The vitamin B6-derived pyridoxal 5-phosphate (PLP) is the cofactor of enzymes catalyzing a large variety of chemical reactions mainly involved in amino acid metabolism. These enzymes have been divided in five families and fold types on the basis of evolutionary relationships and protein structural organization. Almost 1.5% of all genes in prokaryotes code for PLP-dependent enzymes, whereas the percentage is substantially lower in eukaryotes. Although about 4% of enzyme-catalyzed reactions catalogued by the Enzyme Commission are PLP-dependent, only a few enzymes are targets of approved drugs and about twenty are recognised as potential targets for drugs or herbicides. PLP-dependent enzymes for which there are already commercially available drugs are DOPA decarboxylase (involved in the Parkinson disease), GABA aminotransferase (epilepsy), serine hydroxymethyltransferase (tumors and malaria), ornithine decarboxylase (African sleeping sickness and, potentially, tumors), alanine racemase (antibacterial agents), and human cytosolic branched-chain aminotransferase (pathological states associated to the GABA/glutamate equilibrium concentrations). Within each family or metabolic pathway, the enzymes for which drugs have been already approved for clinical use are discussed first, reporting the enzyme structure, the catalytic mechanism, the mechanism of enzyme inactivation or modulation by substrate-like or transition state-like drugs, and on-going research for increasing specificity and decreasing side-effects. Then, PLP-dependent enzymes that have been recently characterized and proposed as drug targets are reported. Finally, the relevance of recent genomic analysis of PLP-dependent enzymes for the selection of drug targets is discussed.

Reprogramming of cellular metabolism towards de novo serine production fuels the growth of cancer cells, providing essential precursors such as amino acids and nucleotides and controlling the antioxidant and methylation capacities of the cell. The enzyme serine hydroxymethyltransferase (SHMT) has a key role in this metabolic shift, and directs serine carbons to one-carbon units metabolism and thymidilate synthesis. While the mitochondrial isoform of SHMT (SHMT2) has recently been identified as an important player in the control of cell proliferation in several cancer types and as a hot target for anticancer therapies, the role of the cytoplasmic isoform (SHMT1) in cancerogenesis is currently less defined. In this paper we show that SHMT1 is overexpressed in tissue samples from lung cancer patients and lung cancer cell lines, suggesting that, in this widespread type of tumor, SHMT1 plays a relevant role. We show that SHMT1 knockdown in lung cancer cells leads to cell cycle arrest and, more importantly, to p53-dependent apoptosis. Our data demonstrate that the induction of apoptosis does not depend on serine or glycine starvation, but is because of the increased uracil accumulation during DNA replication.

The three-dimensional structure of glutamate-1-semialdehyde aminomutase (EC 5.4.3.8 ), an α ₂ -dimeric enzyme from Synechococcus, has been determined by x-ray crystallography using heavy atom derivative phasing. The structure, refined at 2.4-Å resolution to an R-factor of 18.7% and good stereochemistry, explains many of the enzyme’s unusual specificity and functional properties. The overall fold is that of aspartate aminotransferase and related B ₆ enzymes, but it also has specific features. The structure of the complex with gabaculine, a substrate analogue, shows unexpectedly that the substrate binding site involves residues from the N-terminal domain of the molecule, notably Arg-32. Glu-406 is suitably positioned to repel α-carboxylic acids, thereby suggesting a basis for the enzyme’s reaction specificity. The subunits show asymmetry in cofactor binding and in the mobilities of the residues 153–181. In the unliganded enzyme, one subunit has the cofactor bound as an aldimine of pyridoxal phosphate with Lys-273 and, in this subunit, residues 153–181 are disordered. In the other subunit in which the cofactor is not covalently bound, residues 153–181 are well defined. Consistent with the crystallographically demonstrated asymmetry, a form of the enzyme in which both subunits have pyridoxal phosphate bound to Lys-273 through a Schiff base showed biphasic reduction by borohydride in solution. Analysis of absorption spectra during reduction provided evidence of communication between the subunits. The crystal structure of the reduced form of the enzyme shows that, despite identical cofactor binding in each monomer, the structural asymmetry at residues 153–181 remains.

Thiamin (vitamin B1) is a pharmacological agent boosting central metabolism through the action of the coenzyme thiamin diphosphate (ThDP). However, positive effects, including improved cognition, of high thiamin doses in neurodegeneration may be observed without increased ThDP or ThDP-dependent enzymes in brain. Here, we determine protein partners and metabolic pathways where thiamin acts beyond its coenzyme role. Malate dehydrogenase, glutamate dehydrogenase and pyridoxal kinase were identified as abundant proteins binding to thiamin- or thiazolium-modified sorbents. Kinetic studies, supported by structural analysis, revealed allosteric regulation of these proteins by thiamin and/or its derivatives. Thiamin triphosphate and adenylated thiamin triphosphate activate glutamate dehydrogenase. Thiamin and ThDP regulate malate dehydrogenase isoforms and pyridoxal kinase. Thiamin regulation of enzymes related to malate-aspartate shuttle may impact on malate/citrate exchange, responsible for exporting acetyl residues from mitochondria. Indeed, bioinformatic analyses found an association between thiamin- and thiazolium-binding proteins and the term acetylation. Our interdisciplinary study shows that thiamin is not only a coenzyme for acetyl-CoA production, but also an allosteric regulator of acetyl-CoA metabolism including regulatory acetylation of proteins and acetylcholine biosynthesis. Moreover, thiamin action in neurodegeneration may also involve neurodegeneration-related 14-3-3, DJ-1 and β-amyloid precursor proteins identified among the thiamin- and/or thiazolium-binding proteins.