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Human aldehyde oxidase (hAOX1) is a molybdenum dependent enzyme that plays an important role in the metabolism of various compounds either endogenous or xenobiotics. Due to its promiscuity, hAOX1 plays a major role in the pharmacokinetics of many drugs and therefore has gathered a lot of attention from the scientific community and, particularly, from the pharmaceutical industry. In this work, homology modelling, molecular docking and molecular dynamics simulations were used to study the structure of the monomer and dimer of human AOX. The results with the monomer of hAOX1 allowed to shed some light on the role played by thioridazine and two malonate ions that are co-crystalized in the recent X-ray structure of hAOX1. The results show that these molecules endorse several conformational rearrangements in the binding pocket of the enzyme and these changes have an impact in the active site topology as well as in the stability of the substrate (phthalazine). The results show that the presence of both molecules open two gates located at the entrance of the binding pocket, from which results the flooding of the active site. They also endorse several modifications in the shape of the binding pocket (namely the position of Lys893) that, together with the presence of the solvent molecules, favour the release of the substrate to the solvent. Further insights were also obtained with the assembled homodimer of hAOX1. The allosteric inhibitor (THI) binds closely to the region where the dimerization of both monomers occur. These findings suggest that THI can interfere with protein dimerization.

Abstract The knowledge on the binding of vanadium ions and complexes to serum proteins and how vanadium might be transported in blood and up-taken by cells has received much attention during the last decade, particularly as far as the transport of VIVO2+ is concerned. In this work we revise and discuss some relevant aspects of previous research, namely the two main types of binding proposed for transport of VIVO(carrier)2 complexes. New results, obtained by circular dichroism (CD), \{EPR\} and gel electrophoresis, regarding the binding of vanadium to hTF in the oxidation states +5 and +3 are also presented. Namely, evidences for the binding of VV-species to diferric-transferrin, designated by (FeIII)2hTF, as well as to (AlIII)2hTF, are presented and discussed, the possibility of up-take of vanadate by cells through (FeIII)2hTF endocytosis being suggested. It is also confirmed that \{VIII\} binds strongly to hTF, forming di-vanadium(III)-transferrin, designated by (VIII)2hTF, and gel electrophoresis experiments indicate that (VIII)2hTF corresponds to a ‘closed conformation’ similar to (FeIII)2hTF.

Abstract The family 81 glycoside hydrolase (GH81) from Clostridium thermocellum is a β-1,3-glucanase belonging to cellulosomal complex. The gene encoding \{GH81\} from Clostridium thermocellum (CtLam81A) was cloned and expressed displaying a molecular mass of  82 kDa. CtLam81A showed maximum activity against laminarin (100 U/mg), followed by curdlan (65 U/mg), at pH 7.0 and 75 °C. CtLam81A displayed Km, 2.1 ± 0.12 mg/ml and Vmax, 109 ± 1.8 U/mg, against laminarin under optimized conditions. CtLam81A activity was significantly enhanced by Ca2+ or Mg2+ ions. Melting curve analysis of CtLam81A showed an increase in melting temperature from 91 °C to 96 °C by Ca2+ or Mg2+ ions and decreased to 82 °C by EDTA, indicating that Ca2+ and Mg2+ ions may be involved in catalysis and in maintaining structural integrity. \{TLC\} and MALDI-TOF analysis of β-1,3-glucan hydrolysed products released initially, showed β-1,3-glucan-oligosaccharides degree of polymerization (DP) from \{DP2\} to DP7, confirming an endo-mode of action. The catalytically inactive mutant CtLam81A-E515A generated by site-directed mutagenesis was co-crystallized and tetragonal crystals diffracting up to 1.4 Å resolution were obtained. CtLam81A-E515A contained 15 α-helices and 38 β-strands forming a four-domain structure viz. a β-sandwich domain I at N-terminal, an α/β-domain II, an (α/α)6 barrel domain III, and a small 5-stranded β-sandwich domain IV.

Aldehyde oxidase (AOX1) is an enzyme with broad substrate specificity, catalyzing the oxidation of a wide range of endogenous and exogenous aldehydes as well as N-heterocyclic aromatic compounds. In humans, the enzyme’s role in phase I drug metabolism has been established and its importance is now emerging. However, the true physiologic function of AOX1 in mammals is still unknown. Further, numerous single-nucleotide polymorphisms (SNPs) have been identified in human AOX1. SNPs are a major source of interindividual variability in the human population, and SNP-based amino acid exchanges in AOX1 reportedly modulate the catalytic function of the enzyme in either a positive or negative fashion. For the reliable analysis of the effect of amino acid exchanges in human proteins, the existence of reproducible expression systems for the production of active protein in ample amounts for kinetic, spectroscopic, and crystallographic studies is required. In our study we report an optimized expression system for hAOX1 in Escherichia coli using a codon-optimized construct. The codon-optimization resulted in an up to 15-fold increase of protein production and a simplified purification procedure. The optimized expression system was used to study three SNPs that result in amino acid changes C44W, G1269R, and S1271L. In addition, the crystal structure of the S1271L SNP was solved. We demonstrate that the recombinant enzyme can be used for future studies to exploit the role of AOX in drug metabolism, and for the identification and synthesis of new drugs targeting AOX when combined with crystallographic and modeling studies.

Protein crystallization still remains mostly an empirical science, as the production of crystals with the required quality for X-ray analysis is dependent on the intensive screening of the best protein crystallization and crystal’s derivatization conditions. Herein, this demanding step was addressed by the development of a high-throughput and low-budget microfluidic platform consisting of an ion exchange membrane (117 Nafion® membrane) sandwiched between a channel layer (stripping phase compartment) and a wells layer (feed phase compartment) forming 75 independent micro-contactors. This microfluidic device allows for a simultaneous and independent screening of multiple protein crystallization and crystal derivatization conditions, using Hen Egg White Lysozyme (HEWL) as the model protein and Hg2+ as the derivatizing agent. This microdevice offers well-regulated crystallization and subsequent crystal derivatization processes based on the controlled transport of water and ions provided by the 117 Nafion® membrane. Diffusion coefficients of water and the derivatizing agent (Hg2+) were evaluated, showing the positive influence of the protein drop volume on the number of crystals and crystal size. This microfluidic system allowed for crystals with good structural stability and high X-ray diffraction quality and, thus, it is regarded as an efficient tool that may contribute to the enhancement of the proteins’ crystals structural resolution.

AbstractThe current chromatographic approaches used in protein purification are not keeping pace with the increasing biopharmaceutical market demand. With the upstream improvements, the bottleneck shifted towards the downstream process. New approaches rely in Anything But Chromatography methodologies and revisiting former techniques with a bioprocess perspective. Protein crystallization and precipitation methods are already implemented in the downstream process of diverse therapeutic biological macromolecules, overcoming the current chromatographic bottlenecks. Promising work is being developed in order to implement crystallization and precipitation in the purification pipeline of high value therapeutic molecules. This review focuses in the role of these two methodologies in current industrial purification processes, and highlights their potential implementation in the purification pipeline of high value therapeutic molecules, overcoming chromatographic holdups.