote the activation of a water molecule. Notably, the aminoacrylate species present in F125A and F148A was able to react with thiophenol, signifying that serine synthase has biocatalytic potential in the synthesis of noncanonical amino acids.