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BBA - Proteins and Proteomics (v.1814, #11)

Editorial Board (pp. i).

Pyridoxal phosphate enzymology by Michael D. Toney (pp. 1405-1406).

Controlling reaction specificity in pyridoxal phosphate enzymes by Michael D. Toney (pp. 1407-1418).

Pyridoxal 5′-phosphate enzymes are ubiquitous in the nitrogen metabolism of all organisms. They catalyze a wide variety of reactions including racemization, transamination, decarboxylation, elimination, retro-aldol cleavage, Claisen condensation, and others on substrates containing an amino group, most commonly α-amino acids. The wide variety of reactions catalyzed by PLP enzymes is enabled by the ability of the covalent aldimine intermediate formed between substrate and PLP to stabilize carbanionic intermediates at Cα of the substrate. This review attempts to summarize the mechanisms by which reaction specificity can be achieved in PLP enzymes by focusing on three aspects of these reactions: stereoelectronic effects, protonation state of the external aldimine intermediate, and interaction of the carbanionic intermediate with the protein side chains present in the active site. This article is part of a Special Issue entitled: Pyridoxal Phosphate Enzymology.► Stereoelectronic effects in controlling PLP enzyme reaction specificity are discussed. ► The role of various protonation states of the external aldimine intermediate in PLP enzymes is discussed with respect to reaction specificity. ► The role of active site side chains in determining reaction specificity in PLP enzymes is reviewed.

Keywords: Pyridoxal phosphate; Vitamin B6; Reaction specificity; Stereoelectronic effect

The PLP cofactor: Lessons from studies on model reactions by John P. Richard; Tina L. Amyes; Juan Crugeiras; Ana Rios (pp. 1419-1425).

Experimental probes of the acidity of weak carbon acids have been developed and used to determine the carbon acid p K_as of glycine, glycine derivatives and iminium ion adducts of glycine to the carbonyl group, including 5′-deoxypyridoxal (DPL). The high reactivity of the DPL-stabilized glycyl carbanion towards nucleophilic addition to both DPL and the glycine-DPL iminium ion favors the formation of Claisen condensation products at enzyme active sites. The formation of the iminium ion between glycine and DPL is accompanied by a 12-unit decrease in the p K_a of 29 for glycine. The complicated effects of formation of glycine iminium ions to DPL and other aromatic and aliphatic aldehydes and ketones on carbon acid p K_a are discussed. These data provide insight into the contribution of the individual pyridine ring substituents to the catalytic efficiency of DPL. It is suggested that the 5′-phosphodianion group of PLP may play an important role in enzymatic catalysis of carbon deprotonation by providing up to 12kcal/mol of binding energy that is utilized to stabilize the transition state for the enzymatic reaction. This article is part of a Special Issue entitled: Pyridoxal Phospate Enzymology.

Keywords: Pyridoxal; Proton transfer; Carbon acid; Carbanion; Catalysis

Critical hydrogen bonds and protonation states of pyridoxal 5′-phosphate revealed by NMR by Hans-Heinrich Limbach; Monique Chan-Huot; Shasad Sharif; Peter M. Tolstoy; Ilya G. Shenderovich; Gleb S. Denisov; Michael D. Toney (pp. 1426-1437).

In this contribution we review recent NMR studies of protonation and hydrogen bond states of pyridoxal 5′-phosphate (PLP) and PLP model Schiff bases in different environments, starting from aqueous solution, the organic solid state to polar organic solution and finally to enzyme environments. We have established hydrogen bond correlations that allow one to estimate hydrogen bond geometries from¹⁵N chemical shifts. It is shown that protonation of the pyridine ring of PLP in aspartate aminotransferase (AspAT) is achieved by (i) an intermolecular OHN hydrogen bond with an aspartate residue, assisted by the imidazole group of a histidine side chain and (ii) a local polarity as found for related model systems in a polar organic solvent exhibiting a dielectric constant of about 30. Model studies indicate that protonation of the pyridine ring of PLP leads to a dominance of the ketoenamine form, where the intramolecular OHN hydrogen bond of PLP exhibits a zwitterionic state. Thus, the PLP moiety in AspAT carries a net positive charge considered as a pre-requisite to initiate the enzyme reaction. However, it is shown that the ketoenamine form dominates in the absence of ring protonation when PLP is solvated by polar groups such as water. Finally, the differences between acid–base interactions in aqueous solution and in the interior of proteins are discussed. This article is part of a special issue entitled: Pyridoxal Phosphate Enzymology.Display Omitted► The OHN hydrogen bond geometries of pyridoxal 5'-phosphate can be determined by NMR. ► Protonation of the pyridine ring also polarizes the intramolecular OHN hydrogen bond. ► A net positive charge is required to activate the cofactor. ► Hydrogen bond and protonation states strongly depend on the environment. ► Pyridoxal 5'-phosphate behaves in enzymes similar as in polar organic solvents.

Keywords: Pyridoxal 5′-phosphate; Aspartate aminotransferase; Tautomerism; Protonation state; Hydrogen bonding; Solid and liquid state NMR

Molecular dynamics simulations of the intramolecular proton transfer and carbanion stabilization in the pyridoxal 5′-phosphate dependent enzymesl-dopa decarboxylase and alanine racemase by Yen-Lin Lin; Jiali Gao; Amir Rubinstein; Dan Thomas Major (pp. 1438-1446).

Molecular dynamics simulations using a combined quantum mechanical and molecular mechanical (QM/MM) potential have been carried out to investigate the internal proton transfer equilibrium of the external aldimine species inl-dopa decarboxylase, and carbanion stabilization by the enzyme cofactor in the active site of alanine racemase. Solvent effects lower the free energy of the O-protonated PLP tautomer both in aqueous solution and in the active site, resulting a free energy difference of about −1kcal/mol relative to the N-protonated Schiff base in the enzyme. The external aldimine provides the dominant contribution to lowering the free energy barrier for the spontaneous decarboxylation ofl-dopa in water, by a remarkable 16kcal/mol, while the enzymel-dopa decarboxylase further lowers the barrier by 8kcal/mol. Kinetic isotope effects were also determined using a path integral free energy perturbation theory on the primary¹³C and the secondary²H substitutions. In the case of alanine racemase, if the pyridine ring is unprotonated as that in the active site, there is destabilizing contribution to the formation of the α-carbanion in the gas phase, although when the pyridine ring is protonated the contribution is stabilizing. In aqueous solution and in alanine racemase, the α-carbanion is stabilized both when the pyridine ring is protonated and unprotonated. The computational studies illustrated in this article show that combined QM/MM simulations can help provide a deeper understanding of the mechanisms of PLP-dependent enzymes. This article is part of a Special Issue entitled: Pyridoxal Phosphate Enzymology.► The O-protonated hydroxyimine tautomer is preferred inl-dopa decarboxylase. ► Solvent effects play a major role in the carbanion stabilization in water and in alanine decarboxylase. ► Computed kinetic isotope effects provide insight intol-dopa decarboxylation. ► Pyridoxal 5′-phosphate cofactor significantly lower the barrier of amino acid decarboxylation.

Keywords: Abbreviations; AM1; Austin Model 1; AlaR; Alanine racemase; DDC; l; -dopa decarboxylase; KIE; Kinetic isotope effects; NQE; Nuclear quantum effects; PI-FEP/UM; Path integral-free energy perturbation and umbrella sampling; PLP; Pyridoxal 5′-Phosphate; PSB; Protonated Schiff base; QM/MM; quantum mechanical and molecular mechanicalInternal proton transfer of PLP; Carbanion stabilization; Enzymatic decarboxylation; Racemization; QM/MM simulation

³¹P NMR spectroscopy senses the microenvironment of the 5′-phosphate group of enzyme-bound pyridoxal 5′-phosphate by Klaus D. Schnackerz; Babak Andi; Paul F. Cook (pp. 1447-1458).

In this review it is demonstrated that³¹P NMR spectroscopy can be used to elucidate information about the microenvironment around the phosphate group of enzyme-bound pyridoxal 5′-phosphate (PLP). The following information can be obtained for all PLP-dependent enzymes: 1) the protonation state of the 5′-phosphate and its exposure to solvent, and 2) tightness of binding of the 5′-phosphate. In addition, the 5-phosphate can report on the protonation state of the Schiff base lysine in some enzymes. Changes in the 5′-phosphate chemical shift can be used to determine changes in tightness of binding of the phosphate as the reaction pathway is traversed, providing information on the dynamics of the enzyme.³¹P NMR spectroscopy is thus an important probe of structure, dynamics and mechanism in native and site-directed mutations of PLP-dependent enzymes. Examples of all of the above are provided in this review. This article is part of a Special Issue entitled: Pyridoxal Phospate Enzymology.► The pH-dependence of the monoanion to dianion transition of the 5′-phosphate of PLP provides evidence of exposure of the phosphate to solvent. ►³¹P NMR can provide a precise description of the microenvironment of the 5′-phosphate of PLP. ► 5′-phosphate binding sites of PLP-dependent enzymes that catalyze β-elimination reactions are all quite similar.

Pyridoxal-5′-phosphate-dependent enzymes involved in biotin biosynthesis: Structure, reaction mechanism and inhibition by Stéphane Mann; Olivier Ploux (pp. 1459-1466).

The four last steps of biotin biosynthesis, starting from pimeloyl-CoA, are conserved among all the biotin-producing microorganisms. Two enzymes of this pathway, the 8-amino-7-oxononanoate synthase (AONS) and the 7,8-diaminopelargonic acid aminotransferase (DAPA AT) are dependent on pyridoxal-5′-phosphate (PLP). This review summarizes our current understanding of the structure, reaction mechanism and inhibition on these two interesting enzymes. Mechanistic studies as well as the determination of the crystal structure of AONS have revealed a complex mechanism involving an acylation with inversion of configuration and a decarboxylation with retention of configuration. This reaction mechanism is shared by the homologous 5-aminolevulinate synthase and serine palmitoyltransferase. While the reaction catalyzed by DAPA AT is a classical PLP-dependent transamination, the inactivation of this enzyme by amiclenomycin, a natural antibiotic that is active against Mycobacterium tuberculosis, involves the irreversible formation of an adduct between PLP and amiclenomycin. Mechanistic and structural studies allowed the complete description of this unique inactivation mechanism. Several potent inhibitors of these two PLP-dependent enzymes have been prepared and might be useful as starting points for the design of herbicides or antibiotics. This article is part of a Special Issue entitled: Pyridoxal Phospate Enzymology.► Two pyridoxal-5'-phosphate dependent enzymes are involved in biotin biosynthesis. ► AONS reaction mechanism involves two steps with overall inversion of configuration. ► DAPA aminotransferase is irreversibly inactivated by amiclenomycin. ► Amiclenomycin inactivation process was proved by mechanistic and structural studies. ► Inhibitors of these enzymes might lead to herbicides or antibacterial drugs.

Keywords: Abbreviations; AdoMet; S; -adenosyl-L-methionine; ALAS; 5-aminolevulinate synthase, EC 2.3.1.37; AON; 8-amino-7-oxononanoic acid (KAPA was also used in the older literature); AONS; 8-amino-7-oxononanoate synthase (KAPA synthase), EC 2.3.1.47; DAPA; 7,8-diaminononanoic acid; DAPA AT; 7,8-diaminopelargonic acid aminotransferase, EC 2.6.1.62; KBL; 2-amino-3-oxobutyrate CoA ligase, EC 2.3.1.29; KIE; kinetic isotope effect; PLP; pyridoxal-5′-phosphate; PMP; pyridoxamine-5′-phosphate; SPT; serine palmitoyltransferase, EC 2.3.1.508-amino-7-oxononanoate synthase; 7,8-diaminopelargonic acid aminotransferase; Pyridoxal-5′-phosphate; Enzyme reaction mechanism; Inhibition; Amiclenomycin

Molecular enzymology of 5-Aminolevulinate synthase, the gatekeeper of heme biosynthesis by Gregory A. Hunter; Gloria C. Ferreira (pp. 1467-1473).

Pyridoxal-5'-phosphate (PLP) is an obligatory cofactor for the homodimeric mitochondrial enzyme 5-aminolevulinate synthase (ALAS), which controls metabolic flux into the porphyrin biosynthetic pathway in animals, fungi, and the α-subclass of proteobacteria. Recent work has provided an explanation for how this enzyme can utilize PLP to catalyze the mechanistically unusual cleavage of not one but two substrate amino acid α-carbon bonds, without violating the theory of stereoelectronic control of PLP reaction-type specificity. Ironically, the complex chemistry is kinetically insignificant, and it is the movement of an active site loop that defines k_cat and ultimately, the rate of porphyrin biosynthesis. The kinetic behavior of the enzyme is consistent with an equilibrium ordered induced-fit mechanism wherein glycine must bind first and a portion of the intrinsic binding energy with succinyl-Coenzyme A is then utilized to perturb the enzyme conformational equilibrium towards a closed state wherein catalysis occurs. Return to the open conformation, coincident with ALA dissociation, is the slowest step of the reaction cycle. A diverse variety of loop mutations have been associated with hyperactivity, suggesting the enzyme has evolved to be purposefully slow, perhaps as a means to allow for rapid up-regulation of activity in response to an as yet undiscovered allosteric type effector. Recently it was discovered that human erythroid ALAS mutations can be associated with two very different diseases. Mutations that down-regulate activity can lead to X-linked sideroblastic anemia, which is characterized by abnormally high iron levels in mitochondria, while mutations that up-regulate activity are associated with X-linked dominant protoporphyria, which in contrast is phenotypically identified by abnormally high porphyrin levels. This article is part of a Special Issue entitled: Pyridoxal Phosphate Enzymology.► ALAS catalyzes the cleavage of two substrate amino acid α-carbon bonds. ► Succinyl-CoA binding shifts the enzyme conformational equilibrium towards a closed state. ► Movement of the active site loop towards the open conformation controls product release.

Keywords: Pyridoxal-5'-phosphate; Aminolevulinate synthase; Enzyme mechanism; Catalysis; Heme biosynthesis

Mechanistic enzymology of serine palmitoyltransferase by Hiroko Ikushiro; Hideyuki Hayashi (pp. 1474-1480).

Serine palmitoyltransferase, which is one of the α-oxamine synthase family enzymes, catalyzes the condensation reaction of L-serine and palmitoyl-CoA to form 3-ketodihydrosphingosine, the first and rate-determining step of the sphingolipid biosynthesis. As with other α-oxamine synthase family enzymes, the catalytic reaction is composed of multiple elementary steps, and the mechanism to control these steps to avoid side reactions has been the subject of intensive research in recent years. Combined spectroscopic, kinetic, and structural studies have revealed the finely controlled stereochemical mechanism, in which the His residue conserved among the α-oxamine synthase family enzymes plays a central and critical role. This article is part of a Special Issue entitled: Pyridoxal Phosphate Enzymology.Display Omitted

Keywords: Abbreviations; ALAS; δ; -aminolevulinate synthase; AONS; 8-amino-7-oxononanoate synthase; AOS; α; -oxamine synthase; KBL; 2-amino-3-ketobutyrate CoA ligase; KDS; 3-ketodihydrosphingosine; PLP; pyridoxal 5′-phosphate; PMP; pyridoxamine 5′-phosphate; SPT; serine palmitoyltransferaseSerine palmitoyltransferase; α; -Oxamine synthase family; Pyridoxal 5′-phosphate; Stereochemistry; Enzyme mechanism

Structure, mechanism, and substrate specificity of kynureninase by Robert S. Phillips (pp. 1481-1488).

The kynurenine pathway is the major route for tryptophan catabolism in animals and some fungi and bacteria. The procaryotic enzyme preferentially reacts withl-kynurenine, while eucaryotic kynureninases exhibit higher activity with 3-hydroxy-l-kynurenine. Crystallography of kynureninases from Pseudomonas fluorescens (PfKyn) and Homo sapiens (HsKyn) shows that the active sites are nearly identical, except that His-102, Asn-333, and Ser-332 in HsKyn are replaced by Trp-64, Thr-282, and Gly-281 in PfKyn. Site-directed mutagenesis of HsKyn shows that these residues are, at least in part, responsible for the differences in substrate specificity since the H102W/S332G/N333T triple mutant shows activity with kynurenine but not 3-hydroxykynurenine. PfKyn is strongly inhibited by analogs of a proposed gem-diolate intermediate, dihydrokynurenine, and S-(2-aminophenyl)-l-cysteine S,S-dioxide, with K_i values in the low nanomolar range. Stopped-flow kinetic experiments show that a transient quinonoid intermediate is formed on mixing, which decays to a ketimine at 740s−¹. Quench experiments show that anthranilate, the first product, is formed in a stoichiometric burst at 50s−¹ and thus the rate-determining step in the steady-state is the release of the second product,l-Ala. β-Benzoylalanine is also a good substrate for PfKyn but does not show a burst of benzoate formation, indicating that the rate-determining step for this substrate is benzoate release. A Hammett plot of rate constants for substituted β-benzoylalanines is non-linear, suggesting that carbonyl hydration is rate-determining for electron-donating groups, but C_β–C_γ cleavage is rate-determining for electron-withdrawing groups. This article is part of a Special Issue entitled: Pyridoxal phosphate Enzymology.

Keywords: Pyridoxal-5′-phosphate; Tryptophan; Reaction mechanism; Crystallography; Mutagenesis

Serine hydroxymethyltransferase: A model enzyme for mechanistic, structural, and evolutionary studies by Rita Florio; Martino Luigi di Salvo; Mirella Vivoli; Roberto Contestabile . (pp. 1489-1496).

Serine hydroxymethyltransferase is a ubiquitous representative of the family of fold type I, pyridoxal 5′-phosphate-dependent enzymes. The reaction catalyzed by this enzyme, the reversible transfer of the Cβ of serine to tetrahydropteroylglutamate, represents a link between amino acid and folates metabolism and operates as a major source of one-carbon units for several essential biosynthetic processes. Serine hydroxymethyltransferase has been intensively investigated because of the interest aroused by the complex mechanism of the hydroxymethyltransferase reaction and its broad substrate and reaction specificity. Although the increasing availability of crystallographic data and the characterization of several site-specific mutants helped in understanding previous functional and structural studies, they also represent the starting point of novel investigations. This review will focus on recently highlighted catalytic, structural, and evolutionary aspects of serine hydroxymethyltransferase. This article is part of a Special Issue entitled: Pyridoxal phosphate Enzymology.

Keywords: Abbreviations; PLP; pyridoxal 5′-phosphate; H; ₄; PteGlu; tetrahydropteroylglutamate; e; SHMT; Escherichia coli; serine hydroxymethyltransferase; CHCs; conserved hydrophobic contacts; SCRs; structurally conserved regions; e; TA; Escherichia coli; threonine aldolase; AlaRac; alanine racemasePyridoxal phosphate; Serine hydroxymethyltransferase; Fold type I enzymes; Catalytic mechanism; Structural determinants of protein fold; Folding mechanism; Divergent evolution

The multifaceted pyridoxal 5′-phosphate-dependent O-acetylserine sulfhydrylase by Andrea Mozzarelli; Stefano Bettati; Barbara Campanini; Enea Salsi; Samanta Raboni; Ratna Singh; Francesca Spyrakis; Vidya Prasanna Kumar; Paul F. Cook (pp. 1497-1510).

Cysteine is the final product of the reductive sulfate assimilation pathway in bacteria and plants and serves as the precursor for all sulfur-containing biological compounds, such as methionine, S-adenosyl methionine, iron–sulfur clusters and glutathione. Moreover, in several microorganisms cysteine plays a role as a reducing agent, eventually counteracting host oxidative defense strategies. Cysteine is synthesized by the PLP-dependent O-acetylserine sulfhydrylase, a dimeric enzyme belonging to the fold type II, catalyzing a beta-replacement reaction. In this review, the spectroscopic properties, catalytic mechanism, three-dimensional structure, conformational changes accompanying catalysis, determinants of enzyme stability, role of selected amino acids in catalysis, and the regulation of enzyme activity by ligands and interaction with serine acetyltransferase, the preceding enzyme in the biosynthetic pathway, are described. Given the key biological role played by O-acetylserine sulfhydrylase in bacteria, inhibitors with potential antibiotic activity have been developed. This article is part of a Special Issue entitled: Pyridoxal Phospate Enzymology.Display Omitted► In bacteria and plants cysteine is the key amino acid of sulfur assimilation. ► The PLP-dependent O-acetylserine sulfhydrylase catalyzes cysteine synthesis. ► O-acetylserine sulfhydrylase catalysis is accompanied by an open–closed transition. ► Absorption and fluorescence changes allowed to unveil the catalytic mechanism. ► Enzyme mechanism has been determined by steady- and presteady-state kinetics. ► Enzyme inhibitors with potential antibiotic activity have been developed.

Keywords: Abbreviations; AA; α-aminoacrylate external Schiff base; APS; adenosine 5′-phosphosulphate; ATPS; ATP sulfurylase; BCA; β-chloro-; l; -alanine; CS; cysteine synthase; EA; external Schiff base; IA; internal Schiff base; NAS; N; -acetylserine; OAS; O; -acetylserine; OASS; OAS sulfhydrylase; OPS; O; -phosphoserine; OPSS; OPS sulfhydrylase; PAPS; 3′-phosphoadenosine 5′-phosphosulfate; PLP; pyridoxal 5′-phosphate; RSAP; reductive sulfate assimilation pathway; SAT; serine acetyltransferase; TNB; 5-thio-2-nitrobenzoate; GdnHCl; guanidinium hydrochloridePLP catalysis; Enzyme mechanism; Spectroscopy; Vitamin B6

The enzymes of the transsulfuration pathways: Active-site characterizations by Susan M. Aitken; Pratik H. Lodha; Dominique J.K. Morneau (pp. 1511-1517).

The diversity of reactions catalyzed by enzymes reliant on pyridoxal 5′-phosphate (PLP) demonstrates the catalytic versatility of this cofactor and the plasticity of the protein scaffolds of the major fold types of PLP-dependent enzymes. The enzymes of the transsulfuration (cystathionine γ-synthase and cystathionine β-lyase) and reverse transsulfuration (cystathionine β-synthase and cystathionine γ-lyase) pathways interconvertl-cysteine andl-homocysteine, the immediate precursor ofl-methionine, in plants/bacteria and yeast/animals, respectively. These enzymes provide a useful model system for investigation of the mechanisms of substrate and reaction specificity in PLP-dependent enzymes as they catalyze distinct side chain rearrangements of similar amino acid substrates. Exploration of the underlying factors that enable enzymes to control the substrate and reaction specificity of this cofactor will enable the engineering of these properties and the development of therapeutics and antimicrobial compounds. Recent studies probing the role of active-site residues, of the enzymes of the transsulfuration pathways, as determinants of substrate and reaction specificity are the subject of this review. This article is part of a Special Issue entitled: Pyridoxal Phosphate Enzymology.► We present a review of recent work characterizing active-site residues of the enzymes of the transsulfuration pathways. ► Cystathionine β-synthase, a fold-type II, PLP-dependent enzyme, relies upon hydrogen-bonding networks for substrate and cofactor binding. ► Cystathionine β-lyase, typical of the fold-type I, PLP-dependent enzymes of the transsulfuration pathways, employs arginine residues to bind the carboxylate and phosphate groups of the substrate and cofactor, respectively.

Keywords: Abbreviations; AA; aminoacrylate; ALAS; 5-aminolevulineate synthase; AATase; aspartate aminotransferase; l; -Cth; l; -cystathionine; CBL; cystathionine β-lyase; CBS; cystathionine β-synthase; dCBS; drosophila CBS; hCBS; human CBS; yCBS; yeast CBS; ytCBS; truncated yCBS (residues 1–353); CGL; cystathionine γ-lyase; CGS; cystathionine γ-synthase; l; -Hcys; l; -homocysteine; l; -HS; l; -homoserine; OASS; O; -acetylserine sulfhydrylase; PLP; pyridoxal 5′-phosphate; SAM; S; -adenosylmethionine; TS; threonine synthase; TrpS; tryptophan synthasePyridoxal 5'-phosphate; Reaction specificity; Cystathionine; Transsulfuration

PLP-dependent H₂S biogenesis by Sangita Singh; Ruma Banerjee (pp. 1518-1527).

The role of endogenously produced H₂S in mediating varied physiological effects in mammals has spurred enormous recent interest in understanding its biology and in exploiting its pharmacological potential. In these early days in the field of H₂S signaling, large gaps exist in our understanding of its biological targets, its mechanisms of action and the regulation of its biogenesis and its clearance. Two branches within the sulfur metabolic pathway contribute to H₂S production: (i) the reverse transsulfuration pathway in which two pyridoxal 5′-phosphate-dependent (PLP) enzymes, cystathionine β-synthase and cystathionine γ-lyase convert homocysteine successively to cystathionine and cysteine and (ii) a branch of the cysteine catabolic pathway which converts cysteine to mercaptopyruvate via a PLP-dependent cysteine aminotransferase and subsequently, to mercaptopyruvate sulfur transferase-bound persulfide from which H₂S can be liberated. In this review, we present an overview of the kinetics of the H₂S-generating reactions, compare the structures of the PLP-enzymes involved in its biogenesis and discuss strategies for their regulation.This article is part of a Special Issue entitled: Pyridoxal Phospate Enzymology.► H₂S is a gaseous signaling molecule, with neuromodulatory and cardioprotective effects. ► Three of the H₂S-producing enzymes are pyridoxal phosphate-dependent and utilize the sulfur amino acids as substrates. ► The structure, function and regulation of the H₂S producing enzymes are the focus of this review article.

Keywords: PLP; H; ₂; S; Cystathionine β-synthase; Cystathionine γ-lyase; Cysteine aminotransferase

Structural insights for the substrate recognition mechanism ofLL-diaminopimelate aminotransferase by Nobuhiko Watanabe; Michael N.G. James (pp. 1528-1533).

The enzymes involved in the lysine biosynthetic pathway have long been considered to be attractive targets for novel antibiotics due to the absence of this pathway in humans. Recently, a novel pyridoxal 5′-phosphate (PLP) dependent enzyme calledll-diaminopimelate aminotransferase (ll-DAP-AT) was identified in the lysine biosynthetic pathway of plants and Chlamydiae. Understanding its function and substrate recognition mechanism would be an important initial step toward designing novel antibiotics targetingll-DAP-AT. The crystal structures ofll-DAP-AT from Arabidopsis thaliana in complex with various substrates and analogues have been solved recently. These structures revealed howl-glutamate andll-DAP are recognized byll-DAP-AT without significant conformational changes in the enzyme's backbone structure. This review article summarizes the recent developments in the structural characterization and the inhibitor design ofll-DAP-AT from A. thaliana. This article is part of a Special Issue entitled: Pyridoxal Phospate Enzymology.► We reviewed the recent advances inll-DAP-AT research. ► We summarized the recent structural work inll-DAP-AT. ► We illustrated the substrate binding mode ofll-DAP-AT.

Keywords: Abbreviations; DAP; 2,6-diaminopimelic acid; ll; -DAP-AT; ll; -diaminopimelate aminotransferase; AtDAP-AT; Arabidopsis thaliana; ll; -DAP-AT; DHDP; dihydrodipicolinate; PLP; pyridoxal-5′-phosphate; THDP; tetrahydrodipicolinate; AspAT; aspartate aminotransferaseLysine biosynthesis; Aminotransferase; Arabidopsis thaliana; Diaminopimelate; Pyridoxal-5′-phosphate; Chlamydia

Mechanisms and structures of vitamin B₆-dependent enzymes involved in deoxy sugar biosynthesis by Anthony J. Romo; Hung-wen Liu (pp. 1534-1547).

PLP is well-regarded for its role as a coenzyme in a number of diverse enzymatic reactions. Transamination, deoxygenation, and aldol reactions mediated by PLP-dependent enzymes enliven and enrich deoxy sugar biosynthesis, endowing these compounds with unique structures and contributing to their roles as determinants of biological activity in many natural products. The importance of deoxy aminosugars in natural product biosynthesis has spurred several recent structural investigations of sugar aminotransferases. The structure of a PMP-dependent enzyme catalyzing the C-3 deoxygenation reaction in the biosynthesis of ascarylose was also determined. These studies, and the crystal structures they have provided, offer a wealth of new insights regarding the enzymology of PLP/PMP-dependent enzymes in deoxy sugar biosynthesis. In this review, we consider these recent achievements in the structural biology of deoxy sugar biosynthetic enzymes and the important implications they hold for understanding enzyme catalysis and natural product biosynthesis in general. This article is part of a Special Issue entitled: Pyridoxal Phosphate Enzymology.Display Omitted► A survey of PLP-dependent enzymes in prokaryotic deoxy aminosugar biosynthesis. ► Catalysis in the context of recent aminotransferase/dehydratase structural studies. ► Focus on deoxy aminosugars decorating prokaryotic natural products.

Keywords: Vitamin B; ₆; coenzyme; Deoxy sugar; Aminosugar; Biosynthesis; Deoxygenation; Enzyme mechanism

Pyridoxal-5′-phosphate as the catalyst for radical isomerization in reactions of PLP-dependent aminomutases by Perry A. Frey; George H. Reed (pp. 1548-1557).

PLP catalyzes the 1,2 shifts of amino groups in free radical-intermediates at the active sites of amino acid aminomutases. Free radical forms of the substrates are created upon H atom abstractions carried out by the 5′-deoxyadenosyl radical. In most of these enzymes, the 5′-deoxyadenosyl radical is generated by an iron–sulfur cluster-mediated reductive cleavage of S-adenosyl-( S)-methionine. However, in lysine 5,6-aminomutase and ornithine 4,5-aminomutase, the radical is generated by homolytic cleavage of the cobalt–carbon bond of adenosylcobalamin. The imine linkages in the initial radical forms of the external aldimines undergo radical addition to form azacyclopropylcarbinyl radicals as central intermediates in the catalytic cycles. In the case of lysine 2,3-aminomutase, the multistep catalytic mechanism is corroborated by direct spectroscopic observation and characterization of a product radical trapped during steady-state turnover. Analogues of the substrate-related radical having substituents adjacent to the radical center to stabilize the unpaired electron are also observed and characterized spectroscopically. A functional allylic analogue of the 5′-deoxyadenosyl radical is observed spectroscopically. A high-resolution crystal structure fully supports the mechanistic proposals. Evidence for a similar free radical mediated amino group transfer in the adenosylcobalamin-dependent lysine 5,6-aminomutase is provided by spectroscopic detection and characterization of radicals generated from the 4-thia analogues of the lysine substrates. This article is part of a Special Issue entitled: Pyridoxal Phospate Enzymology.Display Omitted

Keywords: Pyridoxal-5'-phosphate; S-Adenosyl-L-methionine; Adenosylcobalamin; Lysine 2,3-aminomutase; Glutamate 2,3-aminomutase; Lysine 5,6-aminomutase; Radical isomerization

Serine racemase and the serine shuttle between neurons and astrocytes by Herman Wolosker (pp. 1558-1566).

d-Serine is a brain-enrichedd-amino acid that works as a transmitter-like molecule by physiologically activating NMDA receptors. Synthesis ofd-serine is carried out by serine racemase (SR), a pyridoxal 5′-phosphate-dependent enzyme. In addition to carry out racemization, SR α,β-eliminates water froml- ord-serine, generating pyruvate and NH₄⁺. Here I review the main mechanisms regulating SR activity andd-serine dynamics in the brain. I propose a role for SR in a novel form of astrocyte-neuron communication—the “serine shuttle”, whereby astrocytes synthesize and exportl-serine required for the synthesis ofd-serine by the predominantly neuronal SR.d-Serine synthesized and released by neurons can be further taken up by astrocytes for storage and activity-dependent release. I discuss how SR α,β-elimination withd-serine itself may limit the achievable intracellulard-serine concentration, providing a mechanistic rationale on why neurons do not store as muchd-serine as astrocytes. The higher content ofd-serine in astrocytes appears to be related to increasedd-serine stability, for their low SR expression will prevent substantiald-serine metabolism via α,β-elimination. SR and the serine shuttle pathway are therapeutic targets in neurodegenerative diseases in which NMDA receptor dysfunction plays pathological roles. This article is part of a Special Issue entitled: Pyridoxal Phospate Enzymology.► Mechanisms ofd-serine synthesis by serine racemase ► Role of serine racemase elimination reaction in limiting the achievable intracellulard-serine concentration and providing a mechanistic rationale on why neurons do not store as muchd-serine as astrocytes. ► I propose the serine shuttle model, whereby astrocytes synthesize and exportl-serine required for the synthesis ofd-serine by the predominantly neuronal serine racemase. ►d-Serine synthesized and released by neurons can be further taken up by astrocytes for storage and activity-dependent release.

Keywords: D-serine; Serine racemase; NMDA; L-serine; Neurotransmission; Astrocytes; Glia; Gliotransmitter; Glycine; Phosphoglycerate; Serine shuttle

PLP-dependent enzymes as potential drug targets for protozoan diseases by Barbara Kappes; Ivo Tews; Alexandra Binter; Peter Macheroux (pp. 1567-1576).

The chemical properties of the B₆ vitamers are uniquely suited for wide use as cofactors in essential reactions, such as decarboxylations and transaminations. This review addresses current efforts to explore vitamin B₆ dependent enzymatic reactions as drug targets. Several current targets are described that are found amongst these enzymes. The focus is set on diseases caused by protozoan parasites. Comparison across a range of these organisms allows insight into the distribution of potential targets, many of which may be of interest in the development of broad range anti-protozoan drugs. This article is part of a Special Issue entitled: Pyridoxal Phosphate Enzymology.► The genomes of fifteen protozoan parasites were analyzed for PLP-dependent enzymes. ► A minimal set of three PLP-dependent enzymes is identified in the order of Piroplasmida. ► This minimal set of PLP-dependent enzymes is observed with the highest frequency among the organisms analyzed. ► The PLP-dependent enzymes identified are potential candidates for the development of anti-protozoan drugs. ► The current state of affairs for some enzymes is discussed and future directions of research are suggested.

Keywords: Serine hydroxymethyltransferase; Aspartate transaminase; Cysteine desulfurase; Ornithine decarboxylase; Cysteine synthase; Antiprotozoan therapy

Human liver peroxisomal alanine:glyoxylate aminotransferase: Characterization of the two allelic forms and their pathogenic variants by Barbara Cellini; Riccardo Montioli; Carla Borri Voltattorni (pp. 1577-1584).

The hepatic peroxisomal alanine:glyoxylate aminotransferase (AGT) is a pyridoxal 5′-phosphate (PLP)-enzyme whose deficiency is responsible for Primary Hyperoxaluria Type 1 (PH1), an autosomal recessive disorder. In the last few years the knowledge of the characteristics of AGT and the transfer of this information into some pathogenic variants have significantly contributed to the improvement of the understanding at the molecular level of the PH1 pathogenesis. In this review, the spectroscopic features, the coenzyme's binding affinity, the steady-state kinetic parameters as well as the sensitivity to thermal and chemical stress of the two allelic forms of AGT, the major (AGT-Ma) and the minor (AGT-Mi) allele, have been described. Moreover, we summarize the characterization obtained by means of biochemical and bioinformatic analyses of the following PH1-causing variants in the recombinant purified forms: G82E associated with the major allele, F152I encoded on the background of the minor allele, and the G41 mutants which co-segregate either with the major allele (G41R-Ma and G41V-Ma) or with the minor allele (G41R-Mi). The data have been correlated with previous clinical and cell biology results, which allow us to (i) highlight the functional differences between AGT-Ma and AGT-Mi, (ii) identify the structural and functional molecular defects of the pathogenic variants, (iii) improve the correlation between the genotype and the enzymatic phenotype, (iv) foresee or understand the molecular basis of the responsiveness to pyridoxine treatment of patients bearing these mutations, and (v) pave the way for new treatment strategies. This article is part of a Special Issue entitled: Pyridoxal Phospate Enzymology.

Keywords: Abbreviations; (PH1); Primary Hyperoxaluria Type 1; (PLP); pyridoxal 5′-phosphate; (PMP); pyridoxamine 5′-phosphate; (AGT); alanine:glyoxylate aminotransferasePrimary Hyperoxaluria Type I; Alanine:glyoxylate aminotransferase; Pyridoxal 5′-phosphate; Pathogenic variants

Pyridoxal phosphate: Biosynthesis and catabolism by Tathagata Mukherjee; Jeremiah Hanes; Ivo Tews; Steven E. Ealick; Tadhg P. Begley (pp. 1585-1596).

Vitamin B₆ is an essential cofactor that participates in a large number of biochemical reactions. Pyridoxal phosphate is biosynthesized de novo by two different pathways (the DXP dependent pathway and the R5P pathway) and can also be salvaged from the environment. It is one of the few cofactors whose catabolic pathway has been comprehensively characterized. It is also known to function as a singlet oxygen scavenger and has protective effects against oxidative stress in fungi. Enzymes utilizing vitamin B₆ are important targets for therapeutic agents. This review provides a concise overview of the mechanistic enzymology of vitamin B₆ biosynthesis and catabolism. This article is part of a Special Issue entitled: Pyridoxal Phosphate Enzymology.

Keywords: Abbreviations; DXP; deoxyxylulose 5-phosphate; DXS; deoxyxylulose 5-phosphate synthase; FAD; flavin adenine dinucleotide; FHMPC; 5-formyl-3-hydroxy-2-methylpyridine 4-carboxylic acid; FMN; flavin adenine mononucleotide; HTP; 4-hydroxy-L-threonine phosphate; MHPC; 2-methyl-3-hydroxypyridine-5-carboxylic acid; MHPCO; 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase; NAD; β-nicotinamide adenine dinucleotide; NADH; β-nicotinamide adenine dinucleotide, reduced form of NAD; NADP; β-nicotinamide adenine dinucleotide phosphate; PL; pyridoxal; PLP; pyridoxal 5′-phosphate; PM; pyridoxamine; PMP; pyridoxamine 5′-phosphate; PN; pyridoxine; PNP; pyridoxine 5′-phosphate; R5P; d; -ribose 5-phosphate; TCEP; tris(2-carboxyethyl)phosphine; α-MG; α-methyl-L-glutamatePLP; Vitamin B; ₆; DXP dependent pathway; R5P pathway; Vitamin B; ₆; catabolism

Vitamin B₆ salvage enzymes: Mechanism, structure and regulation by Martino Luigi di Salvo; Roberto Contestabile; Martin K. Safo (pp. 1597-1608).

Vitamin B₆ is a generic term referring to pyridoxine, pyridoxamine, pyridoxal and their related phosphorylated forms. Pyridoxal 5′-phosphate is the catalytically active form of vitamin B₆, and acts as cofactor in more than 140 different enzyme reactions. In animals, pyridoxal 5′-phosphate is recycled from food and from degraded B₆-enzymes in a “salvage pathway”, which essentially involves two ubiquitous enzymes: an ATP-dependent pyridoxal kinase and an FMN-dependent pyridoxine 5′-phosphate oxidase. Once it is made, pyridoxal 5′-phosphate is targeted to the dozens of different apo-B₆ enzymes that are being synthesized in the cell. The mechanism and regulation of the salvage pathway and the mechanism of addition of pyridoxal 5′-phosphate to the apo-B₆-enzymes are poorly understood and represent a very challenging research field. Pyridoxal kinase and pyridoxine 5′-phosphate oxidase play kinetic roles in regulating the level of pyridoxal 5′-phosphate formation. Deficiency of pyridoxal 5′-phosphate due to inborn defects of these enzymes seems to be involved in several neurological pathologies. In addition, inhibition of pyridoxal kinase activity by several pharmaceutical and natural compounds is known to lead to pyridoxal 5′-phosphate deficiency. Understanding the exact role of vitamin B₆ in these pathologies requires a better knowledge on the metabolism and homeostasis of the vitamin. This article summarizes the current knowledge on structural, kinetic and regulation features of the two enzymes involved in the PLP salvage pathway. We also discuss the proposal that newly formed PLP may be transferred from either enzyme to apo-B₆-enzymes by direct channeling, an efficient, exclusive, and protected means of delivery of the highly reactive PLP. This new perspective may lead to novel and interesting findings, as well as serve as a model system for the study of macromolecular channeling. This article is part of a Special Issue entitled: Pyridoxal Phosphate Enzymology.► In animals, PLP is recycled from food and degraded B6-enzymes in a salvage pathway. ► Two enzymes in this pathway: pyridoxal kinase and pyridoxine 5'-phosphate oxidase. ► Inborn defects of these enzymes are involved in neurological pathologies. ► Regulation mechanism of the salvage pathway enzymes. ► Channeling mechanism for the addition of PLP to apo-B6-enzymes.

Keywords: Abbreviations; PN; pyridoxine; PM; pyridoxamine; PL; pyridoxal; PNP; pyridoxine 5′-phosphate; PMP; pyridoxamine 5′-phosphate; PLP; pyridoxal 5′-phosphate; PLK; pyridoxal kinase coded by; PdxK; gene; PLK2; pyridoxal kinase coded by; PdxY; gene; PNPOx; pyridoxine (pyridoxamine) 5′-phosphate oxidase; NEE; neonatal epileptic encephalopathy; SHMT; serine hydroxymethyltransferase; AAT; aspartate aminotransferaseVitamin B; ₆; Pyridoxal 5′-phosphate; Salvage pathway; Pyridoxal kinase; Pyridoxine 5′-phosphate oxidase

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BBA - Proteins and Proteomics (v.1814, #11)