María Inmaculada García-García

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

ABSTRACT 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.

New stabilized FastPrep-CLEAs for sialic acid synthesis.

N-acetyl-D-neuraminic acid aldolase, a key enzyme in the biotechnological production of N-acetyl-D-neuraminic acid (sialic acid) from N-acetyl-D-mannosamine and pyruvate, was immobilized as cross-linked enzyme aggregates (CLEAs) by precipitation with 90% ammonium sulfate and crosslinking with 1% glutaraldehyde. Because dispersion in a reciprocating disruptor (FastPrep) was only able to recover 40% of the activity, improved CLEAs were then prepared by co-aggregation of the enzyme with 10mg/mL bovine serum albumin followed by a sodium borohydride treatment and final disruption by FastPrep (FastPrep-CLEAs). This produced a twofold increase in activity up to 86%, which is a 30% more than that reported for this aldolase in cross-linked inclusion bodies (CLIBs). In addition, these FastPrep-CLEAs presented remarkable biotechnological features for Neu5Ac synthesis, including, good activity and stability at alkaline pHs, a high K(M) for ManNAc (lower for pyruvate) and good operational stability. These results reinforce the practicability of using FastPrep-CLEAs in biocatalysis, thus reducing production costs and favoring reusability.

Biochemical and Mutational Analysis of a Novel Nicotinamidase from Oceanobacillus iheyensis HTE831

Nicotinamidases catalyze the hydrolysis of nicotinamide to nicotinic acid and ammonia, an important reaction in the NAD+ salvage pathway. This paper reports a new nicotinamidase from the deep-sea extremely halotolerant and alkaliphilic Oceanobacillus iheyensis HTE831 (OiNIC). The enzyme was active towards nicotinamide and several analogues, including the prodrug pyrazinamide. The enzyme was more nicotinamidase (kcat/Km = 43.5 mM−1s−1) than pyrazinamidase (kcat/Km = 3.2 mM−1s−1). Mutational analysis was carried out on seven critical amino acids, confirming for the first time the importance of Cys133 and Phe68 residues for increasing pyrazinamidase activity 2.9- and 2.5-fold, respectively. In addition, the change in the fourth residue involved in the ion metal binding (Glu65) was detrimental to pyrazinamidase activity, decreasing it 6-fold. This residue was also involved in a new distinct structural motif DAHXXXDXXHPE described in this paper for Firmicutes nicotinamidases. Phylogenetic analysis revealed that OiNIC is the first nicotinamidase described for the order Bacillales.

Kinetic study of hydroxytyrosol oxidation and its related compounds by Red Globe grape polyphenol oxidase.

Red Globe grape polyphenol oxidase, partially purified using phase partitioning with Triton-X114, was used to study the oxidation of hydroxytytosol (HT) and its related compounds tyrosol (TS), tyrosol acetate (TSA), and hydroxytyrosol acetate (HTA). The enzyme showed activity toward both monophenols (monophenolase activity) and o-diphenols (diphenolase activity) with a pH optimum (pH 6.5) that was independent of the phenol used. However, the optimal temperature for diphenolase activity was substrate-dependent, with a broad optimum of 25-65 °C for HT, compared with the maximum obtained for HTA (40 °C). Monophenolase activity showed the typical lag period, which was modulated by pH, substrate and enzyme concentrations, and the presence of catalytic amounts of o-diphenols. When the catalytic power (Vmax/K(M)) was determined for both activities, higher values were observed for o-diphenols than for monophenols: 9-fold higher for the HT/TS pair and 4-fold higher for HTA/TSA pair. Surprisingly, this ratio was equally higher for TSA (2.2-fold) compared with that of TS, whereas no such effect was observed for o-diphenols. This higher efficiency of TSA could be related to its greater hydrophobicity. Acetyl modification of these phenols not only changes the kinetic parameters of the enzyme but also affects their antioxidant activity (ORAC-FL assays), which is lower in HTA than in HT.

Properties of BoAGE2, a second N-acetyl-D-glucosamine 2-epimerase from Bacteroides ovatus ATCC 8483.

N-Acyl-D-Glucosamine 2-epimerase (AGE) catalyzes the reversible epimerization between N-acetyl-D-mannosamine (ManNAc) and N-acetyl-D-glucosamine (GlcNAc). Bacteroides ovatus ATCC 8483 shows 3 putative genes for AGE activity (BACOVA_00274, BACOVA_01795 and BACOVA_01816). The BACOVA_00274 gene encodes an AGE (BoAGE1) with strong similarity to the AGE previously characterized in Bacteroides fragilis. Interestingly, the BACOVA_01816 gene (BoAGE2) shares 57% identity with Anabaena sp. CH1 AGE, but has an extra 27-amino acid tag sequence in the N-terminal. When cloned and expressed in Escherichia coli Rosetta (DE3)pLys, BACOVA_01816 was able to convert ManNAc into GlcNAc and vice versa. It was stable over a broad range of pHs and its activity was enhanced by ATP (20 μM). The incubation with ATP stabilized its structure, raising its melting temperature by about 8 °C. In addition, the catalytic efficiency for ManNAc synthesis was higher than that for GlcNAc synthesis. These characteristics make BoAGE2 a promising biocatalyst for sialic acid production using cheap GlcNAc as starting material. BoAGE2 could be considered a Renin-binding Protein and its interaction with renin was studied for the first time in a prokaryotic AGE. Surprisingly, renin activated BoAGE2, an effect which is contrary to that described for mammalian AGE and unrelated with the unique N-terminal tag, since a mutant without this tag was also activated by renin. When BoAGE2 sequence was compared with other related (real and putative) AGE described in the databases, it was seen that AGE enzymes can be divided in 3 different groups. The relationship between these groups is also discussed.

Cimetidine disrupts the renewal of testicular cells and the steroidogenesis in a hermaphrodite fish

The importance of histamine in the physiology of the testis in mammals and reptiles has been recently shown. Histamine receptors (Hrs) are well conserved in fish and are functional in several fish species. We report here for the first time that histamine and the mRNA of Hrh1, Hrh2 and Hrh3 are all present in the gonad of the hermaphrodite teleost fish gilthead seabream. Moreover, cimetidine, which acts in vitro as an agonist of Hrh1 and Hrh2 on this species, was intraperitoneally injected in one and two years old gilthead seabream males. After three and five days of cimetidine injection, we found that this compound differently modified the gonadal hrs transcript levels and affects the testicular cell renewal and the gene expression of steroidogenesis-related molecules as well as the serum steroid levels. Our data point to cimetidine as a reproductive disruptor and elucidate a role for histamine in the gonad of this hermaphrodite fish species through Hr signalling.

First Functional and Mutational Analysis of Group 3 N-Acetylneuraminate Lyases from Lactobacillus antri and Lactobacillus sakei 23K

N-acetyl neuraminate lyases (NALs) catalyze the reversible aldol cleavage of N-acetyl neuraminic acid (Neu5Ac) to pyruvate and N-acetyl-D-mannosamine (ManNAc). Previous phylogenetic studies divided NALs into four different groups. Groups 1 and 2 have been well characterized at both kinetic and molecular levels, but no NAL from group 3 has been studied to date. In this work, a functional characterization of two group 3 members was performed using the recombinant NALs from Lactobacillus antri and Lactobacillus sakei 23K, revealing an optimal pH of between 6.0 and 7.0, low stability at basic pHs (>8.0), low optimal temperatures and, especially, low catalytic efficiency compared with their counterparts in group 1 and 2. The mutational analysis carried out showed that a plausible molecular reason for the low activity shown by Lactobacillus antri and Lactobacillus sakei 23k NALs compared with group 1 and 2 NALs could be the relatively small sugar-binding pocket they contain. A functional divergence analysis concluding that group 3 is more closely related to group 2 than to group 1.

Functional assignment of gene AAC16202.1 from Rhodobacter capsulatus SB1003: New insights into the bacterial SDR sorbitol dehydrogenases family

Short-chain dehydrogenases/reductases (SDR) constitute one of the largest enzyme superfamilies with over 60,000 non-redundant sequences in the database, many of which need a correct functional assignment. Among them, the gene AAC16202.1 (NCBI) from Rhodobacter capsulatus SB1003 has been assigned in Uniprot both as a sorbitol dehydrogenase (#D5AUY1) and, as an N-acetyl-d-mannosamine dehydrogenase (#O66112), both enzymes being of biotechnological interest. When the gene was overexpressed in Escherichia coli Rosetta (DE3)pLys, the purified enzyme was not active toward N-acetyl-d-mannosamine, whereas it was active toward d-sorbitol and d-fructose. However, the relative activities toward xylitol and l-iditol (0.45 and 6.9%, respectively) were low compared with that toward d-sorbitol. Thus, the enzyme could be considered sorbitol dehydrogenase (SDH) with very low activity toward xylitol, which could increase its biotechnological interest for determining sorbitol without the unspecific cross-determination of added xylitol in food and pharma compositions. The tetrameric enzyme (120 kDa) showed similar catalytic efficiency (2.2 × 10(3) M(-1) s(-1)) to other sorbitol dehydrogenases for d-sorbitol, with an optimum pH of 9.0 and an optimum temperature of 37 °C. The enzyme was also more thermostable than other reported SDH, ammonium sulfate being the best stabilizer in this respect, increasing the melting temperature (T(m)) up to 52.9 °C. The enzyme can also be considered as a new member of the Zn(2+) independent SDH family since no effect on activity was detected in the presence of divalent cations or chelating agents. Finally, its in silico analysis enabled the specific conserved sequence blocks that are the fingerprints of bacterial sorbitol dehydrogenases and mainly located at C-terminal of the protein, to be determined for the first time. This knowledge will facilitate future data curation of present databases and a better functional assignment of newly described sequences.

An improved method to measure lipase activity in aqueous media

An improved method based on the p-nitrophenyl long chain esters method is proposed for measuring lipase hydrolytic activity in aqueous media. Using ethylene glycol as co-solvent for hydrophobic p-nitrophenyl substrates in aqueous buffer, lipase activity is measured by following the release of p-nitrophenol. This fast and easy to handle method improves the solubility of both substrate and product, and also the stability of the substrate. It avoids the use of solvents such as ethanol or propanol, permits the comparison of all the p-nitrophenol acyl ester substrates and allows the influence of ions like Ca+2 to be studied, while avoiding turbidity in the reaction medium.

Role of 5α-dihydrotestosterone in testicular development of gilthead seabream following finasteride administration.

In teleosts, spermatogenesis is regulated by pituitary gonadotropins and sex steroids. 5α-dihydrotestosterone (DHT), derived from testosterone (T) through the action of 5α-reductase, has recently been suggested to play a physiologically important role in some fish species. In this study, gilthead seabream, Sparus aurata L., males received an implant of 1μgT/g body mass (bm) or vehicle alone and, 7days later, 1mg finasteride (FIN, an inhibitor of 5α-reductase)/kg bm or vehicle. Serum levels of T, 11-ketotestosterone (11KT), DHT and 17β-estradiol (E2), and the mRNA levels of the main enzymes involved in their synthesis, were analysed. T promoted a transient increase in the serum levels of T, 11KT and E2 but a decrease in those of DHT at day 15 following T injection, in accordance with the up-regulation of mRNA levels of the enzymes involved in T transformation to 11KT (coding genes: cyp11b1 and hsd11b) and the down-regulation of mRNA levels of the enzyme responsible for T transformation to DHT (coding gene: srd5a). Interestingly, a similar effect was observed when FIN was injected. However, when fish were injected with T and FIN successively (T+FIN), control levels were not recovered at the end of the experimental period (28days). DHT seems to regulate E2 serum levels via the down-regulation of mRNA levels of aromatase (coding gene: cyp19a1a), which is needed for the transformation of T into E2. The testis histology, together with the proliferative rates recorded upon T, FIN or T+FIN treatment, suggests that DHT is involved in the onset of the meiotic phase of spermatogenesis.