Characterization of the N-acetylneuraminic acid synthase (NeuB) from the psychrophilic fish pathogen Moritella viscosa. (30th January 2015)

• Record Type: Journal Article
• Authors: Berg, Tor Olav; Gurung, Man Kumari; Altermark, Bjørn; Smalås, Arne O.; Ræder, Inger Lin U.
• Is Part Of: Carbohydrate research. Volume 402(2015)
• Page Start: 133
• Page End: 145
• Publication Date: 2015-01-30
• Subjects: Sias sialic acids -- Neu5Ac N-acetylneuraminic acid -- PSA polysialic acid -- LPS lipopolysaccharide -- LOS lipooligosaccharide -- NeuB N-acetylneuraminic acid synthase/sialic acid synthase -- ManNAc N-acetylmannosamine -- PEP phosphoenolpyruvate -- UDP-N-acetylglucosamine uridine 5′-diphosphate-N-acetylglucosamine -- IPTG isopropyl-1-thio-β-galactopyranoside -- TBA thiobarbituric acid -- SDS–PAGE sodium dodecyl sulfate–polyacrylamide gel electrophoresis -- β-ME β-mercaptoethanol -- DSC differential scanning calorimetry -- PSIPRED Psi-blast based secondary structure prediction -- DMB-HPLC 1, 2-diamino-4, 5-methylenedioxybenzene high-performance liquid chromatography -- MS/MS tandem mass spectrometry -- RT retention time -- +cESI positive ion centroid mode full scan ESI -- +pESI positive ion profile mode full scan ESI -- FTMS Fourier transform mass spectrometry -- MS mass spectrometry -- ITMS ion trap mass spectrometry -- KM the Michaelis constant -- kcat turnover number of an enzyme -- CMP cytidine-5′-monophosphate -- Neu5, 7Ac2 7-O-acetyl-N-acetylneuraminic acid -- Neu neuraminic -- Leg legionaminic -- Pse pseudaminic -- HGT horizontal gene transfer -- GC guanine cytosine -- DNA deoxyribonucleic acid -- NaCl sodium chloride -- dNTP deoxyribonucleotide triphosphate -- EDTA ethylenediaminetetraacetic acid -- PNP purine nucleoside phosphorylase; Moritella viscosa -- Cold active -- Sialic acid synthase -- N-Acetylneuraminic acid synthase -- NeuB; Carbohydrates -- Periodicals; Chemistry, Organic -- Periodicals; Biochemistry -- Periodicals; Carbohydrates -- Periodicals; Chimie organique -- Périodiques; Glucides -- Périodiques; Biochemistry; Carbohydrates; Chemistry, Organic; Periodicals; Electronic journals; 507.78
• DOI: 10.1016/j.carres.2014.10.007
• Languages: English
• ISSNs: 0008-6215

Abstract

Graphical abstract: Highlights: The psychrophilic fish pathogenic bacterium Moritella viscosa produces sialic acids. Presence of sialic acids among fish pathogens points toward an important function. The N -acetylneuraminic acid synthase NeuB from M. viscosa was characterized. The enzyme shows typical cold adapted features. This efficient enzyme can be further explored for production of sialic acids and derivates. Abstract: Moritella viscosa is a Gram-negative psychrophilic bacterium that causes winter ulcer disease in Atlantic salmon and cod. Its genome reveals that it possesses the ability to synthesize sialic acids. Indeed, sialic acid can be isolated from the bacterium and when analyzed using HPLC-MS/MS, the presence of N -acetylneuraminic acid was confirmed. Thus, the N -acetylneuraminic acid synthase NeuB from M. viscosa (MvNeuB) was recombinantly produced and characterized. The optimum pH and temperature for MvNeuB activity are 7.5 and 30 °C, respectively. The K M for N -acetylmannosamine and phosphoenolpyruvate is 18 ± 5 and 0.8 ± 0.2 mM, respectively. The k cat value (∼225 min −1 ) for both N -acetylmannosamine and phosphoenolpyruvate is the highest turnover number found for an enzyme in this class until the date. A calorimetric study of MvNeuB shows that the enzyme has a two-step transition peak probably reflecting the two domains these proteins consist of. MvNeuB is less stable at higher temperature and has a high catalytic activity at lower temperature compared to mesophilic counterparts. Enzymes from psychrophilic organisms are generally cold adapted meaning they can maintain adequate function near the freezing point of water. Cold adapted enzymes are catalytically more efficient at lower temperature and are more thermo-labile compared to their mesophilic counterparts. MvNeuB is a typical cold adapted enzyme and could be further explored for production of sialic acids and derivates at low temperatures.