PLP-dependent Enzymes Research Articles

NMR Studies of the Stability, Protonation States, and Tautomerism of 13C- and 15N-Labeled Aldimines of the Coenzyme Pyridoxal 5′-Phosphate in Water

We have measured the pH-dependent (1)H, (13)C, and (15)N NMR spectra of pyridoxal 5'-phosphate ((13)C(2)-PLP) mixed with equal amounts of either doubly (15)N-labeled diaminopropane, (15)N(α)-labeled l-lysine, or (15)N(ε)-labeled l-lysine as model systems for various intermediates of the transimination reaction in PLP-dependent enzymes. At low pH, only the hydrate and aldehyde forms of PLP and the free protonated diamines are present. Above pH 4, the formation of single- and double-headed aldimines (Schiff bases) with the added diamines is observed, and their (13)C and (15)N NMR parameters have been characterized. For 1:1 mixtures the single-headed aldimines dominate. In a similar way, the NMR parameters of the geminal diamine formed with diaminopropane at high pH are measured. However, no geminal diamine is formed with l-lysine. In contrast to the aldimine formed with the ε-amino group of lysine, the aldimine formed with the α-amino group is unstable at moderately high pH but dominates slightly below pH 10. By analyzing the NMR data, both the mole fractions of the different PLP species and up to 6 different protonation states including their pK(a) values were obtained. Furthermore, the data show that all Schiff bases are subject to a proton tautomerism along the intramolecular OHN hydrogen bond, where the zwitterionic form is favored before deprotonation occurs at high pH. This observation, as well as the observation that around pH 7 the different PLP species are present in comparable amounts, sheds new light on the mechanism of the transimination reaction.

Light-enhanced catalysis by pyridoxal phosphate-dependent aspartate aminotransferase.

The mechanisms of pyridoxal 5'-phosphate (PLP)-dependent enzymes require substrates to form covalent "external aldimine" intermediates, which absorb light strongly between 410 and 430 nm. Aspartate aminotransferase (AAT) is a prototypical PLP-dependent enzyme that catalyzes the reversible interconversion of aspartate and α-ketoglutarate with oxalacetate and glutamate. From kinetic isotope effects studies, it is known that deprotonation of the aspartate external aldimine C(α)-H bond to give a carbanionic quinonoid intermediate is partially rate limiting in the thermal AAT reaction. We show that excitation of the 430-nm external aldimine absorption band increases the steady-state catalytic activity of AAT, which is attributed to the photoenhancement of C(α)-H deprotonation on the basis of studies with Schiff bases in solution. Blue light (250 mW) illumination gives an observed 2.3-fold rate enhancement for WT AAT activity, a 530-fold enhancement for the inactive K258A mutant, and a 58600-fold enhancement for the PLP-Asp Schiff base in water. These different levels of enhancement correlate with the intrinsic reactivities of the C(α)-H bond in the different environments, with the less reactive Schiff bases exhibiting greater enhancement. Time-resolved spectroscopy, ranging from femtoseconds to minutes, was used to investigate the nature of the photoactivation of C(α)-H bond cleavage in PLP-amino acid Schiff bases both in water and bound to AAT. Unlike the thermal pathway, the photoactivation pathway involves a triplet state with a C(α)-H pK(a) that is estimated to be between 11 and 19 units lower than the ground state for the PLP-Val Schiff base in water.

Members of the YjgF/YER057c/UK114 Family of Proteins Inhibit Phosphoribosylamine Synthesis in Vitro

The YjgF/YER057c/UK114 family of proteins is highly conserved across all three domains of life and currently lacks a consensus biochemical function. Analysis of Salmonella enterica strains lacking yjgF has led to a working model in which YjgF functions to remove potentially toxic secondary products of cellular enzymes. Strains lacking yjgF synthesize the thiamine precursor phosphoribosylamine (PRA) by a TrpD-dependent mechanism that is not present in wild-type strains. Here, PRA synthesis was reconstituted in vitro with anthranilate phosphoribosyltransferase (TrpD), threonine dehydratase (IlvA), threonine, and phosphoribosyl pyrophosphate. TrpD-dependent PRA formation in vitro was inhibited by S. enterica YjgF and the human homolog UK114. Thus, the work herein describes the first biochemical assay for diverse members of the highly conserved YjgF/YER057c/UK114 family of proteins and provides a means to dissect the cellular functions of these proteins.

Purification, characterization and amino acid sequence of a novel enzyme, D-threo-3-hydroxyaspartate dehydratase, from Delftia sp. HT23

D-threo-3-hydroxyaspartate dehydratase (D-THA DH) was purified from the cell-free extract of the soil-isolated bacterium Delftia sp. HT23. The enzyme exhibited dehydratase activity towards D-threo-3-hydroxyaspartate, l-threo-3-hydroxyaspartate, l-erythro-3-hydroxyaspartate and d-serine. Absorption of the purified enzyme at 412 nm suggests that it contains pyridoxal 5'-phosphate (PLP) as a cofactor. The NH(2)-terminal and internal amino acid sequences showed significant similarity to hypothetical alanine racemase of genome-sequenced Delftia acidovorans SPH-1; however, the purified enzyme showed no alanine racemase activity. Using the sequence information of D. acidovorans SPH-1, the gene encoding d-THA DH was cloned. The deduced amino acid sequence, which belongs to the alanine racemase family, shows significant (26-36%) similarity to d-serine dehydratase of both Saccharomyces cerevisiae and chicken. In order to obtain purified d-THA DH efficiently, the gene was expressed in Escherichia coli. The recombinant enzyme was highly activated by divalent cations, such as Mn(2+), Co(2+) and Ni(2+). Site-directed mutagenesis experiment revealed that lysine 43 is an important residue involved in PLP binding and catalysis. This is the first reported enzyme that acts on d-THA. In addition, this enzyme is the first example of a prokaryotic dehydratase belonging to the fold-type III PLP-dependent enzyme family.

Crystal Structure-Based Selective Targeting of the Pyridoxal 5′-Phosphate Dependent Enzyme Kynurenine Aminotransferase II for Cognitive Enhancement

Fluctuations in the brain levels of the neuromodulator kynurenic acid may control cognitive processes and play a causative role in several catastrophic brain diseases. Elimination of the pyridoxal 5'-phosphate dependent enzyme kynurenine aminotransferase II reduces cerebral kynurenic acid synthesis and has procognitive effects. The present description of the crystal structure of human kynurenine aminotransferase II in complex with its potent and specific primary amine-bearing fluoroquinolone inhibitor (S)-(-)-9-(4-aminopiperazin-1-yl)-8-fluoro-3-methyl-6-oxo-2,3-dihydro-6H-1-oxa-3a-azaphenalene-5-carboxylic acid (BFF-122) should facilitate the structure-based development of cognition-enhancing drugs. From a medicinal chemistry perspective our results demonstrate that the issue of inhibitor specificity for highly conserved PLP-dependent enzymes could be successfully addressed.

3-Fluoroaspartate and Pyruvoyl-Dependant Aspartate Decarboxylase: Exploiting the Unique Characteristics of Fluorine To Probe Reactivity and Binding

Fluorine-containing amino acids have been used with great success as mechanism-based inhibitors of pyridoxal phosphate (PLP)-dependent enzymes, and the influence of fluorine on the conformation of molecules has also been extensively studied and practically exploited. In this study, we sought to use these unique characteristics to probe the reactivity and binding of aspartate decarboxylase (ADC) enzymes, which are members of the small class of pyruvoyl-dependant decarboxylases. Since ADC activity has been shown to be essential to the virulence of Mycobacterium tuberculosis, information gained in this manner could be used for the development of inhibitors that selectively target pyruvoyl-dependent enzymes such as ADC, without affecting PLP-dependent enzymes in the host. For this purpose, we synthesized the L-erythro and L-threo isomers of 3-fluoroaspartate and tested their ability to act as substrates and/or inhibitors of the M. tuberculosis and Escherichia coli ADC enzymes. Trapping and MS-based binding analysis was additionally used to confirm that both isomers enter the enzymes' active sites. Our studies show that both isomers undergo single turnover decarboxylation and fluorine elimination reactions to give enamine products that can be trapped within the active site. Interestingly, the enamine/ADC complex that forms from the L-erythro (but not the L-threo) isomer is sufficiently stable that it can be observed even without any trapping. This finding suggests that the two 3-fluoroaspartates maintain different conformations within the ADC active site, which leads to the enamine products with configurations of different stabilities. Taken together, our results provide new insights for the development of cofactor-specific inhibitors, and confirm the utility of fluorine as a unique tool for probing reactivity and binding profiles within enzymes.

Mutational Analysis of Substrate Interactions with the Active Site of Dialkylglycine Decarboxylase

Pyridoxal phosphate (PLP)-dependent enzymes catalyze many different types of reactions at the alpha-, beta-, and gamma-carbons of amine and amino acid substrates. Dialkylglycine decarboxylase (DGD) is an unusual PLP-dependent enzyme that catalyzes two reaction types, decarboxylation and transamination, in the same active site. A structurally based, functional model has been proposed for the DGD active site, which maintains that R406 is important in determining substrate specificity through interactions with the substrate carboxylate while W138 provides specificity for short-chain alkyl groups. The mechanistic roles of R406 and W138 were investigated using site-directed mutagenesis, alternate substrates, and analysis of steady-state and half-reaction kinetics. Experiments with the R406M and R406K mutants confirm the importance of R406 in substrate binding. Surprisingly, this work also shows that the positive charge of R406 facilitates catalysis of decarboxylation. The W138F mutant demonstrates that W138 indeed acts to limit the size of the subsite C binding pocket, determining specificity for 2,2-dialkylglycines with small side chains as predicted by the model. Finally, work with the double mutant W138F/M141R shows that these mutations expand substrate specificity to include l-glutamate and lead to an increase in specificity for l-glutamate over 2-aminoisobutyrate of approximately 8 orders of magnitude compared to that of wild-type DGD.

Understanding Catalytic Specificity in Alanine Racemase from Quantum Mechanical and Molecular Mechanical Simulations of the Arginine 219 Mutant

Alanine racemase (AlaR) catalyzes the interconversion between l-Ala and d-Ala with the aid of the cofactor pyridoxal 5'-phosphate (PLP). The pyridine nitrogen in PLP in the wild-type enzyme is unprotonated due to interaction with Arg219, a rare feature among PLP-dependent enzymes. Herein, we performed combined quantum mechanics and molecular mechanics molecular dynamics simulations to study the Arg219Glu mutant AlaR. In this form of the enzyme, the PLP-pyridine nitrogen is protonated. This study suggests that the catalytic effect in the Arg219Glu mutant enzyme is due to a combined solvent and inherent stabilizing effect of the protonated cofactor, in contrast to the wild-type enzyme where the catalytic effect may be ascribed to solvent effects alone. Furthermore, we find that the quinonoid intermediate is greatly stabilized in the mutant enzyme, opening the possibility for side reactions such as transamination. We show that a computed 1,3-proton transfer in PLP due to the catalytic Lys39 is a feasible side reaction en route to transamination.

Targeting the Active Site Gate to Yield Hyperactive Variants of 5-Aminolevulinate Synthase

The rate of porphyrin biosynthesis in mammals is controlled by the activity of the pyridoxal 5'-phosphate-dependent enzyme 5-aminolevulinate synthase (EC 2.3.1.37). Based on the postulate that turnover in this enzyme is controlled by conformational dynamics associated with a highly conserved active site loop, we constructed a variant library by targeting imperfectly conserved noncatalytic loop residues and examined the effects on product and porphyrin production. Functional loop variants of the enzyme were isolated via genetic complementation in Escherichia coli strain HU227. Colony porphyrin fluorescence varied widely when bacterial cells harboring the loop variants were grown on inductive media; this facilitated identification of clones encoding unusually active enzyme variants. Nine loop variants leading to high in vivo porphyrin production were purified and characterized kinetically. Steady state catalytic efficiencies for the two substrates were increased by up to 100-fold. Presteady state single turnover reaction data indicated that the second step of quinonoid intermediate decay, previously assigned as reaction rate-limiting, was specifically accelerated such that in three of the variants this step was no longer kinetically significant. Overall, our data support the postulate that the active site loop controls the rate of product and porphyrin production in vivo and suggest the possibility of an as yet undiscovered means of allosteric regulation.

Entamoeba histolytica Phosphoserine aminotransferase (EhPSAT): insights into the structure-function relationship

BackgroundPresence of phosphorylated Serine biosynthesis pathway upstream to the de novo cysteine biosynthesis pathway makes PSAT a crucial enzyme. Besides this, phoshoserine produced by the enzyme can also be taken up directly by cysteine synthase as a substrate. PSAT is a PLP dependent enzyme where the cofactor serves as an epicenter for functional catalysis with the active site architecture playing crucial role in optimum function of the enzyme.FindingsEhPSAT is a homodimer of molecular mass 86 kDa. To understand the structural modulations associated with pH dependent changes in functional activity of EhPSAT detailed biophysical studies were carried out. pH alterations had no significant effect on the secondary structure, cofactor orientation and oligomeric configuration of the enzyme however, pH dependent compaction in molecular dimensions was observed. Most interestingly, a direct correlation between pH induced modulation of functional activity and orientation of Trp 101 present in the active site of the enzyme was observed. Sodium halides nullified the pH induced global changes in the enzyme, however differential effect of these salts on the active site microenvironment and functional activity of the enzyme was observed.ConclusionsThe study unequivocally demonstrates that pH induced selective modification of active site microenvironment and not global change in structure or oligomeric status of the enzyme is responsible for the pH dependent change in enzymatic activity of PSAT.

Immobilization of PLP-dependent enzymes with cofactor retention and enhanced stability

Immobilization of PLP-dependent enzymes requires specific studies due to their special cofactor-enzyme bond. These enzymes were immobilized using different methods, and the recombinant serine hydroxymethyltransferase (SHMT) was used as a case study. The immobilization of SHMT on glyoxal-agarose resulted in a high retention yield (70%); however, the reduction step caused an enzymatic activity loss of 80%. The immobilization on Eupergit ® C was optimized by considering different ionic strengths, pH and temperatures. SHMT reached 53% retention on this support. Although the enzymatic activity of the derivative decreased by 36% during the treatment with methylamine and washing, it was totally recovered by incubation with the cofactor. SHMT immobilized on Eupergit ® C gained thermal stability with respect to the soluble enzyme. Finally, 6-His-tagged SHMT was adsorbed very rapidly on IMAC supports and reached a 98% immobilization yield and enzymatic retention. The capacity of Eupergit ® C beads to immobilize PLP-dependent enzymes was corroborated by the immobilization of alanine racemase and aspartate aminotransferase. The final immobilization yields were 85 and 74% respectively, and the derivatives were two- and nine-fold more stable than the soluble enzymes.

Conductometric Method for the Rapid Characterization of the Substrate Specificity of Amine-Transaminases

Amine-transaminases (ATAs, omega-transaminases, omega-TA) are PLP-dependent enzymes that catalyze amino group transfer reactions. In contrast to the widespread and well-known amino acid-transaminases, ATAs are able to convert substrates lacking an alpha-carboxylic functional group. They have gained increased attention because of their potential for the asymmetric synthesis of optically active amines, which are frequently used as building blocks for the preparation of numerous pharmaceuticals. Having already introduced a fast kinetic assay based on the conversion of the model substrate alpha-methylbenzylamine for the characterization of the amino acceptor specificity, we now report on a kinetic conductivity assay for investigating the amino donor specificity of a given ATA. The course of an ATA-catalyzed reaction can be followed conductometrically since the conducting substrates, a positively charged amine and a negatively charged keto acid, are converted to nonconducting products, a noncharged ketone and a zwitterionic amino acid. The decrease of conductivity for the investigated reaction systems were determined to be 33-52 microS mM(-1). In contrast to other ATA-assays previously described, with this approach all transamination reactions between any amine and any keto acid can be monitored without the need for an additional enzyme or staining solutions. The assay was used for the characterization of a ATA from Rhodobacter sphaeroides, and the data obtained were in excellent agreement with gas chromatography analysis.

Inhibition of the PLP-dependent enzyme serine palmitoyltransferase by cycloserine: evidence for a novel decarboxylative mechanism of inactivation.

Cycloserine (CS, 4-amino-3-isoxazolidone) is a cyclic amino acid mimic that is known to inhibit many essential pyridoxal 5'-phosphate (PLP)-dependent enzymes. Two CS enantiomers are known; D-cycloserine (DCS, also known as Seromycin) is a natural product that is used to treat resistant Mycobacterium tuberculosis infections as well as neurological disorders since it is a potent NMDA receptor agonist, and L-cycloserine (LCS) is a synthetic enantiomer whose usefulness as a drug has been hampered by its inherent toxicity arising through inhibition of sphingolipid metabolism. Previous studies on various PLP-dependent enzymes revealed a common mechanism of inhibition by both enantiomers of CS; the PLP cofactor is disabled by forming a stable 3-hydroxyisoxazole/pyridoxamine 5'-phosphate (PMP) adduct at the active site where the cycloserine ring remains intact. Here we describe a novel mechanism of CS inactivation of the PLP-dependent enzyme serine palmitoyltransferase (SPT) from Sphingomonas paucimobilis. SPT catalyses the condensation of l-serine and palmitoyl-CoA, the first step in the de novo sphingolipid biosynthetic pathway. We have used a range of kinetic, spectroscopic and structural techniques to postulate that both LCS and DCS inactivate SPT by transamination to form a free pyridoxamine 5'-phosphate (PMP) and beta-aminooxyacetaldehyde that remain bound at the active site. We suggest this occurs by ring opening of the cycloserine ring followed by decarboxylation. Enzyme kinetics show that inhibition is reversed by incubation with excess PLP and that LCS is a more effective SPT inhibitor than DCS. UV-visible spectroscopic data, combined with site-directed mutagenesis, suggest that a mobile Arg(378) residue is involved in cycloserine inactivation of SPT.

Serine 254 Enhances an Induced Fit Mechanism in Murine 5-Aminolevulinate Synthase

5-Aminolevulinate synthase (EC 2.3.1.37) (ALAS), a pyridoxal 5'-phosphate (PLP)-dependent enzyme, catalyzes the initial step of heme biosynthesis in animals, fungi, and some bacteria. Condensation of glycine and succinyl coenzyme A produces 5-aminolevulinate, coenzyme A, and carbon dioxide. X-ray crystal structures of Rhodobacter capsulatus ALAS reveal that a conserved active site serine moves to within hydrogen bonding distance of the phenolic oxygen of the PLP cofactor in the closed substrate-bound enzyme conformation and within 3-4 A of the thioester sulfur atom of bound succinyl-CoA. To evaluate the role(s) of this residue in enzymatic activity, the equivalent serine in murine erythroid ALAS was substituted with alanine or threonine. Although both the K(m)(SCoA) and k(cat) values of the S254A variant increased, by 25- and 2-fold, respectively, the S254T substitution decreased k(cat) without altering K(m)(SCoA). Furthermore, in relation to wild-type ALAS, the catalytic efficiency of S254A toward glycine improved approximately 3-fold, whereas that of S254T diminished approximately 3-fold. Circular dichroism spectroscopy revealed that removal of the side chain hydroxyl group in the S254A variant altered the microenvironment of the PLP cofactor and hindered succinyl-CoA binding. Transient kinetic analyses of the variant-catalyzed reactions and protein fluorescence quenching upon 5-aminolevulinate binding demonstrated that the protein conformational transition step associated with product release was predominantly affected. We propose the following: 1) Ser-254 is critical for formation of a competent catalytic complex by coupling succinyl-CoA binding to enzyme conformational equilibria, and 2) the role of the active site serine should be extended to the entire alpha-oxoamine synthase family of PLP-dependent enzymes.

Molecular and Phylogenetic Analysis of Pyridoxal Phosphate- Dependent Acyltransferase of Exiguobacterium acetylicum

The pyridoxal-5'-phosphate (PLP)-dependent family of enzymes is a very diverse group of proteins that metabolize small molecules like amino acids and sugars, and synthesize cofactors for other metabolic pathways through transamination, decarboxylation, racemization, and substitution reactions. In this study we employed degenerated primer-based PCR amplification, using genomic DNA isolated from the soil bacterium Exiguobacterium acetylicum strain SN as template. We revealed the presence of a PLP-dependent family of enzymes, such as PLP-dependent acyltransferase, and similarity to 8-amino-7-oxononoate synthase. Sequencing analysis and multiple alignment of the thymidine-adenine-cloned PCR amplicon revealed PLP-dependent family enzymes with specific confering codes and consensus amino acid residues specific to this group of functional proteins. Amino acid residues common to the majority of PLP-dependent enzymes were also revealed by the Lasergene MegAlign software. A phylogenetic tree was constructed. Its analysis revealed a close relationship of E. acetylicum to other bacteria isolated from extreme environments suggesting similarities in anabolic adaptability and evolutionary development.

Hydroxamic Acids As a Novel Family of Serine Racemase Inhibitors: Mechanistic Analysis Reveals Different Modes of Interaction with the Pyridoxal-5′-phosphate Cofactor

Mammalian serine racemase (SR) is a pyridoxal-5'-phosphate (PLP) dependent enzyme responsible for the biosynthesis of the neurotransmitter D-serine, which activates N-methyl-D-aspartate (NMDA) receptors in the CNS. Aberrant regulation of NMDA receptor signaling has been implicated in a variety of neuropathologies, and inhibitors of SR would therefore be a worthwhile tool for further investigation or treatment of such conditions. Here, we identify a series of small aliphatic hydroxamic acids (HAs) that act as potent SR inhibitors. However, specificity studies showed that some of these HAs can act as nonspecific inhibitors of PLP-dependent enzymes. We employed NMR, MS, and UV/vis spectroscopic techniques to reveal that the nonspecific effect is likely due to irreversible interaction of the HA moiety with PLP to form aldoxime species. We also characterize L-aspartic acid beta-hydroxamate as a competitive and selective SR inhibitor that could be used as a scaffold for further inhibitor development.

The B6 database: a tool for the description and classification of vitamin B6-dependent enzymatic activities and of the corresponding protein families

Background -Enzymes that depend on vitamin B6 (and in particular on its metabolically active form, pyridoxal 5'-phosphate, PLP) are of great relevance to biology and medicine, as they catalyze a wide variety of biochemical reactions mainly involving amino acid substrates. Although PLP-dependent enzymes belong to a small number of independent evolutionary lineages, they encompass more than 160 distinct catalytic functions, thus representing a striking example of divergent evolution. The importance and remarkable versatility of these enzymes, as well as the difficulties in their functional classification, create a need for an integrated source of information about them.Description -The B6 database contains documented B6-dependent activities and the relevant protein families, defined as monophyletic groups of sequences possessing the same enzymatic function. One or more families were associated to each of 121 PLP-dependent activities with known sequences. Hidden Markov models (HMMs) were built from family alignments and incorporated in the database. These HMMs can be used for the functional classification of PLP-dependent enzymes in genomic sets of predicted protein sequences. An example of such analyses (a census of human genes coding for PLP-dependent enzymes) is provided here, whereas many more are accessible through the database itself.Conclusion -The B6 database is a curated repository of biochemical and molecular information about an important group of enzymes. This information is logically organized and available for computational analyses, providing a key resource for the identification, classification and comparative analysis of B6-dependent enzymes.

Insights into the Biosynthesis of the Vibrio cholerae Major Autoinducer CAI-1 from the Crystal Structure of the PLP-Dependent Enzyme CqsA

CqsA is an enzyme involved in the biosynthesis of cholerae autoinducer-1 (CAI-1), the major Vibrio cholerae autoinducer engaged in quorum sensing. The amino acid sequence of CqsA suggests that it belongs to the family of α-oxoamine synthases that catalyse the condensation of an amino acid to an acyl-CoA substrate. Here we present the apo- and PLP-bound crystal structures of CqsA and confirm that it shares structural homology with the dimeric α-oxoamine synthases, including a conserved PLP-binding site. The chemical structure of CAI-1 suggests that decanoyl-CoA may be one substrate of CqsA and that another substrate may be l-threonine or l-2-aminobutyric acid. A crystal structure of CqsA at 1.9-Å resolution obtained in the presence of PLP and l-threonine reveals an external aldimine that has lost the l-threonine side chain. Similarly, a 1.9-Å-resolution crystal structure of CqsA in the presence of PLP, l-threonine, and decanoyl-CoA shows a trapped external aldimine intermediate, suggesting that the condensation and decarboxylation steps have occurred, again with loss of the l-threonine side chain. It is suggested that this side-chain loss, an observation supported by mass spectrometry, is due to a retro-aldol reaction. Although no structural data have been obtained on CqsA using l-2-aminobutyric acid and decanoyl-CoA as substrates, mass spectrometry confirms the expected product of the enzyme reaction. It is proposed that a region of structure that is disordered in the apo structure is involved in the release of product. While not confirming if CqsA alone is able to synthesize CAI-1, these results suggest possible synthetic routes.

The effects of environmental salinity on trunk kidney proteome of juvenile ayu (Plecoglossus altivelis)

As the life cycle of ayu spans river, brackish and seawater environments, it would be a suitable fish model for studying the responses to salinity changes in aquatic animals. We investigated the effect of salinity on trunk kidney proteome in ayu (Plecoglossus altivelis) using two-dimensional gel electrophoresis and mass spectrometry. The proteins involved in the process of energy metabolism, biosynthesis, DNA methylation and cell differentiation were mainly affected, and 10 significantly changed proteins were identified. Our result showed that isocitrate dehydrogenase (ICD), pyruvate dehydrogenase (E1), O-glycosyl hydrolase, mitochondrial precursor of ATP synthase subunit beta, mitochondrial ferrtin (MtF), retinol binding protein (RBP) were down-regulated, whereas aldehyde dehydrogenase, cytokeratin 1, S-adenosylhomocysteine hydrolase, Cys-Met metabolism PLP-dependent enzyme were up-regulated when ayu transferred from freshwater to brackish water. Partial coding sequences of E1, ICD, MtF and RBP genes were determined, and the effects of salinity on their mRNA expression in ayu trunk kidney were tested by real-time PCR subsequently. Their possible direct or indirect roles in the adaptation of ayu to salinity are discussed.

31P NMR studies of O-acetylserine sulfhydrylase-B from Salmonella typhimurium

O-Acetylserine sulfhydrylase (OASS) is a pyridoxal 5′-phosphate (PLP)-dependent enzyme that catalyzes the conversion of O-acetylserine and bisulfide to l-cysteine and acetate in bacteria and higher plants. Enteric bacteria have two isozymes of OASS, A and B, produced under aerobic and anaerobic growth conditions, respectively, with different substrate specificities. The 31P chemical shift of the internal and external Schiff bases of PLP in OASS-B are further downfield compared to OASS-A, suggesting a tighter binding of the cofactor in the B-isozyme. The chemical shift of the internal Schiff base (ISB) of OASS-B is 6.2 ppm, the highest value reported for the ISB of a PLP-dependent enzyme. Considering the similarity in the binding sites of the PLP cofactor for both isozymes, torsional strain of the C5–C5′ bond (O4′–C5′–C5–C4) of the Schiff base is proposed to contribute to the further downfield shift. The chemical shift of the lanthionine external Schiff base (ESB) of OASS-B is 6.0 ppm, upfield from that of unliganded OASS-B, while that of serine ESB is 6.3 ppm. Changes in chemical shift suggest the torsional strain of PLP changes as the reaction proceeds. The apoenzyme of OASS-B was prepared using hydroxylamine as the resolving reagent. Apoenzyme was reconstituted to holoenzyme by addition of PLP. Reconstitution is pseudo-first order and exhibits a final maximum recovery of 81.4%. The apoenzyme shows no visible absorbance, while the reconstituted enzyme has a UV–visible spectrum that is nearly identical to that of the holoenzyme. Steady-state fluorescence spectra gave tryptophan emission of the apoenzyme that is 3.3-fold higher than the emission of either the native or reconstituted enzyme, suggesting that PLP is a potent quencher of tryptophan emission.