New Insights into the Phylogeny and Molecular Classification of Nicotinamide Mononucleotide Deamidases

Nicotinamide mononucleotide (NMN) deamidase is one of the key enzymes of the bacterial pyridine nucleotide cycle (PNC). It catalyzes the conversion of NMN to nicotinic acid mononucleotide, which is later converted to NAD+ by entering the Preiss-Handler pathway. However, very few biochemical data are available regarding this enzyme. This paper represents the first complete molecular characterization of a novel NMN deamidase from the halotolerant and alkaliphilic bacterium Oceanobacillus iheyensis (OiPncC). The enzyme was active over a broad pH range, with an optimum at pH 7.4, whilst maintaining 90 % activity at pH 10.0. Surprisingly, the enzyme was quite stable at such basic pH, maintaining 61 % activity after 21 days. As regard temperature, it had an optimum at 65 °C but its stability was better below 50 °C. OiPncC was a Michaelian enzyme towards its only substrate NMN, with a Km value of 0.18 mM and a kcat/Km of 2.1 mM-1 s-1. To further our understanding of these enzymes, a complete phylogenetic and structural analysis was carried out taking into account the two Pfam domains usually associated with them (MocF and CinA). This analysis sheds light on the evolution of NMN deamidases, and enables the classification of NMN deamidases into 12 different subgroups, pointing to a novel domain architecture never before described. Using a Logo representation, conserved blocks were determined, providing new insights on the crucial residues involved in the binding and catalysis of both CinA and MocF domains. The analysis of these conserved blocks within new protein sequences could permit the more efficient data curation of incoming NMN deamidases

Caracterizacion molecular de N-acetil-D-glucosamina 2-epimerasa de "bacteroides ovatus" : una enzima relevante en la síntesis de ácido siálico = Molecular characterization of N-acetyl-D-glucosamine 2-epimerase from "bacteroides ovatus" : a relevat enzyme in the sialic acid synthesis / Agustín Sola Carvajal; directores, Alvaro Sánchez Ferrer, Francisco García Carmona.

Texto en inglés, resumen en español.Tesis-Universidad de Murcia.Consulte la tesis en: BCA. GENERAL. ARCHIVO UNIVERSITARIO. TM 4341

Insights into the evolution of sorbitol metabolism: phylogenetic analysis of SDR196C family

<p>Abstract</p> <p>Background</p> <p>Short chain dehydrogenases/reductases (SDR) are NAD(P)(H)-dependent oxidoreductases with a highly conserved 3D structure and of an early origin, which has allowed them to diverge into several families and enzymatic activities. The SDR196C family (<url>http://www.sdr-enzymes.org</url>) groups bacterial sorbitol dehydrogenases (SDH), which are of great industrial interest. In this study, we examine the phylogenetic relationship between the members of this family, and based on the findings and some sequence conserved blocks, a new and a more accurate classification is proposed.</p> <p>Results</p> <p>The distribution of the 66 bacterial SDH species analyzed was limited to Gram-negative bacteria. Six different bacterial families were found, encompassing α-, β- and γ-proteobacteria. This broad distribution in terms of bacteria and niches agrees with that of SDR, which are found in all forms of life. A cluster analysis of sorbitol dehydrogenase revealed different types of gene organization, although with a common pattern in which the SDH gene is surrounded by sugar ABC transporter proteins, another SDR, a kinase, and several gene regulators.</p> <p>According to the obtained trees, six different lineages and three sublineages can be discerned. The phylogenetic analysis also suggested two different origins for SDH in β-proteobacteria and four origins for γ-proteobacteria.</p> <p>Finally, this subdivision was further confirmed by the differences observed in the sequence of the conserved blocks described for SDR and some specific blocks of SDH, and by a functional divergence analysis, which made it possible to establish new consensus sequences and specific fingerprints for the lineages and sub lineages.</p> <p>Conclusion</p> <p>SDH distribution agrees with that observed for SDR, indicating the importance of the polyol metabolism, as an alternative source of carbon and energy. The phylogenetic analysis pointed to six clearly defined lineages and three sub lineages, and great variability in the origin of this gene, despite its well conserved 3D structure. This suggests that SDH are very old and emerged early during the evolution. This study also opens up a new and more accurate classification of SDR196C family, introducing two numbers at the end of the family name, which indicate the lineage and the sublineage of each member, i.e, SDR196C6.3.</p

Overexpression of Lamin B Receptor Results in Impaired Skin Differentiation

<div><p>Hutchinson-Gilford progeria syndrome (HGPS) is a rare segmental progeroid disorder commonly caused by a point mutation in the <i>LMNA</i> gene that results in the increased activation of an intra-exonic splice site and the production of a truncated lamin A protein, named progerin. In our previous work, induced murine epidermal expression of this specific HGPS <i>LMNA</i> mutation showed impaired keratinocyte differentiation and upregulated lamin B receptor (LBR) expression in suprabasal keratinocytes. Here, we have developed a novel transgenic animal model with induced overexpression of LBR in the interfollicular epidermis. LBR overexpression resulted in epidermal hypoplasia, along with the downregulation and mislocalization of keratin 10, suggesting impaired keratinocyte differentiation. Increased LBR expression in basal and suprabasal cells did not coincide with increased proliferation. Similar to our previous report of HGPS mice, analyses of γH2AX, a marker of DNA double-strand breaks, revealed an increased number of keratinocytes with multiple foci in LBR-overexpressing mice compared with wild-type mice. In addition, suprabasal LBR-positive cells showed densely condensed and peripherally localized chromatin. Our results show a moderate skin differentiation phenotype, which indicates that upregulation of LBR is not the sole contributor to the HGPS phenotype.</p></div

Increased DNA damage-related foci and peripheral DNA localization is associated with LBR overexpression.

<p>A-B. Primary keratinocytes from week-13 K5+/LBR+ (a) and K5-/LBR- mice were analyzed for the frequency of γH2AX foci (green), LBR (red) and DAPI (blue). C-F. The intensity profile of DAPI staining in suprabasal cells of the paw skin was examined in week-8 K5+/LBR+ mice. Cells that did not express LBR had a more homogenous intensity (C-D), whereas increased intensity of DAPI staining was seen at the nuclear periphery in cells that expressed LBR (E-F). D and F. The graph indicates fluorescence intensity of DAPI staining along the arrow. The histograms show the measured DAPI intensity. LBR (red). G. Quantification of the frequency of DNA distribution in LBR expressing (LBR high) and non-expressing (LBR low) cells of the suprabasal keratinocytes. Scale bar indicates 20 μm. *<i>p</i><0.05, ** <i>p</i><0.01.</p

Increased S100A9 expression in K5+/LBR+ keratinocytes.

<p>A-G. Relative qPCR analysis of keratinocytes from week-13 K5-/LBR- (blue) and K5+/LBR+ (red) mice (<i>n</i> = 3 per group). (H) Analysis of keratinocyte proliferation, using Ki67 (red), and DNA-stained nuclei (blue) in paw epidermis of week-8 K5-/LBR- (blue) and K5+/LBR+ (red), (<i>n</i> = 3 per group). (I) No significant differences were observed between K5-/LBR- (blue) and K5+/LBR+ mice (red), (<i>n</i> = 3 per group). Error bars indicate SEM (* <i>p</i><0.05). Scale bar indicates 20 μm.</p

Overexpression of LBR results in hypoplasia of paw epidermis.

<p>A-I. Immunofluorescence of plantar paw with LBR (red), keratin 5 (green), and DNA (blue) in 8-week-old K5-/LBR- (A-C) and K5+/LBR+ mice (D-F). Normal expression of LBR in the basal cell layer and a few suprabasal cells (B). LBR overexpression and upregulated keratin 5 expression in basal and suprabasal keratinocytes (G-I). J-L. Measurement of the thickness of the paw epidermis in week-8 K5-/LBR- (blue) and K5+/LBR+ (red) mice. Dashed lines indicate region for measurement of epidermal thickness (basal cell layer to stratum granulosum) in K5-/LBR- (K) and K5+/LBR+ (L) mice, with at least 5 measurements per sample. Error bars indicate SEM (* <i>p</i><0.05). Scale bars indicate 20 μm (C, F) and 10 μm (I).</p

LBR overexpression results in keratin 5 upregulation and keratin 10 downregulation.

<p>A-B. Markers for epidermal differentiation (green) and DNA (blue), in dorsal skin from PD4 and week 8, and paw skin from week 8. Keratin 5 (K5) showed positive cells in the basal layer of the interfollicular epidermis; however, in K5+/LBR+ mice, K5 was also upregulated in cells of the suprabasal layer (A). Keratin 1 (K1) was found in the suprabasal layer, mainly in the spinous layer, while keratin 10 (K10) was located mainly in the suprabasal layer. In the K5-/LBR- mice, some cells of the basal layer showed K10, which was not evident in the K5+/LBR+ mice (B). Keratin 6 (K6). The dashed line marks the basal cell layer. C. A representative Western blot including 8-week-old K5-/LBR- (<i>n</i> = 4) and K5+/LBR+ (<i>n</i> = 3) protein extracts from dorsal skin. Keratin 10 protein expression was quantified by Western blot densitometry in K5-/LBR- (blue bars) and K5+/LBR+ (red bars) mice. Numbers and lines indicate molecular weight markers (kDa). D. Loricin and Filagrin (green) expression in the granular layer of paw skin from 8 weeks old mice. Error bars indicate SEM (* <i>p</i><0.05). Scale bars indicate 20 μm.</p

No changes in lamin A/C expression or signs of senescence upon LBR overexpression.

<p>A. A representative Western blot including 8-week-old K5-/LBR- (<i>n</i> = 3) and K5+/LBR+ (<i>n</i> = 3) protein extracts from dorsal skin. B. Quantification of lamin A/C from Western blots of protein extracts from dorsal skin of week-8 K5-/LBR- (blue) and K5+/LBR+ (red) mice. Numbers and lines indicate molecular weight markers (kDa). C. Relative qPCR of lamin A in primary keratinocytes in week-13 dorsal skin from K5-/LBR- (blue) and K5+/LBR+ (red) mice. D-I. Lamin A/C immunofluorescence (green) was positive in the basal layer and the terminally differentiated cells from the suprabasal layer. J. Lamin B1 expression by qPCR in primary keratinocytes from week-13 K5-/LBR- (blue) and K5+/LBR+ (red) mice. K-L. P16 (green) was assessed by immunofluorescence in keratinocytes extracted from week-13 dorsal skin. DAPI (blue). Scale bars indicate 20 μm (D-E, G-H) and 50 μm (F, I, K-L).</p

Molecular Characterization of a Novel N-Acetylneuraminate Lyase from Lactobacillus plantarum WCFS1 ▿ †

N-Acetylneuraminate lyases (NALs) or sialic acid aldolases catalyze the reversible aldol cleavage of N-acetylneuraminic acid (Neu5Ac) to form pyruvate and N-acetyl-d-mannosamine (ManNAc). In nature, N-acetylneuraminate lyase occurs mainly in pathogens. However, this paper describes how an N-acetylneuraminate lyase was cloned from the human gut commensal Lactobacillus plantarum WCFS1 (LpNAL), overexpressed, purified, and characterized for the first time. This novel enzyme, which reaches a high expression level (215 mg liter−1 culture), shows similar catalytic efficiency to the best NALs previously described. This homotetrameric enzyme (132 kDa) also shows high stability and activity at alkaline pH (pH > 9) and good temperature stability (60 to 70°C), this last feature being further improved by the presence of stabilizing additives. These characteristics make LpNAL a promising biocatalyst. When its sequence was compared with that of other, related (real and putative) NALs described in the databases, it was seen that NAL enzymes could be divided into four structural groups and three subgroups. The relation of these subgroups with human and other mammalian NALs is also discussed