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The cytosolic and mitochondrial isoforms of serine hydroxymethyltransferase (SHMT1 and SHMT2, respectively) are well-recognized targets of cancer research, since their activity is critical for purine and pyrimidine biosynthesis and because of their prominent role in the metabolic reprogramming of cancer cells. Here we show that 3-bromopyruvate (3BP), a potent novel anti-tumour agent believed to function primarily by blocking energy metabolism, differentially inactivates human SHMT1 and SHMT2. SHMT1 is completely inhibited by 3BP, whereas SHMT2 retains a significant fraction of activity. Site directed mutagenesis experiments on SHMT1 demonstrate that selective inhibition relies on the presence of a cysteine residue at the active site of SHMT1 (Cys204) that is absent in SHMT2. Our results show that 3BP binds to SHMT1 active site, forming an enzyme-3BP complex, before reacting with Cys204. The physiological substrate l-serine is still able to bind at the active site of the inhibited enzyme, although catalysis does not occur. Modelling studies suggest that alkylation of Cys204 prevents a productive binding of l-serine, hampering interaction between substrate and Arg402. Conversely, the partial inactivation of SHMT2 takes place without the formation of a 3BP-enzyme complex. The introduction of a cysteine residue in the active site of SHMT2 by site directed mutagenesis (A206C mutation), at a location corresponding to that of Cys204 in SHMT1, yields an enzyme that forms a 3BP-enzyme complex and is completely inactivated. This work sets the basis for the development of selective SHMT1 inhibitors that target Cys204, starting from the structure and reactivity of 3BP., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Pyridoxal 5'-phosphate (PLP), the catalytically active form of vitamin B6, plays a crucial role in several cellular processes. In most organisms, PLP is recycled from nutrients and degraded B6-enzymes in a salvage pathway that involves pyridoxal kinase (PLK), pyridoxine phosphate oxidase and phosphatase activities. Regulation of the salvage pathway is poorly understood. Escherichia coli possesses two distinct pyridoxal kinases, PLK1, which is the focus of the present work, and PLK2. From previous studies dating back to thirty years ago, pyridoxal (PL) was shown to inhibit E. coli PLK1 forming a covalent link with the enzyme. This inhibition was proposed to play a regulative role in vitamin B6 metabolism, although its details had never been clarified. Recently, we have shown that also PLP produced during PLK1 catalytic cycle acts as an inhibitor, forming a Schiff base with Lys229, without being released in the solvent. The question arises as to which is the actual inhibition mechanism by PL and PLP. In the present work, we demonstrated that also PL binds to Lys229 as a Schiff base. However, the isolated covalent PLK1-PL complex is not inactive but, in the presence of ATP, is able to catalyse the single turnover production of PLP, which binds tightly to the enzyme and is ultimately responsible for its inactivation. The inactivation mechanism mediated by Lys229 may play a physiological role in controlling cellular levels of PLP. This article is part of a Special Issue entitled: Cofactor-dependent proteins: evolution, chemical diversity and bio-applications., (Copyright © 2015 Elsevier B.V. All rights reserved.)

L-Threonine aldolases (TAs), a family of enzymes belonging to the fold-type I pyridoxal 5'-phosphate (PLP) dependent enzymes, play a role in catalyzing the reversible cleavage of l-3-hydroxy-α-amino acids to glycine and the corresponding aldehydes. Threonine aldolases have great biotechnological potential for the syntheses of pharmaceutically relevant drug molecules because of their stereospecificity. The pH-dependency of their catalytic activity, affecting reaction intermediates, led us to study the effect of low-pH on Escherichia coli TA (eTA) structure. We report here a low-pH crystal structure of eTA at 2.1 Å resolution, with a non-covalently bound uncleaved l-serine substrate, and a PLP cofactor bound as an internal aldimine. This structure contrasts with other eTA structures obtained at physiological pH that show products or substrates bound as PLP-external aldimines. The non-productive binding at low-pH is due to an unusual substrate serine binding orientation in which the α-amino group and carboxylate group are in the wrong positions (relative to the active site residues) as a result of protonation of the α-amino group of the serine, as well as the active site histidines, His83 and His126. Protonation of these residues prevents the characteristic nucleophilic attack of the α-amino group of substrate serine on C4' of PLP to form the external aldimine. Our study shows that at low pH the change in charge distribution at the active site can result in substrates binding in a non-productive orientation., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Vitamin 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., (Copyright © 2010 Elsevier B.V. All rights reserved.)

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., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Vitamin B(6)-dependent enzymes may be grouped into five evolutionarily unrelated families, each having a different fold. Within fold type I enzymes, L-threonine aldolase (L-TA) and fungal alanine racemase (AlaRac) belong to a subgroup of structurally and mechanistically closely related proteins, which specialised during evolution to perform different functions. In a previous study, a comparison of the catalytic properties and active site structures of these enzymes suggested that they have a catalytic apparatus with the same basic features. Recently, recombinant D-threonine aldolases (D-TAs) from two bacterial organisms have been characterised, their predicted amino acid sequences showing no significant similarities to any of the known B(6) enzymes. In the present work, a comparative structural analysis suggests that D-TA has an alpha/beta barrel fold and therefore is a fold type III B(6) enzyme, as eukaryotic ornithine decarboxylase (ODC) and bacterial AlaRac. The presence of both TA and AlaRac in two distinct evolutionary unrelated families represents a novel and interesting example of convergent evolution. The independent emergence of the same catalytic properties in families characterised by completely different folds may have not been determined by chance, but by the similar structural features required to catalyse pyridoxal phosphate-dependent aldolase and racemase reactions.