Sola-Carvajal A.,University of Murcia |
Gil-Ortiz F.,Institute Biomedicina Of Valencia Consejo Superior Of Investigaciones Cientificas |
Gil-Ortiz F.,ALBA Synchrotron Light Facility |
Garcia-Carmona F.,University of Murcia |
And 2 more authors.
Biochemical Journal | Year: 2014
NAMDH (N-acetyl-D-mannosamine dehydrogenase), from the soil bacteroidete Flavobacterium sp. 141-8, catalyses a rare NAD+ -dependent oxidation of ManNAc (N-acetyl-Dmannosamine) into N-acetylmannosamino-lactone, which spontaneously hydrolyses into N-acetylmannosaminic acid. NAMDH belongs to the SDR (short-chain dehydrogenase/reductase) superfamily and is the only NAMDH characterized to date. Thorough functional, stability, site-directed mutagenesis and crystallographic studies have been carried out to understand better the structural and biochemical aspects of this unique enzyme. NAMDH exhibited a remarkable alkaline pH optimum (pH 9.4) with a high thermal stability in glycine buffer (Tm =64°C) and a strict selectivity towards ManNAc and NAD+ . Crystal structures of ligand-free and ManNAc- and NAD + -bound enzyme forms revealed a compact homotetramer having point 222 symmetry, formed by subunits presenting the characteristic SDR α3β7α3 sandwich fold. A highly developed C-terminal tail used as a latch connecting nearby subunits stabilizes the tetramer. A dense network of polar interactions with the substrate including the encasement of its acetamido group in a specific binding pocket and the hydrogen binding of the sugar 4OH atom ensure specificity for ManNAc. The NAMDH-substrate complexes and site-directed mutagenesis studies identify the catalytic tetrad and provide useful traits for identifying new NAMDH sequences. © 2014 Biochemical Society.
Serrano E.,University of Zaragoza |
Martinez A.B.,University of Murcia |
Martinez A.B.,Murcia Biomedical Research Institute IMIB Arrixaca |
Arruga D.,University of Zaragoza |
And 5 more authors.
PLoS ONE | Year: 2015
Seminal plasma (SP) proteins support the survival of spermatozoa acting not only at the plasma membrane but also by inhibition of capacitation, resulting in higher fertilizing ability. Among SP proteins, BSP (binder of sperm) proteins are the most studied, since they may be useful for the improvement of semen diluents, storage and subsequent fertilization results. However, an updated and detailed phylogenetic analysis of the BSP protein superfamily has not been carried out with all the sequences described in the main databases. The update view shows for the first time an equally distributed number of sequences between the three families: BSP, and their homologs 1 (BSPH1) and 2 (BSPH2). The BSP family is divided in four subfamilies, BSP1 subfamily being the predominant, followed by subfamilies BSP3, BSP5 and BSP2. BSPH proteins were found among placental mammals (Eutheria) belonging to the orders Proboscidea, Primates, Lagomorpha, Rodentia, Chiroptera, Perissodactyla and Cetartiodactyla. However, BSPH2 proteins were also found in the Scandentia order and Metatheria clade. This phylogenetic analysis, when combined with a gene context analysis, showed a completely new evolutionary scenario for the BSP superfamily of proteins with three defined different gene patterns, one for BSPs, one for BSPH1/ BSPH2/ELSPBP1 and another one for BSPH1/BSPH2 without ELSPBP1. In addition, the study has permitted to define concise conserved blocks for each family (BSP, BSPH1 and BSPH2), which could be used for a more reliable assignment for the incoming sequences, for data curation of current databases, and for cloning new BSPs, as the one described in this paper, ram seminal vesicle 20 kDa protein (RSVP20, Ovis aries BSP5b). © 2015 Serrano et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Garcia Garcia M.I.,University of Murcia |
Garcia Garcia M.I.,Murcia Biomedical Research Institute IMIB Arrixaca |
Lau K.,Griffith University |
Von Itzstein M.,Griffith University |
And 4 more authors.
Glycobiology | Year: 2015
N-Acetylneuraminate lyase synthase (NeuB; E.C. 188.8.131.52) is a key enzyme in pathogenic microorganisms for producing N-acetylneuraminic acid through the irreversible condensation of N-acetylmannosamine (ManNAc) and phosphoenolpyruvate (PEP). However, nothing is known about this enzyme in non-pathogenic bacteria. This paper describes, for the first time, one of the two putative N-acetylneuraminate synthases from the halophilic non-pathogenic gamma-proteobacterium Idiomarina loihiensis NeuB1 (IlNeuB1). The obtained 95-kDa dimeric enzyme showed maximal activity at pH 7.0 and 40°C and was more stable at pH 8.0 (8 h half-life) than the previously described NeuB. Its catalytic efficiency towards ManNAc and PEP was 10- and 40-fold higher, respectively, than that determined for Campylobacter jejuni NeuB, but only half that found for Neisseria meningitidis NeuB towards PEP. The phylogenetic and structural analyses of NeuB enzymes revealed the new domain architecture 4 has no cystathionine-β-synthase domain (cystathionine-β-synthetase domain), unlike domain architecture 3. In addition, 10 conserved blocks (I-X) were found, and surprisingly, this study showed that the arginine essential for catalysis that is present in antifreeze-like domain (block X) was not fully conserved in NeuB, but is replaced by a serine in a long sequence (>700 residues) NeuB, such as that existing in domain architectures 3 and 4. © 2014 The Author 2014. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: firstname.lastname@example.org.