A rat liver system that catalyses a pyridoxal phosphate-independent alpha beta-eliminto.
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ABSTRACT:
SUBMITTER: Tudballn
PROVIDER: S-EPMC1184855 | biostudies-other | 1969 Sep
REPOSITORIES: biostudies-other
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Project description:Pyridoxal-5'-phosphate (PLP) is a versatile cofactor that assists in different types of enzymatic reactions. PLP has also been reported to react with substrates and catalyze some of these reactions independent of enzymes. One such catalytic reaction is the breakdown of cysteine to produce hydrogen sulfide (H2S) in the presence of multivalent metal ions. However, the enzyme-independent catalytic activity of PLP in catabolizing cysteine in the absence of multivalent ions is unknown. In this study, we show that PLP reacts with cysteine to form a thiazolidine product, which is supported by quantum chemical calculations of the absorption spectrum. The reaction of PLP with cysteine is dependent on ionic strength and pH. The thiazolidine product slowly decomposes to produce H2S and the PLP regenerates to its active form with longer reaction times (> 24 h), suggesting that PLP can act as a catalyst. We propose an enzyme-independent plausible reaction mechanism for PLP catalyzed cysteine breakdown to produce H2S, which proceeds through the formation of thiazolidine ring intermediates that later hydrolyzes slowly to regenerate the PLP. This work demonstrates that PLP catalyzes cysteine breakdown in the absence of enzymes, base, and multivalent metal ions to produce H2S.
| S-EPMC9822980 | biostudies-literature
Project description:L-serine dehydratase (SDH), a member of the beta-family of pyridoxal phosphate-dependent (PLP) enzymes, catalyzes the deamination of L-serine and L-threonine to yield pyruvate or 2-oxobutyrate. The crystal structure of L-serine dehydratase from human liver (hSDH) has been solved at 2.5 A-resolution by molecular replacement. The structure is a homodimer and reveals a fold typical for beta-family PLP-dependent enzymes. Each monomer serves as an active unit and is subdivided into two distinct domains: a small domain and a PLP-binding domain that covalently anchors the cofactor. Both domains show the typical open alpha/beta architecture of PLP enzymes. Comparison with the rSDH-(PLP-OMS) holo-enzyme reveals a large structural difference in active sites caused by the artifical O-methylserine. Furthermore, the activity of hSDH-PLP was assayed and it proved to show catalytic activity. That suggests that the structure of hSDH-PLP is the first structure of the active natural holo-SDH.
| S-EPMC2279282 | biostudies-literature
Project description:The RidA protein (PF01042) from Salmonella enterica is a deaminase that quenches 2-aminoacrylate (2AA) and other reactive metabolites. In the absence of RidA, 2AA accumulates, damages cellular enzymes, and compromises the metabolic network. In vitro, RidA homologs from all domains of life deaminate 2AA, and RidA proteins from plants, bacteria, yeast, and humans complement the mutant phenotype of a ridA mutant strain of S. enterica In the present study, a methanogenic archaeon, Methanococcus maripaludis S2, was used to probe alternative mechanisms to restore metabolic balance. M. maripaludis MMP0739, which is annotated as an aspartate/glutamate racemase, complemented a ridA mutant strain and reduced the intracellular 2AA burden. The aspartate/glutamate racemase YgeA from Escherichia coli or S. enterica, when provided in trans, similarly restored wild-type growth to a ridA mutant. These results uncovered a new mechanism to ameliorate metabolic stress, and they suggest that direct quenching by RidA is not the only strategy to quench 2AA.IMPORTANCE 2-Aminoacrylate is an endogenously generated reactive metabolite that can damage cellular enzymes if not directly quenched by the conserved deaminase RidA. This study used an archaeon to identify a RidA-independent mechanism to prevent metabolic stress caused by 2AA. The data suggest that a gene product annotated as an aspartate/glutamate racemase (MMP0739) produces a metabolite that can quench 2AA, expanding our understanding of strategies available to quench reactive metabolites.
| S-EPMC5892117 | biostudies-literature
Project description:Cystathionine beta-synthase (CBS) is a unique heme- containing enzyme that catalyzes a pyridoxal 5'-phosphate (PLP)-dependent condensation of serine and homocysteine to give cystathionine. Deficiency of CBS leads to homocystinuria, an inherited disease of sulfur metabolism characterized by increased levels of the toxic metabolite homocysteine. Here we present the X-ray crystal structure of a truncated form of the enzyme. CBS shares the same fold with O-acetylserine sulfhydrylase but it contains an additional N-terminal heme binding site. This heme binding motif together with a spatially adjacent oxidoreductase active site motif could explain the regulation of its enzyme activity by redox changes.
| S-EPMC149156 | biostudies-literature
Project description:D-aspartate (D-Asp) is found in specific neurons, transported to neuronal terminals and released in a stimulation-dependent manner. Because D-Asp formation is not well understood, determining its function has proved challenging. Significant levels of D-Asp are present in the cerebral ganglion of the F- and C-clusters of the invertebrate Aplysia californica, and D-Asp appears to be involved in cell-cell communication in this system. Here, we describe a novel protein, DAR1, from A. californica that can convert aspartate and serine to their other chiral form in a pyridoxal 5'-phosphate (PLP)-dependent manner. DAR1 has a predicted length of 325 amino acids and is 55% identical to the bivalve aspartate racemase, EC 5.1.1.13, and 41% identical to the mammalian serine racemase, EC 5.1.1.18. However, it is only 14% identical to the recently reported mammalian aspartate racemase, DR, which is closely related to glutamate-oxaloacetate transaminase, EC 2.6.1.1. Using whole-mount immunohistochemistry staining of the A. californica central nervous system, we localized DAR1-like immunoreactivity to the medial region of the cerebral ganglion where the F- and C-clusters are situated. The biochemical and functional similarities between DAR1 and other animal serine and aspartate racemases make it valuable for examining PLP-dependent racemases, promising to increase our knowledge of enzyme regulation and ultimately, D-serine and D-Asp signaling pathways.
| S-EPMC3075720 | biostudies-literature
Project description:The first synthesis of 1-deaza-pyridoxal 5'-phosphate (2-formyl-3-hydroxy-4-methylbenzyl phosphate) is described. The chemoenzymatic approach described here is a reliable route to this important isosteric pyridoxal phosphate analogue. This work enables elucidation of the role of the pyridine nitrogen in pyridoxal 5'-phosphate dependent enzymes.
| S-EPMC3357107 | biostudies-literature
Project description:Enzymes that use the cofactor pyridoxal phosphate (PLP) constitute a ubiquitous class of biocatalysts. Here, we analyse their variety and genomic distribution as an example of the current opportunities and challenges for the study of protein families. In many free-living prokaryotes, almost 1.5% of all genes code for PLP-dependent enzymes, but in higher eukaryotes the percentage is substantially lower, consistent with these catalysts being involved mainly in basic metabolism. Assigning the function of PLP-dependent enzymes simply on the basis of sequence criteria is not straightforward because, as a consequence of their common mechanistic features, these enzymes have intricate evolutionary relationships. Thus, many genes for PLP-dependent enzymes remain functionally unclassified, and several of them might encode undescribed catalytic activities. In addition, PLP-dependent enzymes often show catalytic promiscuity (that is, a single enzyme catalyses different reactions), implying that an organism can have more PLP-dependent activities than it has genes for PLP-dependent enzymes. This observation presumably applies to many other classes of protein-encoding genes.
| S-EPMC1326353 | biostudies-literature
Project description:Proteins that fold as (β/α)8 barrels are thought to have evolved from half-barrels that underwent duplication and fusion events. The evidence is particularly clear for small barrels, which have almost identical halves. Additionally, computational calculations of the thermodynamic stability of these structures in the presence of denaturants have revealed that (β/α)8 barrels contain two subunits or domains corresponding to half-barrels. Hence, within (β/α)8 barrels, half-barrels are self-contained units. Here, we tested this hypothesis using β-glucosidase from the bacterium Thermotoga maritima (bglTm), which has a (β/α)8 barrel structure. Mutations were introduced to disrupt the noncovalent contacts between its halves and reveal the presence of two domains within bglTm, thus resulting in the creation of mutants T1 (containing W12A and I217A mutations) and T2 (containing W12A, H195A, I217A and F404A mutations). Mutants T1 and T2 were properly folded, as indicated by their fluorescence spectra and enzyme kinetic parameters. T1 and wild-type bglTm were equally stable, as shown by the results of thermal inactivation, differential scanning fluorimetry and guanidine hydrochloride denaturation experiments. However, T2 showed a first-order inactivation at 80°C, a single melting temperature of 82°C and only one transition concentration (c50) in 2.4 M guanidine hydrochloride. Additionally, T1 and T2 exhibited a cooperative denaturation process that followed a two-state model (m-values equal to 1.4 and 1.6 kcal/mol/M, respectively), similar to that of wild-type bglTm (1.2 kcal/mol/M). Hence, T1 and T2 each denatured as a single unit, although they contained different degrees of disruption between their halves. In conclusion, bglTm halves are equivalent in terms of their thermal and chemical stability; thus, their separate contributions to (β/α)8 barrel unfolding cannot be disentangled.
| S-EPMC5770038 | biostudies-literature
Project description: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 ?-ketoglutarate with oxalacetate and glutamate. From kinetic isotope effects studies, it is known that deprotonation of the aspartate external aldimine C(?)-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(?)-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(?)-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(?)-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(?)-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.
| S-EPMC3021986 | biostudies-literature
Project description:Vitamin B6 is an essential metabolic cofactor that has more functions in humans than any other single nutrient. Its de novo biosynthesis occurs through two mutually exclusive pathways that are absent in animals. The predominant pathway found in most prokaryotes, fungi, and plants has only recently been discovered. It is distinguished by a glutamine amidotransferase, which is remarkable in that it alone can synthesize the cofactor form, pyridoxal 5'-phosphate (PLP), directly from a triose and a pentose saccharide and glutamine. Here we report the 3D structure of the PLP synthase complex with substrate glutamine bound as well as those of the individual synthase and glutaminase subunits Pdx1 and Pdx2, respectively. The complex is made up of 24 protein units assembled like a cogwheel, a dodecameric Pdx1 to which 12 Pdx2 subunits attach. In contrast to the architecture of previously determined glutamine amidotransferases, macromolecular assembly is directed by an N-terminal alpha-helix on the synthase. Interaction with the synthase subunit leads to glutaminase activation, resulting in formation of an oxyanion hole, a prerequisite for catalysis. Mutagenesis permitted identification of the remote glutaminase and synthase catalytic centers and led us to propose a mechanism whereby ammonia shuttles between these active sites through a methionine-rich hydrophobic tunnel.
| S-EPMC1748218 | biostudies-literature