Pyridoxal Phosphate Binding Research Articles

Incorporation of pyridoxal-5′-phosphate into the apoenzyme: A structural study of D-amino acid transaminase from Haliscomenobacter hydrossis

Pyridoxal-5′-phosphate (PLP)-dependent transaminases are key enzymes of amino acid metabolism in cells and remarkable biocatalysts of stereoselective amination for process chemistry applications. As cofactor-dependent enzymes, transaminases are prone to cofactor leakage. Here we discuss the holoenzyme-apoenzyme interconversion and the kinetics of PLP incorporation into the apo form of a PLP-dependent transaminase from Haliscomenobacter hydrossis. PLP binding to the apoenzyme was slow in buffer, but was accelerated in the presence of substrates. Two crystal structures of the apoenzyme were obtained: the directly obtained apoenzyme (PDB ID: 7P8O) and the one obtained by soaking crystals of the holoenzyme in a phenylhydrazine solution (PDB ID: 8YRU). The mechanism of PLP association with the apoenzyme was proposed on the basis of structural analysis of these apo forms. Three rearrangement steps, including (I) anchoring of the PLP via the phosphate group, (II) displacement of two loops, and (III) Schiff-bonding between the PLP and the ε-amino group of the catalytic lysine residue, reconstituted the active holo form of the transaminase from H. hydrossis. The results obtained allowed us to determine in the active site a permanent part and elements that are assembled by PLP, these findings may be useful for transaminase engineering for biocatalysis.

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.

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.

Identification of the pyridoxal 5'-phosphate allosteric site in human pyridox(am)ine 5'-phosphate oxidase.

Adequate levels of pyridoxal 5'-phosphate (PLP), the catalytically active form of vitamin B6 , and its proper distribution in the body are essential for human health. The PLP recycling pathway plays a crucial role in these processes and its defects cause severe neurological diseases. The enzyme pyridox(am)ine 5'-phosphate oxidase (PNPO), whose catalytic action yields PLP, is one of the key players in this pathway. Mutations in the gene encoding PNPO are responsible for a severe form of neonatal epilepsy. Recently, PNPO has also been described as a potential target for chemotherapeutic agents. Our laboratory has highlighted the crucial role of PNPO in the regulation of PLP levels in the cell, which occurs via a feedback inhibition mechanism of the enzyme, exerted by binding of PLP at an allosteric site. Through docking analyses and site-directed mutagenesis experiments, here we identified the allosteric PLP binding site of human PNPO. This site is located in the same protein region as the allosteric site we previously identified in the Escherichia coli enzyme homologue. However, the identity and arrangement of the amino acid residues involved in PLP binding are completely different and resemble those of the active site of PLP-dependent enzymes. The identification of the PLP allosteric site of human PNPO paves the way for the rational design of enzyme inhibitors as potential anti-cancer compounds.

Protein-protein interfaces as druggable targets: a common motif of the pyridoxal-5′-phosphate-dependent enzymes to receive the coenzyme from its producer

Pyridoxal-5′-phosphate (PLP), a phosphorylated form of vitamin B6, acts as a coenzyme for numerous reactions, including those changed in cancer and/or associated with the disease prognosis. Since highly reactive PLP may modify cellular proteins, it is hypothesized to be directly transferred from its donors to acceptors. Our goal is to validate the hypothesis by finding common motif(s) in a multitude of the PLP-dependent enzymes for binding the limited number of the PLP donors, namely pyridoxal kinase (PdxK), pyridox(am)in-5′-phosphate oxidase (PNPO) and the PLP-binding protein (PLPBP). Experimentally confirmed interactions between the PLP donors and acceptors reveal that PdxK and PNPO interact with the PLP acceptors of folds I and II, while PLPBP - with those of folds III and V. Aligning the sequences and 3D structures of the identified interactors of PdxK and PNPO, we have found a common motif in the PLP-dependent enzymes of folds I and II. The motif extends from the enzyme surface to the neighborhood of the PLP binding site, represented by an exposed alfa-helix, a partially buried beta-strand and residual loops. Pathogenicity of mutations in human PLP-dependent enzymes within or in the vicinity of the motif, but outside of the active sites, supports functional significance of the motif that may provide an interface for the direct transfer of PLP from the sites of its synthesis to those of the coenzyme binding. The enzyme-specific amino acid residues of the common motif may be used to develop selective inhibitors blocking PLP delivery to the PLP-dependent enzymes critical for proliferation of malignant cells.

Crystal structure of an aspartate aminotransferase Lpg0070 from Legionella pneumophila

Legionella pneumophila aspartate aminotransferase (Lpg0070) is a member of the transaminase and belongs to the pyridoxal 5′-phosphate (PLP)-dependent superfamily. It is responsible for the transfer of α-amino between aspartate and α-ketoglutarate to form glutamate and oxaloacetate. Here, we report the crystal structure of Lpg0070 at the resolution of 2.14 Å and 1.7 Å, in apo-form and PLP-bound, respectively. Our structural analysis revealed the specific residues involved in the PLP binding and free form against PLP-bound supported conformational changes before substrate recognition. In vitro enzyme activity proves that the absence of the N-terminal arm reduces the enzyme activity of Lpg0070. These data provide further evidence to support the N-terminal arm plays a crucial role in catalytic activity.

Identification of high-affinity pyridoxal kinase inhibitors targeting cancer therapy: an integrated docking and molecular dynamics simulation approach

Pyridoxal kinase (PDXK) is a vitamin B6-dependent transferase enzyme encoded by the PDXK gene, crucial for leukemic cell proliferation. Disruption of its activity causes altered metabolism and reduced levels of nucleotides and polyamines. PDXK and pyridoxal 5'-phosphate (PLP) are overexpressed in various carcinomas, making them promising targets for drug design against cancer. Targeting PDXK may hold promise as a therapeutic approach for cancer treatment. This study focused on discovering potential inhibitors that could selectively interrupt the binding of pyridoxal phosphate (PLP) to pyridoxal kinase (PDXK). A commercially available library of 7,28,747 natural and druglike compounds was virtually screened using a molecular docking approach to target the substrate binding pocket of PDXK. Six promising inhibitors were identified, and all-atom molecular dynamics simulations were conducted on the PDXK-ligand complexes for 100 ns to assess their binding conformational stability. The simulation results indicated that the binding of ZINC095099376, ZINC01612996, ZINC049841390, ZINC095098959, ZINC01482077, and ZINC03830976 induced a slight structural change and stabilized the PDXK structure. This analysis provided valuable information about the critical residues involved in the PDXK-PLP complex formation and can be utilized in designing specific and effective PDXK inhibitors. According to this study, these compounds could be developed as anticancer agents targeting PDXK as a potential candidate for further study. Communicated by Ramaswamy H. Sarma

Protein–Protein Interfaces as Druggable Targets: A Common Motif of the Pyridoxal-5′-Phosphate-Dependent Enzymes to Receive the Coenzyme from Its Producers

Pyridoxal-5′-phosphate (PLP), a phosphorylated form of vitamin B6, acts as a coenzyme for numerous reactions, including those changed in cancer and/or associated with the disease prognosis. Since highly reactive PLP can modify cellular proteins, it is hypothesized to be directly transferred from its donors to acceptors. Our goal is to validate the hypothesis by finding common motif(s) in the multitude of PLP-dependent enzymes for binding the limited number of PLP donors, namely pyridoxal kinase (PdxK), pyridox(am)in-5′-phosphate oxidase (PNPO), and PLP-binding protein (PLPBP). Experimentally confirmed interactions between the PLP donors and acceptors reveal that PdxK and PNPO interact with the most abundant PLP acceptors belonging to structural folds I and II, while PLPBP – with those belonging to folds III and V. Aligning sequences and 3D structures of the identified interactors of PdxK and PNPO, we have identified a common motif in the PLP-dependent enzymes of folds I and II. The motif extends from the enzyme surface to the neighborhood of the PLP binding site, represented by an exposed alfa-helix, a partially buried beta-strand, and residual loops. Pathogenicity of mutations in the human PLP-dependent enzymes within or in the vicinity of the motif, but outside of the active sites, supports functional significance of the motif that may provide an interface for the direct transfer of PLP from the sites of its synthesis to those of coenzyme binding. The enzyme-specific amino acid residues of the common motif may be useful to develop selective inhibitors blocking PLP delivery to the PLP-dependent enzymes critical for proliferation of malignant cells.

Crystal Structure and Functional Characterization of Acetylornithine Aminotransferase from Corynebacterium glutamicum.

The amino acids l-arginine and l-ornithine are widely used in animal feed and as health supplements and pharmaceutical compounds. In arginine biosynthesis, acetylornithine aminotransferase (AcOAT) uses pyridoxal-5'-phosphate (PLP) as a cofactor for amino group transfer. Here, we determined the crystal structures of the apo and PLP complex forms of AcOAT from Corynebacterium glutamicum (CgAcOAT). Our structural observations revealed that CgAcOAT undergoes an order-to-disorder conformational change upon binding with PLP. Additionally, we observed that unlike other AcOATs, CgAcOAT exists as a tetramer. Subsequently, we identified the key residues involved in PLP and substrate binding based on structural analysis and site-directed mutagenesis. This study might provide structural insights on CgAcOAT, which can be utilized for the development of improved l-arginine production enzymes.

Molecular Responses of Red Ripe Tomato Fruit to Copper Deficiency Stress.

Fruit nutritional value, plant growth, and yield can be compromised by deficient copper (Cu) bioavailability, which often appears in arable lands. This condition causes low Cu content and modifications in the ripening-associated processes in tomato fruit. This research studies the transcriptomic changes that occur in red ripe tomato fruit grown under suboptimal Cu conditions to shed light on the molecular mechanisms underlying this stress. Comparative RNA-sequencing and functional analyses revealed that Cu deficiency during cultivation activates signals for metal ion transport, cellular redox homeostasis, pyridoxal phosphate binding, and amino acid metabolism while repressing the response to phosphate starvation in harvested fruit. Transcriptomic analyses highlighted a number of novel Cu stress-responsive genes of unknown function and indicated that Cu homeostasis regulation in tomato fruit may involve additional components than those described in model plants. It also studied the regulation of high-affinity Cu transporters and a number of well-known Cu stress-responsive genes during tomato fruit ripening depending on Cu availability, which allowed potential candidates to be targeted for biotechnological improvements in reproductive tissues. We provide the first study characterizing the molecular responses of fruit to Cu deficiency stress for any fruit crop.

Biochemical and Bioinformatic Studies of Mutations of Residues at the Monomer-Monomer Interface of Human Ornithine Aminotransferase Leading to Gyrate Atrophy of Choroid and Retina.

Deficit of human ornithine aminotransferase (hOAT), a mitochondrial tetrameric pyridoxal-5'-phosphate (PLP) enzyme, leads to gyrate atrophy of the choroid and retina (GA). Although 70 pathogenic mutations have been identified, only few enzymatic phenotypes are known. Here, we report biochemical and bioinformatic analyses of the G51D, G121D, R154L, Y158S, T181M, and P199Q pathogenic variants involving residues located at the monomer-monomer interface. All mutations cause a shift toward a dimeric structure, and changes in tertiary structure, thermal stability, and PLP microenvironment. The impact on these features is less pronounced for the mutations of Gly51 and Gly121 mapping to the N-terminal segment of the enzyme than those of Arg154, Tyr158, Thr181, and Pro199 belonging to the large domain. These data, together with the predicted ΔΔG values of monomer-monomer binding for the variants, suggest that the proper monomer-monomer interactions seem to be correlated with the thermal stability, the PLP binding site and the tetrameric structure of hOAT. The different impact of these mutations on the catalytic activity was also reported and discussed on the basis of the computational information. Together, these results allow the identification of the molecular defects of these variants, thus extending the knowledge of enzymatic phenotypes of GA patients.

Biochemical and cellular effects of a novel missense mutation of the AGXT gene associated with Primary Hyperoxaluria Type 1

Primary Hyperoxaluria Type 1 (PH1) is a rare autosomal disease caused by mutations in AGXT that lead to the deficiency of alanine:glyoxylate aminotransferase (AGT). AGT is a liver pyridoxal 5′-phosphate (PLP)-dependent enzyme that detoxifies glyoxylate inside peroxisomes. The lack of AGT activity results in a build-up of glyoxylate that is oxidized to oxalate, then culminating in hyperoxaluria often leading to kidney failure. Most pathogenic mutations reduce AGT specific activity because of catalytic defects, improper folding, mistargeting to mitochondria, reduced intracellular stability, dimerization, and/or aggregation. Administration of pyridoxine (PN), a precursor of PLP, is a therapeutic option available for PH1 patients carrying responsive genotypes through the ability of the coenzyme to behave as a chaperone.Here, we report the clinical and biochemical characterization of the novel mutation c.1093G > T (p.Gly365Cys) identified in a Japanese patient. In silico studies predict that the p.Gly365Cys mutation causes a steric clash resulting in a local rearrangement of the region surrounding the active site, thus possibly affecting PLP binding and catalysis. Indeed, the purified p.Gly365Cys mutant displays proper folding but shows an extensive decrease of catalytic efficiency due to an altered PLP-binding. When expressed in AGXT1-KO HepG2 cells the variant shows reduced specific activity and protein levels in comparison with wild type AGT that cannot be rescued by PN treatment.Overall, our data indicate that the mutation of Gly365 induces a conformational change at the AGT active site translating into a functional and structural defect and allow to predict that the patients will not be responsive to vitamin B6, thus supporting the usefulness of preclinical studies to guide therapeutic decisions in the era of precision medicine.

Characterization of the Escherichia coli pyridoxal 5'-phosphate homeostasis protein (YggS): Role of lysine residues in PLP binding and protein stability.

The pyridoxal 5′‐phosphate (PLP) homeostasis protein (PLPHP) is a ubiquitous member of the COG0325 family with apparently no catalytic activity. Although the actual cellular role of this protein is unknown, it has been observed that mutations of the PLPHP encoding gene affect the activity of PLP‐dependent enzymes, B6 vitamers and amino acid levels. Here we report a detailed characterization of the Escherichia coli ortholog of PLPHP (YggS) with respect to its PLP binding and transfer properties, stability, and structure. YggS binds PLP very tightly and is able to slowly transfer it to a model PLP‐dependent enzyme, serine hydroxymethyltransferase. PLP binding to YggS elicits a conformational/flexibility change in the protein structure that is detectable in solution but not in crystals. We serendipitously discovered that the K36A variant of YggS, affecting the lysine residue that binds PLP at the active site, is able to bind PLP covalently. This observation led us to recognize that a number of lysine residues, located at the entrance of the active site, can replace Lys36 in its PLP binding role. These lysines form a cluster of charged residues that affect protein stability and conformation, playing an important role in PLP binding and possibly in YggS function.

Rewiring of the protein–protein–metabolite interactome during the diauxic shift in yeast

In budding yeast Saccharomyces cerevisiae, the switch from aerobic fermentation to respiratory growth is separated by a period of growth arrest, known as the diauxic shift, accompanied by a significant metabolic rewiring, including the derepression of gluconeogenesis and the establishment of mitochondrial respiration. Previous studies reported hundreds of proteins and tens of metabolites accumulating differentially across the diauxic shift transition. To assess the differences in the protein–protein (PPIs) and protein–metabolite interactions (PMIs) yeast samples harvested in the glucose-utilizing, fermentative phase, ethanol-utilizing and early stationary respiratory phases were analysed using isothermal shift assay (iTSA) and a co-fractionation mass spectrometry approach, PROMIS. Whereas iTSA monitors changes in protein stability and is informative towards protein interaction status, PROMIS uses co-elution to delineate putative PPIs and PMIs. The resulting dataset comprises 1627 proteins and 247 metabolites, hundreds of proteins and tens of metabolites characterized by differential thermal stability and/or fractionation profile, constituting a novel resource to be mined for the regulatory PPIs and PMIs. The examples discussed here include (i) dissociation of the core and regulatory particle of the proteasome in the early stationary phase, (ii) the differential binding of a co-factor pyridoxal phosphate to the enzymes of amino acid metabolism and (iii) the putative, phase-specific interactions between proline-containing dipeptides and enzymes of central carbon metabolism.

Transcriptomic Analysis of the Porcine Gut in Response to Heat Stress and Dietary Soluble Fiber from Beet Pulp

This study aimed to investigate the impact of heat stress (HS) and the effects of dietary soluble fiber from beet pulp (BP) on gene expression (differentially expressed genes, DEGs) of the porcine jejunum. Out of the 82 DEGs, 47 genes were up-regulated, and 35 genes were downregulated between treatments. The gene ontology (GO) enrichment analysis showed that the DEGs were related mainly to the actin cytoskeleton organization and muscle structure development in biological processes, cytoplasm, stress fibers, Z disc, cytoskeleton, and the extracellular regions in cellular composition, and actin binding, calcium ion binding, actin filament binding, and pyridoxal phosphate binding in the molecular function. The KEGG pathway analysis showed that the DEGs were involved in hypertrophic cardiomyopathy, dilated cardiomyopathy, vascular smooth muscle contraction, regulation of actin cytoskeleton, mucin type O-glycan biosynthesis, and African trypanosomiasis. Several of the genes (HSPB6, HSP70, TPM1, TAGLN, CCL4) in the HS group were involved in cellular oxidative stress, immune responses, and cellular differentiation. In contrast, the DEGs in the dietary BP group were related to intestinal epithelium integrity and immune response to pathogens, including S100A2, GCNT3, LYZ, SCGB1A1, SAA3, and ST3GAL1. These findings might help understand the HS response and the effect of dietary fiber (DF) regarding HS and be a valuable reference for future studies.

Structural and Functional Analysis of the Pyridoxal Phosphate Homeostasis Protein YggS from Fusobacterium nucleatum.

Pyridoxal 5′-phosphate (PLP) is the active form of vitamin B6, but it is highly reactive and poisonous in its free form. YggS is a PLP-binding protein found in bacteria and humans that mediates PLP homeostasis by delivering PLP to target enzymes or by performing a protective function. Several biochemical and structural studies of YggS have been reported, but the mechanism by which YggS recognizes PLP has not been fully elucidated. Here, we report a functional and structural analysis of YggS from Fusobacterium nucleatum (FnYggS). The PLP molecule could bind to native FnYggS, but no PLP binding was observed for selenomethionine (SeMet)-derivatized FnYggS. The crystal structure of FnYggS showed a type III TIM barrel fold, exhibiting structural homology with several other PLP-dependent enzymes. Although FnYggS exhibited low (<35%) amino acid sequence similarity with previously studied YggS proteins, its overall structure and PLP-binding site were highly conserved. In the PLP-binding site of FnYggS, the sulfate ion was coordinated by the conserved residues Ser201, Gly218, and Thr219, which were positioned to provide the binding moiety for the phosphate group of PLP. The mutagenesis study showed that the conserved Ser201 residue in FnYggS was the key residue for PLP binding. These results will expand the knowledge of the molecular properties and function of the YggS family.

Biochemical characterization of the first step in sulfonolipid biosynthesis in Alistipes finegoldii

Sulfonolipids are unusual lipids found in the outer membranes of Gram-negative bacteria in the phylum Bacteroidetes. Sulfonolipid and its deacylated derivative, capnine, are sulfur analogs of ceramide-1-phosphate and sphingosine-1-phosphate, respectively; thus, sulfonolipid biosynthesis is postulated to be similar to the sphingolipid biosynthetic pathway. Here, we identify the first enzyme in sulfonolipid synthesis in Alistipes finegoldii as the product of the alfi_1224 gene, cysteate acyl-acyl carrier protein (ACP) transferase (SulA). We show SulA catalyzes the condensation of acyl-ACP and cysteate (3-sulfo-alanine) to form 3-ketocapnine. Acyl-CoA is a poor substrate. We show SulA has a bound pyridoxal phosphate (PLP) cofactor that undergoes a spectral redshift in the presence of cysteate, consistent with the transition of the lysine–aldimine complex to a substrate–aldimine complex. Furthermore, the SulA crystal structure shows the same prototypical fold found in bacterial serine palmitoyltransferases (Spts), enveloping the PLP cofactor bound to Lys251. We observed the SulA and Spt active sites are identical except for Lys281 in SulA, which is an alanine in Spt. Additionally, SulA(K281A) is catalytically inactive but binds cysteate and forms the external aldimine normally, highlighting the structural role of the Lys281 side chain in walling off the active site from bulk solvent. Finally, the electropositive groove on the protein surface adjacent to the active site entrance provides a landing pad for the electronegative acyl-ACP surface. Taken together, these data identify the substrates, products, and mechanism of SulA, the PLP-dependent condensing enzyme that catalyzes the first step in sulfonolipid synthesis in a gut commensal bacterium.

Structure of cysteate acyltransferase, the condensation enzyme that initiates sulfonolipid biosynthesis by inhabitants of the gut microbiome

Sulfonolipids (SL) are unusual lipids found in the outer membranes of Gram‐negative bacteria in the phylum Bacteroidetes. SL and its deacylated derivative, capnine, are sulfur analogs of ceramide‐1‐phosphate and sphingosine‐1‐phosphate, respectively. The structural similarity to key mammalian signaling molecules led us to investigate the biochemistry and distribution of SL production in inhabitants of the gut microbiome. Alistipes finegoldii, an anaerobic gut commensal contains two genes related to the bacterial serine palmitoyl transferase (Spt) identified in Sphingobacteriia. The two proteins were purified and the Afl_1224 gene product catalyzed the condensation of [14C]acyl‐acyl carrier protein and cysteate (3‐sulfo‐alanine). The product of cysteate acyltransferase (AfCat) was identified as 3‐ketocapnine by mass spectrometry. AfCat has a bound pyridoxal phosphate cofactor (PLP) that undergoes a spectral blue shift when exposed to cysteate consistent with the transition of a lysine‐aldimine complex to a substrate‐aldimine complex. We solved the crystal structure of dimeric AfCat using SAD phasing. AfCat has the prototypical fold enveloping the PLP cofactor bound to Lys‐251 found in Spt and has an electrostatic surface patch for ACP binding. AfCat is distinguished from Spt by its carboxy terminal domain that extends to interact with the amino terminus, an enlarged substrate binding site to accommodate and stabilize the negatively charged sulfono head group of cysteate, and the HAS motif in Spt is HAC in AfCat. These unique structural elements were used to identify SL producing organisms in metagenomic sequencing data from the gut microbiome that are responsible for SL and capnine in mammalian stool samples. Supported by GM034496.

A Computational Model for the PLP-Dependent Enzyme Methionine γ-Lyase.

Pyridoxal-5′-phosphate (PLP) is a cofactor in the reactions of over 160 enzymes, several of which are implicated in diseases. Methionine γ-lyase (MGL) is of interest as a therapeutic protein for cancer treatment. It binds PLP covalently through a Schiff base linkage and digests methionine, whose depletion is damaging for cancer cells but not normal cells. To improve MGL activity, it is important to understand and engineer its PLP binding. We develop a simulation model for MGL, starting with force field parameters for PLP in four main states: two phosphate protonation states and two tautomeric states, keto or enol for the Schiff base moiety. We used the force field to simulate MGL complexes with each form, and showed that those with a fully-deprotonated PLP phosphate, especially keto, led to the best agreement with MGL structures in the PDB. We then confirmed this result through alchemical free energy simulations that compared the keto and enol forms, confirming a moderate keto preference, and the fully-deprotonated and singly-protonated phosphate forms. Extensive simulations were needed to adequately sample conformational space, and care was needed to extrapolate the protonation free energy to the thermodynamic limit of a macroscopic, dilute protein solution. The computed phosphate pK a was 5.7, confirming that the deprotonated, −2 form is predominant. The PLP force field and the simulation methods can be applied to all PLP enzymes and used, as here, to reveal fine details of structure and dynamics in the active site.

A novel concept of Pyridoxal 5'-phosphate permeability in E.coli for modulating the heterologous expression of PLP dependent proteins

PLP (Pyridoxal 5′-phosphate) dependent proteins are important drug targets and effector molecules, and thus, their heterologous overexpression is of industrial importance and has commercial value. We have predicted the docking site of PLP on O-acetylserine sulphdrylase (OASS) of H.contortus and determined that the lysine-47 is very important for the binding of PLP. We have used this protein as a model protein for testing the effect of PLP on the expression of PLP dependent proteins by E.coli. Soluble recombinant protein could be purified from each of the culture vials grown with variable amount of PLP [0 mM (Group I), 0.01 mM (Group II), 0.025 mM (Group III), 0.05 mM (Group IV) and 0.1 mM (Group V)]. There was approximately 4.2 %, 7.2 %, 10.5 % and 18 % increase in purified protein yield in Group II, III, IV and V, respectively, in comparison to group I. There was a significant quenching of tryptophan fluorescence emission in groups II, III, IV and V compared to group I. We could find a PLP concentration-dependent increase in expression and catalytic activity signifying the probable transport of PLP across the membrane. E.coli does not secrete any dephosphorylation factor for PLP in the immediate microenvironment. There was a significant reduction in levels of inorganic phosphate after PLP dephosphorylation in induced group in comparison to uninduced E.coli and BL21 groups, signifying PLP internalization in E.coli. The mechanism of transport of PLP in the light of the current experiment is still unknown and should be a subject of future studies.