Phosphate-dependent Enzyme Research Articles

Heterologous gene expression and characterization of two serine hydroxymethyltransferases from Thermoplasma acidophilum.

Serine hydroxymethyltransferase (SHMT) and threonine aldolase are classified as fold type I pyridoxal-5'-phosphate-dependent enzymes and engaged in glycine biosynthesis from serine and threonine, respectively. The acidothermophilic archaeon Thermoplasma acidophilum possesses two distinct SHMT genes, while there is no gene encoding threonine aldolase in its genome. In the present study, the two SHMT genes (Ta0811 and Ta1509) were heterologously expressed in Escherichia coli and Thermococcus kodakarensis, respectively, and biochemical properties of their products were investigated. Ta1509 protein exhibited dual activities to catalyze tetrahydrofolate (THF)-dependent serine cleavage and THF-independent threonine cleavage, similar to other SHMTs reported to date. In contrast, the Ta0811 protein lacks amino acid residues involved in the THF-binding motif and catalyzes only the THF-independent cleavage of threonine. Kinetic analysis revealed that the threonine-cleavage activity of the Ta0811 protein was 3.5times higher than the serine-cleavage activity of Ta1509 protein. In addition, mRNA expression of Ta0811 gene in T. acidophilum was approximately 20times more abundant than that of Ta1509. These observations suggest that retroaldol cleavage of threonine, mediated by the Ta0811 protein, has a major role in glycine biosynthesis in T. acidophilum.

Characterization of an aminotransferase from Acanthamoeba polyphaga Mimivirus.

Acanthamoeba polyphaga Mimivirus, a complex virus that infects amoeba, was first reported in 2003. It is now known that its DNA genome encodes for nearly 1,000 proteins including enzymes that are required for the biosynthesis of the unusual sugar 4-amino-4,6-dideoxy-d-glucose, also known as d-viosamine. As observed in some bacteria, the pathway for the production of this sugar initiates with a nucleotide-linked sugar, which in the Mimivirus is thought to be UDP-d-glucose. The enzyme required for the installment of the amino group at the C-4' position of the pyranosyl moiety is encoded in the Mimivirus by the L136 gene. Here, we describe a structural and functional analysis of this pyridoxal 5'-phosphate-dependent enzyme, referred to as L136. For this analysis, three high-resolution X-ray structures were determined: the wildtype enzyme/pyridoxamine 5'-phosphate/dTDP complex and the site-directed mutant variant K185A in the presence of either UDP-4-amino-4,6-dideoxy-d-glucose or dTDP-4-amino-4,6-dideoxy-d-glucose. Additionally, the kinetic parameters of the enzyme utilizing either UDP-d-glucose or dTDP-d-glucose were measured and demonstrated that L136 is efficient with both substrates. This is in sharp contrast to the structurally related DesI from Streptomyces venezuelae, whose three-dimensional architecture was previously reported by this laboratory. As determined in this investigation, DesI shows a profound preference in its catalytic efficiency for the dTDP-linked sugar substrate. This difference can be explained in part by a hydrophobic patch in DesI that is missing in L136. Notably, the structure of L136 reported here represents the first three-dimensional model for a virally encoded PLP-dependent enzyme and thus provides new information on sugar aminotransferases in general.

Discovery of a New, Recurrent Enzyme in Bacterial Phosphonate Degradation: (R)-1-Hydroxy-2-aminoethylphosphonate Ammonia-lyase.

Phosphonates represent an important source of bioavailable phosphorus in certain environments. Accordingly, many microorganisms (particularly marine bacteria) possess catabolic pathways to degrade these molecules. One example is the widespread hydrolytic route for the breakdown of 2-aminoethylphosphonate (AEP, the most common biogenic phosphonate). In this pathway, the aminotransferase PhnW initially converts AEP into phosphonoacetaldehyde (PAA), which is then cleaved by the hydrolase PhnX to yield acetaldehyde and phosphate. This work focuses on a pyridoxal 5′-phosphate-dependent enzyme that is encoded in >13% of the bacterial gene clusters containing the phnW–phnX combination. This enzyme (which we termed PbfA) is annotated as a transaminase, but there is no obvious need for an additional transamination reaction in the established AEP degradation pathway. We report here that PbfA from the marine bacterium Vibrio splendidus catalyzes an elimination reaction on the naturally occurring compound (R)-1-hydroxy-2-aminoethylphosphonate (R-HAEP). The reaction releases ammonia and generates PAA, which can be then hydrolyzed by PhnX. In contrast, PbfA is not active toward the S enantiomer of HAEP or other HAEP-related compounds such as ethanolamine and d,l-isoserine, indicating a very high substrate specificity. We also show that R-HAEP (despite being structurally similar to AEP) is not processed efficiently by the PhnW–PhnX couple in the absence of PbfA. In summary, the reaction catalyzed by PbfA serves to funnel R-HAEP into the hydrolytic pathway for AEP degradation, expanding the scope and the usefulness of the pathway itself.

Nanoparticles: Mechanism of biosynthesis using plant extracts, bacteria, fungi, and their applications

The synthesis of eco-friendly nanoparticles (NPs) is a crucial step in nanotechnology. In recent years, to avoid environmental contamination, many efforts have been made many efforts are made to develop green methods for this purpose. For this reason, natural resources including plant extract, fungi and bacteria have been used to synthesize NPs. Plant extract contains active compounds of terpenoids, alkaloids, phenols, tannins, and vitamins that act as capping and reducing agents. The size, shape and applications of NPs are determined by the source from which they are manufactured. While active compounds of bacteria and fungi are intracellular enzymes, sugar molecules, canonical membrane proteins, Nicotinamide Adenine Dinucleotide (NADH) and Nicotinamide Adenine Dinucleotide Phosphate (NADPH) dependent enzymes play a crucial part as reducing agents. The characterization of NPs can be done by Fourier-Transform Infrared Spectroscopy (FTIR), X-ray Crystallography (XRD), Energy-dispersive X-ray Spectroscopy (EDX), Scanning Electron Microscope (SEM), Transmission Electron microscopy (TEM) and UV–Visible spectroscopy. This review paper elaborates major natural resources with detailed mechanisms involved in NPs synthesis and their few applications in biomedical science, life science and electronics.

Gamma cleavage is a rate-determining step in the gamma-elimination reaction of L-methionine analogues catalyzed by methionine-gamma-lyase

Methionine-γ-lyase (MGL) is a pyridoxal-5′-phosphate dependent enzyme found in bacteria and protozoa that catalyzes a variety of reactions, including the γ-elimination of L-methionine (L-Met). Here we report experimental kinetic data and density functional theory (DFT) computational data for the γ-elimination reaction of L-Met and several other substrate analogues by a recombinant MGL from P. gingivalis (MGL_Pg). UV–Visible spectrophotometry experiments revealed a heavily populated species with maximum absorbance at 478 nm during steady-state catalysis of L-Met, L-ethionine, L-methionine sulfone and L-homoserine, which we assign to a late crotonate intermediate formed after the γ-cleavage step in the reaction and thus common to all substrates. A more red-shifted (498 nm) species was observed during the reaction of L-homoserine lactone, which we assign to an early quinonoid intermediate with the aid of time-dependent self-consistent field calculations. Significant differences in both binding and the rate of turnover were observed for the substrates. MGL_Pg's highest catalytic efficiency was recorded for L-vinylglycine (kcat/Km = 6455 s−1 M−1), exceeding that of L-Met (kcat/Km = 4211 s−1 M−1), while L-Met sulfone displayed the largest turnover number (kcat = 1638 min−1). A direct correlation between experimental kcat values and DFT-calculated γ-cleavage Gibbs activation energies was identified for the various substrates. In light of these data, we propose that the γ-cleavage step in the catalytic reaction pathway is rate-limiting. This conclusion has direct implications for the rational design of substrates or inhibitors aimed at regulating MGL activity.

Characterization of Cystathionine β-Synthase TtCbs1 and Cysteine Synthase TtCsa1 Involved in Cysteine Biosynthesis in Tetrahymena thermophila.

Cysteine is implicated in important biological processes. It is synthesized through two different pathways. Cystathionine β-synthase and cystathionine γ-lyase participate in the reverse transsulfuration pathway, while serine acetyltransferase and cysteine synthase function in the de novo pathway. Two evolutionarily related pyridoxal 5'-phosphate-dependent enzymes, cystathionine β-synthase TtCBS1 (TTHERM_00558300) and cysteine synthase TtCSA1 (TTHERM_00239430), were identified from a freshwater protozoan Tetrahymena thermophila. TtCbs1 contained the N-terminal heme binding domain, catalytic domain, and C-terminal regulatory domain, whereas TtCsa1 consisted of two α/β domains. The catalytic core of the two enzymes is similar. TtCBS1 and TtCSA1 showed high expression levels in the vegetative growth stage and decreased during the sexual developmental stage. TtCbs1 and TtCsa1 were localized in the cytoplasm throughout different developmental stages. His-TtCbs1 and His-TtCsa1 were expressed and purified invitro. TtCbs1 catalyzed the canonical reaction with the highest velocity and possessed serine sulfhydrylase activity. TtCsa1 showed cysteine synthase activity with high Km for O-acetylserine and low Km for sulfide and also had serine sulfhydrylase activity toward serine. Both TtCbs1 and TtCsa1 catalyzed hydrogen sulfide producing. TtCBS1 knockdown and TtCSA1 knockout mutants affected cysteine and glutathione synthesis. TtCbs1 and TtCsa1 are involved in cysteine synthesis through two different pathways in T. thermophila.

KYNU, a novel potential target that underpins CD44-promoted breast tumour cell invasion.

Using a validated tetracycline‐off‐inducible CD44 expression system in mouse model, we have previously demonstrated that the hyaluronan (HA) receptor CD44 promotes breast cancer (BC) metastasis to the liver. To unravel the mechanisms that underpin CD44‐promoted BC cell invasion, RNA samples were isolated from two cell models: (a) a tetracycline (Tet)‐Off‐regulated expression system of the CD44s in MCF‐7 cells and; (b) as a complementary approach, the highly metastatic BC cells, MDA‐MB‐231, were cultured in the presence and absence of 50 µg/mL of HA. Kynureninase (KYNU), identified by Microarray analysis, was up‐regulated by 3‐fold upon induction and activation of CD44 by HA; this finding suggests that KYNU is a potential novel transcriptional target of CD44‐downtstream signalling. KYNU is a pyridoxal phosphate (PLP) dependent enzyme involved in the biosynthesis of NAD cofactors from tryptophan that has been associated with the onset and development of BC. This review will attempt to identify and discuss the findings supporting this hypothesis and the mechanisms linking KYNU cell invasion via CD44.

Structures and kinetics of Thermotoga maritima MetY reveal new insights into the predominant sulfurylation enzyme of bacterial methionine biosynthesis

Bacterial methionine biosynthesis can take place by either the trans-sulfurylation route or direct sulfurylation. The enzymes responsible for trans-sulfurylation have been characterized extensively because they occur in model organisms such as Escherichia coli. However, direct sulfurylation is actually the predominant route for methionine biosynthesis across the phylogenetic tree. In this pathway, most bacteria use an O-acetylhomoserine aminocarboxypropyltransferase (MetY) to catalyze the formation of homocysteine from O-acetylhomoserine and bisulfide. Despite the widespread distribution of MetY, this pyridoxal 5′-phosphate–dependent enzyme remains comparatively understudied. To address this knowledge gap, we have characterized the MetY from Thermotoga maritima (TmMetY). At its optimal temperature of 70 °C, TmMetY has a turnover number (apparent kcat = 900 s−1) that is 10- to 700-fold higher than the three other MetY enzymes for which data are available. We also present crystal structures of TmMetY in the internal aldimine form and, fortuitously, with a β,γ-unsaturated ketimine reaction intermediate. This intermediate is identical to that found in the catalytic cycle of cystathionine γ-synthase (MetB), which is a homologous enzyme from the trans-sulfurylation pathway. By comparing the TmMetY and MetB structures, we have identified Arg270 as a critical determinant of specificity. It helps to wall off the active site of TmMetY, disfavoring the binding of the first MetB substrate, O-succinylhomoserine. It also ensures a strict specificity for bisulfide as the second substrate of MetY by occluding the larger MetB substrate, cysteine. Overall, this work illuminates the subtle structural mechanisms by which homologous pyridoxal 5′-phosphate–dependent enzymes can effect different catalytic, and therefore metabolic, outcomes.

L-Threonine Transaldolase Activity Is Enabled by a Persistent Catalytic Intermediate.

l-Threonine transaldolases (lTTAs) are a poorly characterized class of pyridoxal-5'-phosphate (PLP) dependent enzymes responsible for the biosynthesis of diverse β-hydroxy amino acids. Here, we study the catalytic mechanism of ObiH, an lTTA essential for biosynthesis of the β-lactone natural product obafluorin. Heterologously expressed ObiH purifies as a mixture of chemical states including a catalytically inactive form of the PLP cofactor. Photoexcitation of ObiH promotes the conversion of the inactive state of the enzyme to the active form. UV-vis spectroscopic analysis reveals that ObiH catalyzes the retro-aldol cleavage of l-threonine to form a remarkably persistent glycyl quinonoid intermediate, with a half-life of ∼3 h. Protonation of this intermediate is kinetically disfavored, enabling on-cycle reactivity with aldehydes to form β-hydroxy amino acids. We demonstrate the synthetic potential of ObiH via the single step synthesis of (2S,3R)-β-hydroxyleucine. To further understand the structural features underpinning this desirable reactivity, we determined the crystal structure of ObiH bound to PLP as the Schiff's base at 1.66 Å resolution. This high-resolution model revealed a unique active site configuration wherein the evolutionarily conserved Asp that traditionally H-bonds to the cofactor is swapped for a neighboring Glu. Molecular dynamics simulations combined with mutagenesis studies indicate that a structural rearrangement is associated with l-threonine entry into the catalytic cycle. Together, these data explain the basis for the unique reactivity of lTTA enzymes and provide a foundation for future engineering and mechanistic analysis.

Glutamate Decarboxylase from Lactic Acid Bacteria-A Key Enzyme in GABA Synthesis.

Glutamate decarboxylase (l-glutamate-1-carboxylase, GAD; EC 4.1.1.15) is a pyridoxal-5’-phosphate-dependent enzyme that catalyzes the irreversible α-decarboxylation of l-glutamic acid to γ-aminobutyric acid (GABA) and CO2. The enzyme is widely distributed in eukaryotes as well as prokaryotes, where it—together with its reaction product GABA—fulfils very different physiological functions. The occurrence of gad genes encoding GAD has been shown for many microorganisms, and GABA-producing lactic acid bacteria (LAB) have been a focus of research during recent years. A wide range of traditional foods produced by fermentation based on LAB offer the potential of providing new functional food products enriched with GABA that may offer certain health-benefits. Different GAD enzymes and genes from several strains of LAB have been isolated and characterized recently. GABA-producing LAB, the biochemical properties of their GAD enzymes, and possible applications are reviewed here.

Bioreduction of Gold Ions from Anisotropic to Isotropic Nanostructures by NADPH‐Dependent Reductase from Bipolaris oryzae

AbstractHere, we report an eco‐friendly, benign and rapid mycogenic synthesis of gold nanoparticles of various shapes using cell‐free extract of the fungus Bipolaris oryzae (B. oryzae). Three different volumes of cell‐free extract of the fungus were exposed to an aqueous solution of gold chloride and in all three cases, there is a gradual color change from light yellow to pink indicating the formation of gold nanoparticle. Reduced nicotinamide adenine dinucleotide phosphate (NADPH) dependent enzyme present in the cell‐free extract of the fungus B. oryzae leads to the rapid reduction of Au3+ to Au0 within 5–10 min when incubated with an aqueous solution of gold chloride. UV‐visible and fourier transform infra red (FTIR) spectroscopy measurements show the presence of aminoacids, such as tyrosine/phenylalanine and proteins as the possible capping agent. High‐resolution transmission electron microscopy (HR‐TEM) revealed the morphology of the structures were anisotropic triangles, hexagons, decahedrals and isotropic spheres. Isotropic spherical nanoparticles and anisotropic nanoparticles such as triangles, hexagons and decahedrals were produced at higher and lower volumes of the fungal extract, respectively. Rapid and sustainable approach for the production of gold nanoparticles of various shapes such as the one reported has a plethora of applications in the field of life‐sciences and medicine.

Substrate Binding Stiffens Aspartate Aminotransferase by Altering the Enzyme Picosecond Vibrational Dynamics

Protein dynamics on various time scales from femtoseconds to milliseconds impacts biological function by driving proteins to conformations conducive to ligand binding and creating functional states in enzyme catalysis. Neutron vibrational spectroscopy carried out by measuring inelastic neutron scattering from protein molecules in combination with molecular simulations has the unique ability of detecting and visualizing changes in the picosecond protein vibrational dynamics due to ligand binding. Here we present neutron vibrational spectra of a homodimeric pyridoxal 5′-phosphate-dependent enzyme, aspartate aminotransferase, obtained from the open internal aldimine and closed external aldimine conformational states. We observe that in the external aldimine state the protein structure stiffens relative to the internal aldimine state, indicating rigidified vibrational dynamics on the picosecond time scale in the low-frequency regime of 5–50 cm–1. Our molecular dynamics simulations indicate substantial changes in the picosecond dynamics of the enzyme secondary structure elements upon substrate binding, with the largest contributions from just two helices and the β-sheet.

Hydrogen Sulfide and Pathophysiology of the CNS

The literature data and results of our research team concerning the physiological and pathological effects of hydrogen sulfide, a gas transmitter that has recently attracted significant and increasing attention of neurophysiologists, are analyzed. Hydrogen sulfide is a gaseous signaling molecule, the effects of which were discovered later than those of NO and CO; H2S was found to play great pathophysiological roles in various diseases. This compound is synthesized in the body by enzymatic and non-enzymatic pathways from cysteine, using the pyridoxal 5’-phosphate-dependent enzymes cystathionine β-synthase (CBS) or cystathionine γ-lyase (CSE); 3-mercaptopyruvate sulfurtransferase (3MST) plays a considerable role in H2S catabolism. Abnormal deviations in H2S metabolism are important factors involved in the development of a number of dangerous neurological pathologies, in particular of Parkinson’s disease (PD), Alzheimer’s disease (AD), Huntington’s disease (HD), and Down’s syndrome (DS). This compound was also demonstrated to be involved in various biological and pathophysiological processes in the brain resulting from traumatic brain injury (TBI), stroke, oxidative stress, and cerebral edema. In the case of traumatic injury in the trout brain, the activation of CBS expression in the radial glia phenotypes occurs in the aNSC.

Birth of a pathway for sulfur metabolism in early amniote evolution.

Among amniotes, reptiles and mammals are differently adapted to terrestrial life. It is well appreciated that terrestrialization required adaptive changes of vertebrate metabolism, particularly in the mode of nitrogen excretion. However, the current paradigm is that metabolic adaptation to life on land did not involve synthesis of enzymatic pathways de novo but repurposing of existing ones. Here, by comparing the inventory of pyridoxal phosphate-dependent enzymes (PLPome) in different amniotes, we identify in silico a pathway for sulfur metabolism present in chick embryos but not in mammals. Cysteine lyase (CL) contains heme and PLP cofactors and converts cysteine and sulfite into cysteic acid and hydrogen sulfide. A specific cysteic acid decarboxylase (CAD) produces taurine while hydrogen sulfide is recycled into cysteine by cystathionine beta-synthase (CBS). This reaction sequence enables the formation of sulfonated amino acids during embryo development in the egg at no cost of reduced sulfur. The pathway originated around 300 million years ago in a proto-reptile by CBS duplication, CL neofunctionalization, and CAD co-option. Our findings indicate that adaptation to terrestrial life involved innovations in metabolic pathways and reveal the molecular mechanisms by which such innovations arose in amniote evolution.

Structural and kinetic properties of serine hydroxymethyltransferase from the halophytic cyanobacterium Aphanothece halophytica provide a rationale for salt tolerance

Serine hydroxymethyltransferase (SHMT) is a pyridoxal 5′-phosphate-dependent enzyme that plays a pivotal role in cellular one‑carbon metabolism. In plants and cyanobacteria, this enzyme is also involved in photorespiration and confers salt tolerance, as in the case of SHMT from the halophilic cyanobacterium Aphanothece halophytica (AhSHMT). We have characterized the catalytic properties of AhSHMT in different salt and pH conditions. Although the kinetic properties of AhSHMT correlate with those of the mesophilic orthologue from Escherichia coli, AhSHMT appears more catalytically efficient, especially in presence of salt. Our studies also reveal substrate inhibition, previously unobserved in AhSHMT. Furthermore, addition of the osmoprotectant glycine betaine under salt conditions has a distinct positive effect on AhSHMT activity. The crystal structures of AhSHMT in three forms, as internal aldimine, as external aldimine with the l-serine substrate, and as a covalent complex with malonate, give structural insights on the possible role of specific amino acid residues implicated in the halophilic features of AhSHMT. Importantly, we observed that overexpression of the gene encoding SHMT, independently from its origin, increases the capability of E. coli to grow in high salt conditions, suggesting that the catalytic activity of this enzyme in itself plays a fundamental role in salt tolerance.

Quorum Sensing System-Regulated Proteins Affect the Spoilage Potential of Co-cultured Acinetobacter johnsonii and Pseudomonas fluorescens From Spoiled Bigeye Tuna (Thunnus obesus) as Determined by Proteomic Analysis.

Food spoilage by certain species of bacteria is reported to be regulated by quorum sensing (QS). Acinetobacter johnsonii and Pseudomonas fluorescens, the major specific spoilage organisms, are found to be limited in their QS and co-culture interactions. The aim of this study was to determine how QS-regulated proteins affect the spoilage potential of co-cultured A. johnsonii and P. fluorescens obtained from spoiled bigeye tuna (Thunnus obesus) using a proteomics approach. The A. johnsonii, P. fluorescens, and their co-culture tested the N-acyl-homoserine lactone (AHL) activities using reporter Chromobacterium violaceum CV026 and LC-MS/MS in qualitative and quantitative approaches, respectively. These latter showed that, of the 470 proteins and 444 proteins in A. johnsonii (A) and P. fluorescens (P), respectively, 80 were significantly up-regulated and 97 were significantly down-regulated in A vs. AP, whereas 90 were up-regulated and 65 were down-regulated in P vs. AP. The differentially expressed proteins included the AI-2E family transporter OS, 50S ribosomal protein L3, thioredoxin reductase OS, cysteine synthase CysM OS, DNA-binding response regulator, and amino acid ABC transporter ATPase OS. The cellular process (GO:0009987), metabolic process (GO:0008152), and single-organism process (GO:0044699) were classified into the gene ontology (GO) term. In addition, energy production and conversion, amino acid transport and metabolism, translation, ribosomal structure and biogenesis, post-translational modification, protein turnover, and chaperones were distributed into the clusters of orthologous groups of proteins (COG) terms. The KEGG pathways revealed that 84 and 77 differentially expressed proteins were divided into 20 KEGG pathways in A vs. AP and P vs. AP, respectively, and amino acid metabolism, carbohydrate metabolism, energy metabolism, and translation were significantly enriched. Proteins that correlated with the spoilage-related metabolic pathways, including thioredoxin reductase OS, cysteine synthase OS, and pyridoxal phosphate-dependent enzyme family protein OS, were identified. AI-2E family transporter OS and LuxR family transcriptional regulator OS were identified that related to the QS system. These findings provide a differential proteomic profile of co-culture in A. johnsonii and P. fluorescens, and have potential applications in QS and the regulation of spoilage potential.

Structural characterization of human O-phosphoethanolamine phospho-lyase.

Human O-phosphoethanolamine phospho-lyase (hEtnppl; EC 4.2.3.2) is a pyridoxal 5'-phosphate-dependent enzyme that catalyzes the degradation of O-phosphoethanolamine (PEA) into acetaldehyde, phosphate and ammonia. Physiologically, the enzyme is involved in phospholipid metabolism, as PEA is the precursor of phosphatidylethanolamine in the CDP-ethanolamine (Kennedy) pathway. Here, the crystal structure of hEtnppl in complex with pyridoxamine 5'-phosphate was determined at 2.05 Å resolution by molecular replacement using the structure of A1RDF1 from Arthrobacter aurescens TC1 (PDB entry 5g4i) as the search model. Structural analysis reveals that the two proteins share the same general fold and a similar arrangement of active-site residues. These results provide novel and useful information for the complete characterization of the human enzyme.

Molecular Mechanism of ISC Iron-Sulfur Cluster Biogenesis Revealed by High-Resolution Native Mass Spectrometry.

Iron-sulfur (Fe-S) clusters are ubiquitous protein cofactors that are required for many important biological processes including oxidative respiration, nitrogen fixation, and photosynthesis. Biosynthetic pathways assemble Fe-S clusters with different iron-to-sulfur stoichiometries and distribute these clusters to appropriate apoproteins. In the ISC pathway, the pyridoxal 5'-phosphate-dependent cysteine desulfurase enzyme IscS provides sulfur to the scaffold protein IscU, which templates the Fe-S cluster assembly. Despite their functional importance, mechanistic details for cluster synthesis have remained elusive. Recent advances in native mass spectrometry (MS) have allowed proteins to be preserved in native-like structures and support applications in the investigation of protein structure, dynamics, ligand interactions, and the identification of protein-associated intermediates. Here, we prepared samples under anaerobic conditions and then applied native MS to investigate the molecular mechanism for Fe-S cluster synthesis. This approach was validated by the high agreement between native MS and traditional visible circular dichroism spectroscopic assays. Time-dependent native MS experiments revealed potential iron- and sulfur-based intermediates that decay as the [2Fe-2S] cluster signal developed. Additional experiments establish that (i) Zn(II) binding stabilizes IscU and protects the cysteine residues from oxidation, weakens the interactions between IscU and IscS, and inhibits Fe-S cluster biosynthesis; and (ii) Fe(II) ions bind to the IscU active site cysteine residues and another lower affinity binding site and promote the intermolecular sulfur transfer reaction from IscS to IscU. Overall, these results support an iron-first model for Fe-S cluster synthesis and highlight the power of native MS in defining protein-associated intermediates and elucidating mechanistic details of enzymatic processes.

A Remarkable Difference That One Fluorine Atom Confers on the Mechanisms of Inactivation of Human Ornithine Aminotransferase by Two Cyclohexene Analogues of γ-Aminobutyric Acid.

Human ornithine aminotransferase (hOAT), a pyridoxal 5'-phosphate-dependent enzyme, plays a critical role in the progression of hepatocellular carcinoma (HCC). Pharmacological selective inhibition of hOAT has been shown to be a potential therapeutic approach for HCC. Inspired by the discovery of the nonselective aminotransferase inactivator (1R,3S,4S)-3-amino-4-fluoro cyclopentane-1-carboxylic acid (1), in this work, we rationally designed, synthesized, and evaluated a novel series of fluorine-substituted cyclohexene analogues, thereby identifying 8 and 9 as novel selective hOAT time-dependent inhibitors. Intact protein mass spectrometry and protein crystallography demonstrated 8 and 9 as covalent inhibitors of hOAT, which exhibit two distinct inactivation mechanisms resulting from the difference of a single fluorine atom. Interestingly, they share a similar turnover mechanism, according to the mass spectrometry-based analysis of metabolites and fluoride ion release experiments. Molecular dynamics (MD) simulations and electrostatic potential (ESP) charge calculations were conducted, which elucidated the significant influence of the one-fluorine difference on the corresponding intermediates, leading to two totally different inactivation pathways. The novel addition-aromatization inactivation mechanism for 9 contributes to its significantly enhanced potency, along with excellent selectivity over other aminotransferases.

Impaired folate binding of serine hydroxymethyltransferase 8 from soybean underlies resistance to the soybean cyst nematode

Management of the agricultural pathogen soybean cyst nematode (SCN) relies on the use of SCN-resistant soybean cultivars, a strategy that has been failing in recent years. An underutilized source of resistance in the soybean genotype Peking is linked to two polymorphisms in serine hydroxy-methyltransferase 8 (SHMT8). SHMT is a pyridoxal 5'-phosphate-dependent enzyme that converts l-serine and (6S)-tetrahydrofolate to glycine and 5,10-methylenetetrahydrofolate. Here, we determined five crystal structures of the 1884-residue SHMT8 tetramers from the SCN-susceptible cultivar (cv.) Essex and the SCN-resistant cv. Forrest (whose resistance is derived from the SHMT8 polymorphisms in Peking); the crystal structures were determined in complex with various ligands at 1.4-2.35 Å resolutions. We find that the two Forrest-specific polymorphic substitutions (P130R and N358Y) impact the mobility of a loop near the entrance of the (6S)-tetrahydrofolate-binding site. Ligand-binding and kinetic studies indicate severely reduced affinity for folate and dramatically impaired enzyme activity in Forrest SHMT8. These findings imply widespread effects on folate metabolism in soybean cv. Forrest that have implications for combating the widespread increase in virulent SCN.