A two-component protease in Methylorubrum extorquens with high activity toward the peptide precursor of the redox cofactor pyrroloquinoline quinone

Pyrroloquinoline quinone is a prominent redox cofactor in many prokaryotes, produced from a ribosomally synthesized and post-translationally modified peptide PqqA via a pathway comprising four conserved proteins PqqB?E. These four proteins are now fairly well-characterized and span radical SAM activity (PqqE), aided by a peptide chaperone (PqqD), a dual hydroxylase (PqqB), and an eight-electron, eight-proton oxidase (PqqC). A full description of this pathway has been hampered by a lack of information regarding a protease/peptidase required for the excision of an early, cross-linked di-amino acid precursor to pyrroloquinoline quinone. Herein, we isolated and characterized a two-component heterodimer protein from the ?-proteobacterium Methylobacterium (Methylorubrum) extorquens that can rapidly catalyze cleavage of PqqA into smaller peptides. Using pulldown assays, surface plasmon resonance, and isothermal calorimetry, we demonstrated the formation of a complex PqqF/PqqG, with a K-D of 300 nm. We created a molecular model of the heterodimer by comparison with the Sphingomonas sp. A1 M16B Sph2681/Sph2682 protease. Analysis of time-dependent patterns for the appearance of proteolysis products indicates high specificity of PqqF/PqqG for serine side chains. We hypothesize that PqqF/PqqG initially cleaves between the PqqE/PqqD-generated cross-linked form of PqqA, with nonspecific cellular proteases completing the release of a suitable substrate for the downstream enzyme PqqB. The finding of a protease that specifically targets serine side chains is rare, and we propose that this activity may be useful in proteomic analyses of the large family of proteins that have undergone post-translational phosphorylation at serine.National Institutes of HealthUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA [GM118117, GM124002, 1S10OD020062-01

The Role of Protein Crystallography in Defining the Mechanisms of Biogenesis and Catalysis in Copper Amine Oxidase

Copper amine oxidases (CAOs) are a ubiquitous group of enzymes that catalyze the conversion of primary amines to aldehydes coupled to the reduction of O2 to H2O2. These enzymes utilize a wide range of substrates from methylamine to polypeptides. Changes in CAO activity are correlated with a variety of human diseases, including diabetes mellitus, Alzheimer’s disease, and inflammatory disorders. CAOs contain a cofactor, 2,4,5-trihydroxyphenylalanine quinone (TPQ), that is required for catalytic activity and synthesized through the post-translational modification of a tyrosine residue within the CAO polypeptide. TPQ generation is a self-processing event only requiring the addition of oxygen and Cu(II) to the apoCAO. Thus, the CAO active site supports two very different reactions: TPQ synthesis, and the two electron oxidation of primary amines. Crystal structures are available from bacterial through to human sources, and have given insight into substrate preference, stereospecificity, and structural changes during biogenesis and catalysis. In particular both these processes have been studied in crystallo through the addition of native substrates. These latter studies enable intermediates during physiological turnover to be directly visualized, and demonstrate the power of this relatively recent development in protein crystallography

Host-parasite co-metabolic activation of antitrypanosomal aminomethyl-benzoxaboroles

<div><p>Recent development of benzoxaborole-based chemistry gave rise to a collection of compounds with great potential in targeting diverse infectious diseases, including human African Trypanosomiasis (HAT), a devastating neglected tropical disease. However, further medicinal development is largely restricted by a lack of insight into mechanism of action (MoA) in pathogenic kinetoplastids. We adopted a multidisciplinary approach, combining a high-throughput forward genetic screen with functional group focused chemical biological, structural biology and biochemical analyses, to tackle the complex MoAs of benzoxaboroles in <i>Trypanosoma brucei</i>. We describe an oxidative enzymatic pathway composed of host semicarbazide-sensitive amine oxidase and a trypanosomal aldehyde dehydrogenase TbALDH3. Two sequential reactions through this pathway serve as the key underlying mechanism for activating a series of 4-aminomethylphenoxy-benzoxaboroles as potent trypanocides; the methylamine parental compounds as pro-drugs are transformed first into intermediate aldehyde metabolites, and further into the carboxylate metabolites as effective forms. Moreover, comparative biochemical and crystallographic analyses elucidated the catalytic specificity of TbALDH3 towards the benzaldehyde benzoxaborole metabolites as xenogeneic substrates. Overall, this work proposes a novel drug activation mechanism dependent on both host and parasite metabolism of primary amine containing molecules, which contributes a new perspective to our understanding of the benzoxaborole MoA, and could be further exploited to improve the therapeutic index of antimicrobial compounds.</p></div

Determination of nutrient salts by automatic methods both in seawater and brackish water: the phosphate blank

9 páginas, 2 tablas, 2 figurasThe main inconvenience in determining nutrients in seawater by automatic methods is simply solved: the preparation of a suitable blank which corrects the effect of the refractive index change on the recorded signal. Two procedures are proposed, one physical (a simple equation to estimate the effect) and the other chemical (removal of the dissolved phosphorus with ferric hydroxide).Support for this work came from CICYT (MAR88-0245 project) and Conselleria de Pesca de la Xunta de GaliciaPeer reviewe