Internal Aldimine Research Articles

Pyridoxal 5'-phosphate dependent reactions: Analyzing the mechanism of aspartate aminotransferase.

Enzyme catalysis is the primary activity in energy and information metabolism and enzyme cofactors are key to the catalytic ability of most enzymes. Pyridoxal 5'-phosphate (PLP) cofactor, derived from Vitamin B6, is widely distributed in nature and has significant latitude in catalytic diversity. X-ray crystallography has revealed the structures of diverse PLP dependent enzymes from multiple families. But these structures are incomplete, lacking the positions of protons essential for understanding enzymatic mechanisms. Here, we review the diversity of PLP and discuss the use of neutron crystallography and joint X-ray/neutron refinement of Fold Type I aspartate aminotransferase to visualize the positions of protons in both the internal and external aldimine forms. Strategies used to prepare extremely large crystals required for neutron diffraction and the approach to data refinement including the PLP cofactor are discussed. The observed positions of protons, including one located in a previously unknown low-barrier hydrogen bond, have been used to create more accurate models for computational analysis. The results revealed a new mechanism for the transaminase reaction where hyperconjugation is key to reducing the energy barrier which finally provides a clear explanation of the Dunathan alignment.

Structural insights into the mechanism of internal aldimine formation and catalytic loop dynamics in an archaeal Group II decarboxylase

Formation of the internal aldimine (LLP) is the first regulatory step that activates pyridoxal 5′-phosphate (PLP) dependent enzymes. The process involves a nucleophilic attack on PLP by an active site Lys residue, followed by proton transfers resulting in a carbinolamine (CBA) intermediate that undergoes dehydration to form the aldimine. Despite a general understanding of the pathway, the structural basis of the mechanistic roles of specific residues in each of these steps is unclear. Here we determined the crystal structure of the LLP form (holo-form) of a Group II PLP-dependent decarboxylase from Methanocaldococcus jannaschii (MjDC) at 1.7 Å resolution. By comparing the crystal structure of MjDC in the LLP form with that of the pyridoxal-P (non-covalently bound aldehyde) form, we demonstrate structural evidence for a water-mediated mechanism of LLP formation. A conserved extended hydrogen-bonding network around PLP coupled to the pyridinyl nitrogen influences activation and catalysis by affecting the electronic configuration of PLP. Furthermore, the two cofactor bound forms revealed open and closed conformations of the catalytic loop (CL) in the absence of a ligand, supporting a hypothesis for a regulatory link between LLP formation and CL dynamics. The evidence suggests that activation of Group II decarboxylases involves a complex interplay of interactions between the electronic states of PLP, the active site micro-environment and CL dynamics.

Transimination Reaction at the Active Site of Aspartate Aminotransferase: A Proton Hopping Mechanism through Pyridoxal 5′-Phosphate

The transimination reaction involves conversion of an internal aldimine involving pyridoxal 5′-phosphate (PLP) and an enzyme to an external aldimine involving PLP and a substrate amino acid and it ...

Elucidating the Heme Domain's Regulatory Role on Human Cystathionine β‐Synthase (hCBS) Product Formation

Human cystathionine β‐synthase (hCBS) is a vital enzyme found within the human transulfuration pathway and is responsible for the biotransformation of serine and homocysteine into cystathionine. hCBS at high cysteine concentrations has been shown to release hydrogen sulfide as a byproduct via an alternate reaction, suggesting hCBS to be closely involved with regulating metabolic sulfur. Catalysis in both the primary and alternate reactions proceeds via a PLP dependent β‐replacement ping‐pong mechanism, with the last step before product dissociation from the internal aldimine (Schiff base) intermediate being the condensation of H2O or H2S, respectively. Consequently, mis‐regulation of substrate levels or loss of CBS activity has been causally linked to hyperhomocysteinemia, a biological condition signifying abnormally high levels of homocysteine within the circulatory system; this condition being one of many causes leading to cardiovascular disease. A highly conserved PLP catalytic and SAM regulatory domain can be seen when comparing yeast analogue (yCBS) to mammalian variants. However, the mammalian variants, specifically the human analog hCBS, differs by containing a unique heme binding domain not found in yCBS. Given that the heme prosthetic group is not required for catalytic activity, the role that this additional domain plays in regulating cystathionine production in hCBS is not yet clearly understood. Current methods for measuring CBS catalytic activity involve using radioactively labeled substrates and typically have focused only on yeast and truncated hCBS variants. We have adapted a previously established colorimetric coupled enzyme assay to measure hCBS kinetics, coupling CBS with cystathionine β‐lyase (CBL), and lactate dehydrogenase (LDH); where the stoichiometric oxidation of NADH, monitored spectrophotometrically at 340 nm, is correlated directly to the formation of cystathionine. Preliminary results suggest this coupled assay is a viable method for measuring product formation kinetics of full length wild type hCBS. Current and future work aims to 1) asses catalytic rate activity for wild type hCBS and 2) asses the activity of single point mutant variants with mutations localized within the heme binding domain. Through these experiments, we hope to elucidate a deeper understanding of how the heme binding domain affects cystathionine product formation.Support or Funding InformationNIH MARC: T34‐GM008574This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Crystal structures of aminotransferases Aro8 and Aro9 from Candida albicans and structural insights into their properties

Aminotransferases catalyze reversibly the transamination reaction by a ping-pong bi-bi mechanism with pyridoxal 5′-phosphate (PLP) as a cofactor. Various aminotransferases acting on a range of substrates have been reported. Aromatic transaminases are able to catalyze the transamination reaction with both aromatic and acidic substrates. Two aminotransferases from C. albicans, Aro8p and Aro9p, have been identified recently, exhibiting different catalytic properties. To elucidate the multiple substrate recognition of the two enzymes we determined the crystal structures of an unliganded CaAro8p, a complex of CaAro8p with the PLP cofactor bound to a substrate, forming an external aldimine, CaAro9p with PLP in the form of internal aldimine, and CaAro9p with a mixture of ligands that have been interpreted as results of the enzymatic reaction. The crystal structures of both enzymes contains in the asymmetric unit a biologically relevant dimer of 55 kDa for CaAro8 and 59 kDa for CaAro9p protein subunits. The ability of the enzymes to process multiple substrates could be related to a feature of their architecture in which the active site resides on one subunit while the substrate-binding site is formed by a long loop extending from the other subunit of the dimeric molecule. The separation of the two functions to different chemical entities could facilitate the evolution of the substrate-binding part and allow it to be flexible without destabilizing the conservative catalytic mechanism.

Activity Improvements of an Engineered ω-transaminase for Ketones Are Positively Correlated with Those for Cognate Amines

Chiral amines are broadly used as essential building blocks of diverse pharmaceutical drugs. ω-Transaminase (ω-TA) is one of the promising enzymes for biocatalytic preparation of chiral amines owing to its capability for reductive amination of ketones. However, industrial application of ω-TA to asymmetric synthesis of chiral amines is often limited by marginal activities for ketones. Here, we explore whether activity improvements of ω-TA for ketones caused by active site engineering are correlated with those for cognate amines. We examined amino donor reactivities of 20 amines with ω-TA from Ochrobactrum anthropi (OATA) and its engineered variant carrying a W58L mutation (OATAW58L). Consistent with the previously observed activity increases for ketones, OATAW58L showed 3 to 79-fold activity improvements for amines. Docking simulations suggested that the activity improvements resulted from increased proximity of a bound amine to an internal aldimine owing to relocation of a small binding pocket. Activity comparison indicated that the W58L mutation induced activity increases for ketones better than it did those for amines. This result motivated us to carry out Pearson correlation analysis among the activity improvements for amines and ketones, revealing a strong positive correlation (r = 0.84). This suggests that active site engineering beneficial for activity of ω-TA toward a target amine is likely to evoke an activity improvement for its cognate ketone. Our finding is expected to provide a clue to design a robust high-throughput screening method required for creation of an engineered mutant displaying a better activity toward a target ketone.

Thermostable Branched-Chain Amino Acid Transaminases From the Archaea Geoglobus acetivorans and Archaeoglobus fulgidus: Biochemical and Structural Characterization.

Two new thermophilic branched chain amino acid transaminases have been identified within the genomes of different hyper-thermophilic archaea, Geoglobus acetivorans, and Archaeoglobus fulgidus. These enzymes belong to the class IV of transaminases as defined by their structural fold. The enzymes have been cloned and over-expressed in Escherichia coli and the recombinant enzymes have been characterized both biochemically and structurally. Both enzymes showed high thermostability with optimal temperature for activity at 80 and 85°C, respectively. They retain good activity after exposure to 50% of the organic solvents, ethanol, methanol, DMSO and acetonitrile. The enzymes show a low activity to (R)-methylbenzylamine but no activity to (S)-methylbenzylamine. Both enzymes have been crystallized and their structures solved in the internal aldimine form, to 1.9 Å resolution for the Geoglobus enzyme and 2.0 Å for the Archaeoglobus enzyme. Also the Geoglobus enzyme structure has been determined in complex with the amino acceptor α-ketoglutarate and the Archaeoglobus enzyme in complex with the inhibitor gabaculine. These two complexes have helped to determine the conformation of the enzymes during enzymatic turnover and have increased understanding of their substrate specificity. A comparison has been made with another (R) selective class IV transaminase from the fungus Nectria haematococca which was previously studied in complex with gabaculine. The subtle structural differences between these enzymes has provided insight regarding their different substrate specificities.

Biochemical Characterization of Aspergillus fumigatus AroH, a Putative Aromatic Amino Acid Aminotransferase.

The rise in the frequency of nosocomial infections is becoming a major problem for public health, in particular in immunocompromised patients. Aspergillus fumigatus is an opportunistic fungus normally present in the environment directly responsible for lethal invasive infections. Recent results suggest that the metabolic pathways related to amino acid metabolism can regulate the fungus-host interaction and that an important role is played by enzymes involved in the catabolism of L-tryptophan. In particular, in A. fumigatus L-tryptophan regulates Aro genes. Among them, AroH encodes a putative pyridoxal 5'-phosphate-dependent aminotransferase. Here we analyzed the biochemical features of recombinant purified AroH by spectroscopic and kinetic analyses corroborated by in silico studies. We found that the protein is dimeric and tightly binds the coenzyme forming a deprotonated internal aldimine in equilibrium with a protonated ketoenamine form. By setting up a new rapid assay method, we measured the kinetic parameters for the overall transamination of substrates and we demonstrated that AroH behaves as an aromatic amino acid aminotransferase, but also accepts L-kynurenine and α-aminoadipate as amino donors. Interestingly, computational approaches showed that the predicted overall fold and active site topology of the protein are similar to those of its yeast ortholog, albeit with some differences in the regions at the entrance of the active site, which could possibly influence substrate specificity. Should targeting fungal metabolic adaptation be of therapeutic value, the results of the present study may pave the way to the design of specific AroH modulators as potential novel agents at the host/fungus interface.

Structural Analysis of Phosphoserine Aminotransferase (Isoform 1) From Arabidopsis thaliana– the Enzyme Involved in the Phosphorylated Pathway of Serine Biosynthesis

Phosphoserine aminotransferase (PSAT) is a pyridoxal 5′-phosphate (PLP)-dependent enzyme that catalyzes the conversion of 3-phosphohydroxypyruvate (3-PHP) to 3-phosphoserine (PSer) in an L-glutamate (Glu)-linked reversible transamination reaction. This process proceeds through a bimolecular ping–pong mechanism and in plants takes place in plastids. It is a part of the phosphorylated pathway of serine biosynthesis, one of three routes recognized in plant organisms that yield serine. In this three-step biotransformation, 3-phosphoglycerate (3-PGA) delivered from plastidial glycolysis and Calvin cycle is oxidized by 3-PGA dehydrogenase. Then, 3-PHP is subjected to transamination with Glu to yield PSer and α-ketoglutarate (AKG). In the last step of the pathway, serine is produced by the action of phosphoserine phosphatase. Here we present the structural characterization of PSAT isoform 1 from Arabidopsis thaliana (AtPSAT1), a dimeric S-shaped protein that truncated of its 71-residue-long chloroplast-targeting signal peptide. Three crystal structures of AtPSAT1 captured at different stages of the reaction: (i) internal aldimine state with PLP covalently bound to the catalytic K265, (ii) holoenzyme in complex with pyridoxamine-5′-phosphate (PMP) after transfer of the amino group from glutamate and (iii) the geminal diamine intermediate state wherein the cofactor is covalently bound to both, K265 and PSer. These snapshots over the course of the reaction present detailed architecture of AtPSAT1 and allow for the comparison of this plant enzyme with other PSATs. Conformational changes of the protein during the catalytic event concern (i) the neighborhood of K265 when the amino group is transferred to the cofactor to form PMP and (ii) movement of the gate-keeping loop (residues 391–401) upon binding of 3-PHP and PSer. The latter conformational change of the loop may likely be one of key elements that regulate catalytic activity of PSATs.

Free energy landscapes of prototropic tautomerism in pyridoxal 5'-phosphate schiff bases at the active site of an enzyme in aqueous medium.

We have performed hybrid quantum-classical metadynamics simulations and quantum chemical calculations to investigate the free energy landscapes of intramolecular proton transfer and associated tautomeric equilibrium in pyridoxal 5 '-phosphate (PLP) Schiff Bases, namely the internal and external aldimines, at the active site of serine hydroxymethyltransferase (SHMT) enzyme in aqueous medium. It is important to determine the relative stability of the two tautomers (ketoenamine and enolimine) of the PLP aldimines to study the catalytic activity of the concerned enzyme. Both the internal PLP aldimine (PLP-LYS) and the external PLP aldimine (PLP-SER) of SHMT are found to have a higher stability for the ketoenamine tautomer over the enolimine form. The higher stability of the ketoenamine tautomer can be attributed to the more number of favorable interactions of the ketoenamine form with its surroundings at the active site of the enzyme. The ketoenamine is found to be stabilized by about 2.5 kcal/mol in the PLP-LYS internal aldimine, while this stabilization is about 6.7 kcal/mol for the PLP-SER external aldimine at the active site of the enzyme compared to the corresponding enolimine forms. The interactions faced by the PLP aldimines at the active site pocket determine the relative dominance of the tautomers and could possibly alter the tautomeric shift in different PLP dependent enzymes. © 2018 Wiley Periodicals, Inc.

Chloroplastic Serine Hydroxymethyltransferase From Medicago truncatula: A Structural Characterization.

Serine hydroxymethyltransferase (SHMT, EC 2.1.2.1) is a pyridoxal 5′-phosphate (PLP)-dependent enzyme which catalyzes the reversible serine-to-glycine conversion in either a tetrahydrofolate-dependent or -independent manner. The enzyme is also responsible for the tetrahydrofolate-independent cleavage of other β-hydroxy amino acids. In addition to being an essential player in the serine homeostasis, SHMT action is the main source of activated one-carbon units, which links SHMT activity with the control of cell proliferation. In plants, studies of SHMT enzymes are more complicated than of those of, e.g., bacterial or mammalian origins because plant genomes encode multiple SHMT isozymes that are targeted to different subcellular compartments: cytosol, mitochondria, plastids, and nucleus. Here we report crystal structures of chloroplast-targeted SHMT from Medicago truncatula (MtSHMT3). MtSHMT3 is a tetramer in solution, composed of two tight and obligate dimers. Our complexes with PLP internal aldimine, PLP-serine and PLP-glycine external aldimines, and PLP internal aldimine with a free glycine reveal structural details of the MtSHMT3-catalyzed reaction. Capturing the enzyme in different stages along the course of the slow tetrahydrofolate-independent serine-to-glycine conversion allowed to observe a unique conformation of the PLP-serine γ-hydroxyl group, and a concerted movement of two tyrosine residues in the active site.

Direct visualization of critical hydrogen atoms in a pyridoxal 5\u2032-phosphate enzyme

Enzymes dependent on pyridoxal 5′-phosphate (PLP, the active form of vitamin B6) perform a myriad of diverse chemical transformations. They promote various reactions by modulating the electronic states of PLP through weak interactions in the active site. Neutron crystallography has the unique ability of visualizing the nuclear positions of hydrogen atoms in macromolecules. Here we present a room-temperature neutron structure of a homodimeric PLP-dependent enzyme, aspartate aminotransferase, which was reacted in situ with α-methylaspartate. In one monomer, the PLP remained as an internal aldimine with a deprotonated Schiff base. In the second monomer, the external aldimine formed with the substrate analog. We observe a deuterium equidistant between the Schiff base and the C-terminal carboxylate of the substrate, a position indicative of a low-barrier hydrogen bond. Quantum chemical calculations and a low-pH room-temperature X-ray structure provide insight into the physical phenomena that control the electronic modulation in aspartate aminotransferase.

Radiation damage at the active site of human alanine:glyoxylate aminotransferase reveals that the cofactor position is finely tuned during catalysis

The alanine:glyoxylate aminotransferase (AGT), a hepatocyte-specific pyridoxal-5′-phosphate (PLP) dependent enzyme, transaminates L-alanine and glyoxylate to glycine and pyruvate, thus detoxifying glyoxylate and preventing pathological oxalate precipitation in tissues. In the widely accepted catalytic mechanism of the aminotransferase family, the lysine binding to PLP acts as a catalyst in the stepwise 1,3-proton transfer, interconverting the external aldimine to ketimine. This step requires protonation by a conserved aspartate of the pyridine nitrogen of PLP to enhance its ability to stabilize the carbanionic intermediate. The aspartate residue is also responsible for a significant geometrical distortion of the internal aldimine, crucial for catalysis. We present the structure of human AGT in which complete X-ray photoreduction of the Schiff base has occurred. This result, together with two crystal structures of the conserved aspartate pathogenic variant (D183N) and the molecular modeling of the transaldimination step, led us to propose that an interplay of opposite forces, which we named spring mechanism, finely tunes PLP geometry during catalysis and is essential to move the external aldimine in the correct position in order for the 1,3-proton transfer to occur.

Mechanistic insights into the γ-elimination reaction of l-methionine catalyzed by methionine γ-lyase (MGL)

The γ-elimination mechanism of l-methionine catalyzed by methionine γ-lyase was studied by employing the combined quantum mechanics and molecular (QM/MM) calculations. Based on the two crystal structures of methionine γ-lyase from Clostridium sporogenes and Citrobacter freundii, a computational model that contains the enzyme and the external aldimine intermediate was constructed. According to the results of our calculations, the whole catalytic reaction can be divided into two parts. Part I describes the formation of external aldimine intermediate from the internal aldimine intermediate, in which the key protonation step within the tetrahedral intermediate (IM2) should require the assistance of one water molecule, and the dissociation of Lys210 from the internal aldimine intermediate corresponds to an overall energy barrier of 13.5 kcal/mol. In Part II, the external aldimine intermediate converts to the aminocrotonate intermediate, which contains complex asynchronous and concerted proton transfer processes, including those of Cα-proton and Cβ-proton of the substrate as well as the C4′-proton of PLP, in which the pocket residues Tyr113 and Lys210 play key roles. The abstraction of Cβ-proton and the transfer of C4′-proton correspond to very similar barrier heights (16.5 and 16.2 kcal/mol) on the potential energy surface, implying that both steps are possibly rate-limiting. Besides, the calculated overall energy barrier is close to the free energy barrier estimated from the experiment (16.3 kcal/mol). These results may provide useful information for further understanding the catalysis of pyridoxal 5′-phosphate (PLP)-dependent lyase.

Streptomyces wadayamensis MppP: A Novel PLP‐Dependent L‐Arginine Hydroxylase in L‐Enduracididine Biosynthesis

The nonproteinogenic amino acid L‐enduracididine (L‐End) is a critical component of the mannopeptimycins, a family of cyclic glycopeptide antibiotics with potent activities against drug‐resistant pathogens like MRSA. Creating derivatives of mannopeptimycin and other L‐End‐containing natural products is hampered by the limited availability of L‐End. We are investigating the L‐End biosynthetic pathway in order to develop an efficient enzymatic or chemo‐enzymatic route to produce this unusual amino acid. Recently, we found that MppP is a PLP‐dependent L‐Arg hydroxylase in L‐End biosynthesis in Streptomyces wadayamensis. It reacts with L‐Arg and dioxygen to yield two products, 2‐ketoarginine and 4‐hydroxy‐2‐ketoarginine in a ratio of ~1.7:1. Surprisingly, 1 equivalent of H2O2 is produced for each equivalent of O2 consumed. In addition, the ratio of O2 consumption to L‐Arg consumption was ~1.4:1, suggesting that one product requires 2 equivalents of O2, and the other only 1 equivalent. We have determined the structures of MppP in four states: the internal aldimine, the external aldimine with the substrate, L‐Arg, the product complex with 4‐hydroxy‐2‐ketoarginine, and the product complex with 2‐ketoarginine. According to the structures, the N‐terminal helix is disordered in the internal aldimine and covers the active site only when the substrate is bound. A glutamate residue in the N‐terminal helix, E15, makes a hydrogen bonding interaction with the carboxylate of the substrate, L‐Arg. This observation prompted us to make three N‐terminal variants of SwMppP: E15A, E15Q, and the truncation mutant, SwMppP23‐276. Steady state kinetics showed that the two point mutants had no effect on kcat or KM,L‐Arg. The truncation mutant, however, showed an approximately 10‐fold increase in KM and a 3‐fold decrease in kcat, which reduced the pseudo‐second order rate constant by almost 25‐fold. Interestingly, although the steady state kinetics (as measured by dioxygen consumption) are indistinguishable for the E15A and E15Q variants, the E15A variant does not produce 4‐hydroxy‐2‐ketoarginine, only the abortive product 2‐ketoarginine. Likewise, the truncation mutant also produced only 2‐ketoarginine. Our structural and kinetic characterization of the wild‐type and variant forms of SwMppP have allowed us to propose a revised model where the oxygen incorporated in the hydroxy‐arginine product is derived from water rather than from dioxygen.Support or Funding InformationNational Science Foundation CHE‐1606842

High resolution crystal structures of human kynurenine aminotransferase-I bound to PLP cofactor, and in complex with aminooxyacetate.

In this study, we report two high-resolution structures of the pyridoxal 5' phosphate (PLP)-dependent enzyme kynurenine aminotransferase-I (KAT-I). One is the native structure with the cofactor in the PLP form bound to Lys247 with the highest resolution yet available for KAT-I at 1.28 Å resolution, and the other with the general PLP-dependent aminotransferase inhibitor, aminooxyacetate (AOAA) covalently bound to the cofactor at 1.54 Å. Only small conformational differences are observed in the vicinity of the aldimine (oxime) linkage with which the PLP forms the Schiff base with Lys247 in the 1.28 Å resolution native structure, in comparison to other native PLP-bound structures. We also report the inhibition of KAT-1 by AOAA and aminooxy-phenylpropionic acid (AOPP), with IC50s of 13.1 and 5.7 μM, respectively. The crystal structure of the enzyme in complex with the inhibitor AOAA revealed that the cofactor is the PLP form with the external aldimine linkage. The location of this oxime with the PLP, which forms in place of the native internal aldimine linkage of PLP of the native KAT-I, is away from the position of the native internal aldimine, with the free Lys247 substantially retaining the orientation of the native structure. Tyr101, at the active site, was observed in two conformations in both structures.

Structural Snapshots of an Engineered Cystathionine-γ-lyase Reveal the Critical Role of Electrostatic Interactions in the Active Site.

Enzyme therapeutics that can degrade l-methionine (l-Met) are of great interest as numerous malignancies are exquisitely sensitive to l-Met depletion. To exhaust the pool of methionine in human serum, we previously engineered an l-Met-degrading enzyme based on the human cystathionine-γ-lyase scaffold (hCGL-NLV) to circumvent immunogenicity and stability issues observed in the preclinical application of bacterially derived methionine-γ-lyases. To gain further insights into the structure-activity relationships governing the chemistry of the hCGL-NLV lead molecule, we undertook a biophysical characterization campaign that captured crystal structures (2.2 Å) of hCGL-NLV with distinct reaction intermediates, including internal aldimine, substrate-bound, gem-diamine, and external aldimine forms. Curiously, an alternate form of hCGL-NLV that crystallized under higher-salt conditions revealed a locally unfolded active site, correlating with inhibition of activity as a function of ionic strength. Subsequent mutational and kinetic experiments pinpointed that a salt bridge between the phosphate of the essential cofactor pyridoxal 5'-phosphate (PLP) and residue R62 plays an important role in catalyzing β- and γ-eliminations. Our study suggests that solvent ions such as NaCl disrupt electrostatic interactions between R62 and PLP, decreasing catalytic efficiency.

Catalytic Intermediate Crystal Structures of Cysteine Desulfurase from the Archaeon Thermococcus onnurineus NA1.

Thermococcus onnurineus NA1 is an anaerobic archaeon usually found in a deep-sea hydrothermal vent area, which can use elemental sulfur (S0) as a terminal electron acceptor for energy. Sulfur, essential to many biomolecules such as sulfur-containing amino acids and cofactors including iron-sulfur cluster, is usually mobilized from cysteine by the pyridoxal 5′-phosphate- (PLP-) dependent enzyme of cysteine desulfurase (CDS). We determined the crystal structures of CDS from Thermococcus onnurineus NA1 (ToCDS), which include native internal aldimine (NAT), gem-diamine (GD) with alanine, internal aldimine structure with existing alanine (IAA), and internal aldimine with persulfide-bound Cys356 (PSF) structures. The catalytic intermediate structures showed the dihedral angle rotation of Schiff-base linkage relative to the PLP pyridine ring. The ToCDS structures were compared with bacterial CDS structures, which will help us to understand the role and catalytic mechanism of ToCDS in the archaeon Thermococcus onnurineus NA1.

Structural Basis for Phospholyase Activity of a Class III Transaminase Homologue.

Pyridoxal-phosphate (PLP)-dependent enzymes catalyse a remarkable diversity of chemical reactions in nature. A1RDF1 from Arthrobacter aurescens TC1 is a fold type I, PLP-dependent enzyme in the class III transaminase (TA) subgroup. Despite sharing 28 % sequence identity with its closest structural homologues, including β-alanine:pyruvate and γ-aminobutyrate:α-ketoglutarate TAs, A1RDF1 displayed no TA activity. Activity screening revealed that the enzyme possesses phospholyase (E.C. 4.2.3.2) activity towards O-phosphoethanolamine (PEtN), an activity described previously for vertebrate enzymes such as human AGXT2L1, enzymes for which no structure has yet been reported. In order to shed light on the distinctive features of PLP-dependent phospholyases, structures of A1RDF1 in complex with PLP (internal aldimine) and PLP⋅PEtN (external aldimine) were determined, revealing the basis of substrate binding and the structural factors that distinguish the enzyme from class III homologues that display TA activity.

Molecular and Spectroscopic Characterization of Aspergillus flavipes and Pseudomonas putida L-Methionine γ-Lyase in Vitro.

Pseudomonas putida L-methionine γ-lyase (PpMGL) has been recognized as an efficient anticancer agent, however, its antigenicity and stability remain as critical challenges for its clinical use. From our studies, Aspergillus flavipes L-methionine γ-lyase (AfMGL) displayed more affordable biochemical properties than PpMGL. Thus, the objective of this work was to comparatively assess the functional properties of AfMGL and PpMGL via stability of their internal aldimine linkage, tautomerism of pyridoxal 5'-phosphate (PLP) and structural stability responsive to physicochemical factors. The internal Schiff base of AfMGL and PpMGL have the same stability to hydroxylamine and human serum albumin. Acidic pHs resulted in strong cleavage of the internal Schiff base, inducing the unfolding of MGLs, compared to neutral-alkaline pHs. At λ 280nm excitation, both AfMGL and PpMGL have identical fluorescence emission spectra at λ 335nm for the intrinsic tryptophan and λ 560nm for the internal Schiff base. The maximum PLP tautomeric shift of ketoenamine to enolimine was detected at acidic pH causing complete enzyme unfolding, subunits dissociation and tautomeric shift of intrinsic PLP, rather than neutral-alkaline ones. The T m of AfMGL and PpMGL in presence of thermal stabilizer/ destabilizer was assayed by DSF. The T m of AfMGL and PpMGL was 73.1°C and 74.4°C, respectively, suggesting the higher proximity to the tertiary structure of both enzymes. The T m of AfMGL and PpMGL was slightly increased by trehalose and EDTA in contrast to guanidine HCl and urea. The active site and PLP-binding domains are identically conserved in both AfMGL and PpMGL.