Aurelio A. Moya-García

Mapping of catalytically important residues in the rat L-histidine decarboxylase enzyme using bioinformatic and site-directed mutagenesis approaches.

HDC (L-histidine decarboxylase), the enzyme responsible for the catalytic production of histamine from L-histidine, belongs to an evolutionarily conserved family of vitamin B6-dependent enzymes known as the group II decarboxylases. Yet despite the obvious importance of histamine, mammalian HDC enzymes remain poorly characterized at both the biochemical and structural levels. By comparison with the recently described crystal structure of the homologous enzyme L-DOPA decarboxylase, we have been able to identify a number of conserved domains and motifs that are important also for HDC catalysis. This includes residues that were proposed to mediate events within the active site, and HDC proteins carrying mutations in these residues were inactive when expressed in reticulocyte cell lysates reactions. Our studies also suggest that a significant change in quartenary structure occurs during catalysis. This involves a protease sensitive loop, and incubating recombinant HDC with an L-histidine substrate analogue altered enzyme structure so that the loop was no longer exposed for tryptic proteolysis. In total, 27 mutant proteins were used to test the proposed importance of 34 different amino acid residues. This is the most extensive mutagenesis study yet to identify catalytically important residues in a mammalian HDC protein sequence and it provides a number of novel insights into the mechanism of histamine biosynthesis.

Study by optical spectroscopy and molecular dynamics of the interaction of acridine-spermine conjugate with DNA

We report a spectroscopic and theoretical study of the interaction between double-stranded oligonucleotides containing either adenine-thymine or guanine-cytosine alternating sequences and N1-(Acridin-9-ylcarbonyl)-1,5,9,14,18-pentazaoctadecane, or ASC, which is formed by the covalent bonding of spermine and 9-amidoacridine moieties via a trimethylene chain. Solutions containing the oligonucleotides and the conjugate at different molar ratios were studied using complementary spectroscopic techniques, including electronic absorption, fluorescence emission, circular dichroism, and Raman spectroscopy. The spectroscopical properties of ASC at both the vibrational and the electronic levels were described by means of ab initio quantum-chemical calculations on 9-amidoacridine, used as a model compound. Molecular dynamics calculations, based on the QM/MM methodology, were also performed using previously docked structures of two oligonucleotide-ASC complexes containing the A-T and the G-C sequence. Our data, taken all together, allowed us to demonstrate that conjugation of spermine to acridine modulates and gives additional properties to the interaction of the latter with DNA. As the ASC molecule has a high affinity by the polyamine transport system, these results are promising for their application in the development of new anti-tumour drugs.

Analysis of the Decarboxylation Step in Mammalian Histidine Decarboxylase A COMPUTATIONAL STUDY

We report a hybrid quantum mechanics/molecular mechanics theoretical study on the reaction mechanism of mammalian histidine decarboxylase that allows us to obtain valuable insights on the structure of the cofactor-substrate adduct (external aldimine) in the active site of rat histidine decarboxylase. By means of molecular dynamics simulations, we traced the potential of mean force corresponding to the decarboxylation reaction of the adduct both in the active site of the enzyme and in aqueous solution. By comparing this process in both media, we have identified the key electrostatic interactions that explain the lowering of the free energy barrier in the enzyme. Our analysis also offers a validation of Dunathans hypothesis (Dunathan, H. (1966) Proc. Natl. Acad. Sci. U. S. A. 55, 712–716) regarding the role of stereoelectronic effects in the enzymatic decarboxylation process.

Structural features of mammalian histidine decarboxylase reveal the basis for specific inhibition

For a long time the structural and molecular features of mammalian histidine decarboxylase (EC 4.1.1.22), the enzyme that produces histamine, have evaded characterization. We overcome the experimental problems for the study of this enzyme by using a computer‐based modelling and simulation approach, and have now the conditions to use histidine decarboxylase as a target in histamine pharmacology. In this review, we present the recent (last 5 years) advances in the structure–function relationship of histidine decarboxylase and the strategy for the discovery of new drugs.

Molecular characterization of five patients with homocystinuria due to severe methylenetetrahydrofolate reductase deficiency

Urreizti R, Moya‐García AA, Pino‐ Ángeles A, Cozar M, Langkilde A, Fanhoe U, Esteves C, Arribas J, Vilaseca MA, Pérez‐Dueñas B, Pineda M, González V, Artuch R, Baldellou, A, Vilarinho L, Fowler B, Ribes A, Sánchez‐Jiménez F, Grinberg D, Balcells S. Molecular characterization of five patients with homocystinuria due to severe MTHFR deficiency.

Molecular Modeling and Site-Directed Mutagenesis Reveal Essential Residues for Catalysis in a Prokaryote-Type Aspartate Aminotransferase

We recently reported that aspartate (Asp) biosynthesis in plant chloroplasts is catalyzed by two different Asp aminotransferases (AAT): a previously characterized eukaryote type and a prokaryote type (PT-AAT) similar to bacterial and archaebacterial enzymes. The available molecular and kinetic data suggest that the eukaryote-type AAT is involved in the shuttling of reducing equivalents through the plastidic membrane, whereas the PT-AAT could be involved in the biosynthesis of the Asp-derived amino acids inside the organelle. In this work, a comparative modeling of the PT-AAT enzyme from Pinus pinaster (PpAAT) was performed using x-ray structures of a bacterial AAT (Thermus thermophilus; Protein Data Bank accession nos. 1BJW and 1BKG) as templates. We computed a three-dimensional folding model of this plant homodimeric enzyme that has been used to investigate the functional importance of key amino acid residues in its active center. The overall structure of the model is similar to the one described for other AAT enzymes, from eukaryotic and prokaryotic sources, with two equivalent active sites each formed by residues of both subunits of the homodimer. Moreover, PpAAT monomers folded into one large and one small domain. However, PpAAT enzyme showed unique structural and functional characteristics that have been specifically described in the AATs from the prokaryotes Phormidium lapideum and T. thermophilus, such as those involved in the recognition of the substrate side chain or the “open-to-closed” transition following substrate binding. These predicted characteristics have been substantiated by site-direct mutagenesis analyses, and several critical residues (valine-206, serine-207, glutamine-346, glutamate-210, and phenylalanine-450) were identified and functionally characterized. The reported data represent a valuable resource to understand the function of this enzyme in plant amino acid metabolism.

Insights into Polypharmacology from Drug-Domain Associations

MOTIVATION Polypharmacology (the ability of a single drug to affect multiple targets) is a key feature that may explain part of the decreasing success of conventional drug discovery strategies driven by the quest for drugs to act selectively on a single target. Most drug targets are proteins that are composed of domains (their structural and functional building blocks). RESULTS In this work, we model drug-domain networks to explore the role of protein domains as drug targets and to explain drug polypharmacology in terms of the interactions between drugs and protein domains. We find that drugs are organized around a privileged set of druggable domains. CONCLUSIONS Protein domains are a good proxy for drug targets, and drug polypharmacology emerges as a consequence of the multi-domain composition of proteins. CONTACT amoyag@uma.es SUPPLEMENTARY INFORMATION Supplementary data are available at Bioinformatics online.

Structural Perspective on the Direct Inhibition Mechanism of EGCG on Mammalian Histidine Decarboxylase and DOPA Decarboxylase

Histidine decarboxylase (HDC) and l-aromatic amino acid decarboxylase (DDC) are homologous enzymes that are responsible for the synthesis of important neuroactive amines related to inflammatory, neurodegenerative, and neoplastic diseases. Epigallocatechin-3-gallate (EGCG), the most abundant catechin in green tea, has been shown to target histamine-producing cells and to promote anti-inflammatory, antitumor, and antiangiogenic effects. Previous experimental work has demonstrated that EGCG has a direct inhibitory effect on both HDC and DDC. In this study, we investigated the binding modes of EGCG to HDC and DDC as a first step for designing new polyphenol-based HDC/DDC-specific inhibitors.

Substrate uptake and protein stability relationship in mammalian histidine decarboxylase.

There is some evidence linking the substrate entrance in the active site of mammalian histidine decarboxylase and an increased stability against proteolytic degradation. In this work, we study the basis of this relationship by means of protein structure network analysis and molecular dynamics simulations. We find that the substrate binding to the active site influences the conformation of a flexible region sensible to proteolytic degradation and observe how formation of the Michaelis–Menten complex increases stability in the conformation of this region. Proteins 2010.

New structural insights to help in the search for selective inhibitors of mammalian pyridoxal 5’-phosphate-dependent histidine decarboxylase

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