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VITAMIN B(6) SALVAGE ENZYMES: MECHANISM, STRUCTURE AND REGULATION.
No full textVitamin B(6) is a generic term referring to pyridoxine, pyridoxamine, pyridoxal and their related phosphorylated forms. Pyridoxal 5'-phosphate is the catalytically active form of vitamin B(6), and acts as cofactor in more than 140 different enzyme reactions. In animals, pyridoxal 5'-phosphate is recycled from food and from degraded B(6)-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-B(6) 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-B(6)-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 B(6) 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-B(6)-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
Biomedical aspects of pyridoxal 5’-phosphate availability
No full textThe biologically active form of vitamin B6, pyridoxal 5'-phosphate (PLP), is a cofactor in over 160 enzyme activities involved in a number of metabolic pathways, including neurotransmitter synthesis and degradation. In humans, PLP is recycled from food and from degraded PLP-dependent enzymes in a salvage pathway requiring the action of pyridoxal kinase, pyridoxine 5'-phosphate oxidase and phosphatases. Once pyridoxal 5'-phosphate is made, it is targeted to the dozens different apoenzymes that need it as a cofactor. The regulation of the salvage pathway and the mechanism of addition of PLP to the apoenzymes are poorly understood and represent a very challenging research field. Severe neurological disorders, such as convulsions and epileptic encephalopathy, result from a reduced availability of pyridoxal 5'-phosphate in the cell, due to inborn errors in the enzymes of the salvage pathway or other metabolisms and to interactions of drugs with PLP or pyridoxal kinase. Multifactorial neurological pathologies, such as autism, schizophrenia, Alzheimer's disease, Parkinson's disease and epilepsy have also been correlated to inadequate intracellular levels of PLP
COMUNICAZIONE ORALE A CONGRESSO SU INVITO “On the mechanism of addition of pyridoxal 5’-phosphate to serine hydroxymethyltransferase”, in The Third International Conference on Cofactors (ICC03), Finland, Turku, 10-15 July 2011.
No full textON THE MECHANISM OF PYRIDOXAL 5’-PHOSPHATE ADDITION TO SERINE HYDROXYMETHYLTRANSFERASE.
Roberto Contestabile1, Rita Florio1, Isabel Noguès2, Martin K. Safo3, Verne Schirch3 and Martino Luigi di Salvo1
1Dipartimento di Scienze Biochimiche and Istituto Pasteur-Fondazione Cenci Bolognetti, Sapienza Università di Roma, Italy, w3.uniroma1.it/bio_chem/sito_biochimica/EN/index.html
2Istituto di Biologia Agroambientale e Forestale, Centro Nazionale delle Ricerche, Monterotondo Scalo, Roma, Italy.
3Department of Medicinal Chemistry and Institute for Structural Biology and Drug Discovery, Virginia Commonwealth University, Richmond, VA, USA.
The addition of cofactors to newly synthesized apo-proteins in the cell is a crucial event that can occur at different stages of polypeptide folding and affect it to a variable extent. The form in which the cofactor is available in the intracellular medium may also differ according to its reactivity or toxicity. Although much work has been done on the mechanism and structure of vitamin B6–dependent enzymes, little is known on how pyridoxal 5’phosphate (PLP) is supplied to the apoenzymes in order to meet their requirement in terms of cofactor. Free PLP availability in the cell is significantly limited by the high reactivity of its aldehyde group, forming aldimines with amino groups on non-B6 enzymes and amino acids, as well as by dephosphorylation by phosphatases. This raises the intriguing question of how the cell supplies sufficient PLP, with high specificity, to the dozens of B6 enzymes. The traditionally proposed mechanism involves a release of PLP from the enzymes that catalyze its formation into solution, where it finds its way to the active site of apo-B6 enzymes. An obvious problem with this mode of PLP addition is the potential interactions of the cofactor with nucleophiles and its dephosphorylation by phosphatases, which would significantly deplete the free PLP availability. An alternative mechanism for PLP addition involves a direct channeling from the enzymes that produce it in the cell to the acceptor B6 enzymes, thus avoiding its release into solution. This mechanism offers an efficient, exclusive, and protected means of delivery of the reactive PLP.
As we approached this matter, we chose Escherichia coli as a model system. To start with, we decided to use serine hydroxymethyltransferase (eSHMT) as PLP acceptor, since this is a well characterized fold type I B6 enzyme. A first step was the understanding of how free PLP reacts in vitro with apo-eSHMT to form the active holoenzyme. Thereafter, we focused on the mechanism of PLP transfer from the enzymes that catalyse its formation in the E. coli cell, pyridoxine phosphate oxidase and pyridoxal kinase, to eSHMT. The transfer kinetic studies were performed by means of UV-visible spectroscopy, circular dichroism measurements and chemical quenched-flow experiments. Our presentation will The results obtained are in favor of a channeling mechanism
Active Site Structure and Sterospecificity of Escherichia coli Pyridoxine 5'-phosphate Oxidase
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Serine hydroxymethyltransferase: role of glu75 and evidence that serine is cleaved by a retroaldol mechanism
No full textSerine hydroxymethyltransferase (SHMT) catalyzes the reversible interconversion of serine and glycine with tetrahydrofolate serving as the one-carbon carrier. SHMT also catalyzes the folate-independent retroaldol cleavage of allothreonine and 3-phenylserine and the irreversible conversion of 5,10-methenyltetrahydrofolate to 5-formyltetrahydrofolate. Studies of wild-type and site mutants of SHMT have failed to clearly establish the mechanism of this enzyme. The cleavage of 3-hydroxy amino acids to glycine and an aldehyde occurs by a retroaldol mechanism. However, the folate-dependent cleavage of serine can be described by either the same retroaldol mechanism with formaldehyde as an enzyme-bound intermediate or by a nucleophilic displacement mechanism in which N5 of tetrahydrofolate displaces the C3 hydroxyl of serine, forming a covalent intermediate. Glu75 of SHMT is clearly involved in the reaction mechanism; it is within hydrogen bonding distance of the hydroxyl group of serine and the formyl group of 5-formyltetrahydrofolate in complexes of these species with SHMT. This residue was changed to Leu and Gln, and the structures, kinetics, and spectral properties of the site mutants were determined. Neither mutation significantly changed the structure of SHMT, the spectral properties of its complexes, or the kinetics of the retroaldol cleavage of allothreonine and 3-phenylserine. However, both mutations blocked the folate-dependent serine-to-glycine reaction and the conversion of methenyltetrahydrofolate to 5-formyltetrahydrofolate. These results clearly indicate that interaction of Glu75 with folate is required for folate-dependent reactions catalyzed by SHMT. Moreover, we can now propose a promising modification to the retroaldol mechanism for serine cleavage. As the first step, N5 of tetrahydrofolate makes a nucleophilic attack on C3 of serine, breaking the C2-C3 bond to form N5-hydroxymethylenetetrahydrofolate and an enzyme-bound glycine anion. The transient formation of formaldehyde as an intermediate is possible, but not required. This mechanism explains the greatly enhanced rate of serine cleavage in the presence of folate, and avoids some serious difficulties presented by the nucleophilic displacement mechanism involving breakage of the C3-OH bond
Unintended consequences? Water molecules at biological and crystallographic protein–protein interfaces
No full texttThe importance of protein–protein interactions (PPIs) is becoming increasingly appreciated, as theseinteractions lie at the core of virtually every biological process. Small molecule modulators that targetPPIs are under exploration as new therapies. One of the greatest obstacles faced in crystallographicallydetermining the 3D structures of proteins is coaxing the proteins to form “artificial” PPIs that lead touniform crystals suitable for X-ray diffraction. This work compares interactions formed naturally, i.e.,“biological”, with those artificially formed under crystallization conditions or “non-biological”. In partic-ular, a detailed analysis of water molecules at the interfaces of high-resolution (≤2.30 ̊A) X-ray crystalstructures of protein–protein complexes, where 140 are biological protein–protein complex structuresand 112 include non-biological protein–protein interfaces, was carried out using modeling tools basedon the HINT forcefield. Surprisingly few and relatively subtle differences were observed between thetwo types of interfaces: (i) non-biological interfaces are more polar than biological interfaces, yet there isbetter organized hydrogen bonding at the latter; (ii) biological associations rely more on water-mediatedinteractions with backbone atoms compared to non-biological associations; (iii) aromatic/planar residuesplay a larger role in biological associations with respect to water, and (iv) Lys has a particularly large roleat non-biological interfaces. A support vector machines (SVMs) classifier using descriptors from this studywas devised that was able to correctly classify 84% of the two interface type
On the catalytic mechanism and stereospecificity of Escherichia coli L-threonine aldolase
No full textL-Threonine aldolases (L-TAs) represent a family of homologous pyridoxal 5′-phosphate-dependent enzymes found in bacteria and fungi, and catalyse the reversible cleavage of several l-3-hydroxy-α-amino acids. L-TAs have great biotechnological potential, as they catalyse the formation of carbon-carbon bonds, and therefore may be exploited for the bioorganic synthesis of l-3-hydroxyamino acids that are biologically active or constitute building blocks for pharmaceutical molecules. Many L-TAs, showing different stereospecificity towards the Cβ configuration, have been isolated. Because of their potential to carry out diastereoselective syntheses, L-TAs have been subjected to structural, functional and mechanistic studies. Nevertheless, their catalytic mechanism and the structural bases of their stereospecificity have not been elucidated. In this study, we have determined the crystal structure of low-specificity L-TA from Escherichia coli at 2.2Å resolution, in the unliganded form and cocrystallized with l-serine and l-threonine. Furthermore, several active site mutants have been functionally characterized in order to elucidate the reaction mechanism and the molecular bases of stereospecificity. No active site catalytic residue was revealed, and a structural water molecule was assumed to act as the catalytic base in the retro-aldol cleavage reaction. Interestingly, the very large active site opening of E. Coli L-TA suggests that much larger molecules than L-threonine isomers may be easily accommodated, and L-TAs may actually have diverse physiological functions in different organisms. Substrate recognition and reaction specificity seem to be guided by the overall microenvironment that surrounds the substrate at the enzyme active site, rather than by one ore more specific residues
Characterization of hemoglobin bassett (alpha94Asp-->Ala), a variant with very low oxygen affinity.
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