title: New N-acyl-D-glucosamine 2-epimerases from cyanobacteria with high activity in the absence of ATP and low inhibition by pyruvate.
abstract: N-Acetylneuraminic acid, an important component of glycoconjugates with various biological functions, can be produced from N-acetyl-D-glucosamine (GlcNAc) and pyruvate using a one-pot, two-enzyme system consisting of N-acyl-D-glucosamine 2-epimerase (AGE) and N-acetylneuraminate lyase (NAL). In this system, the epimerase catalyzes the conversion of GlcNAc into N-acetyl-D-mannosamine (ManNAc). However, all currently known AGEs have one or more disadvantages, such as a low specific activity, substantial inhibition by pyruvate and strong dependence on allosteric activation by ATP. Therefore, four novel AGEs from the cyanobacteria Acaryochloris marina MBIC 11017, Anabaena variabilis ATCC 29413, Nostoc sp. PCC 7120, and Nostoc punctiforme PCC 73102 were characterized. Among these enzymes, the AGE from the Anabaena strain showed the most beneficial characteristics. It had a high specific activity of 117±2 U mg(-1) at 37 °C (pH 7.5) and an up to 10-fold higher inhibition constant for pyruvate as compared to other AGEs indicating a much weaker inhibitory effect. The investigation of the influence of ATP revealed that the nucleotide has a more pronounced effect on the Km for the substrate than on the enzyme activity. At high substrate concentrations (≥200 mM) and without ATP, the enzyme reached up to 32% of the activity measured with ATP in excess.
authors.affiliation: Institute of Biochemical Engineering, Technische Universität München, Boltzmannstr. 15, 85748 Garching, Germany.
authors.forename: Ludwig
authors.initials: L
authors.lastname: Klermund
authors.forename: Anna
authors.initials: A
authors.lastname: Groher
authors.forename: Kathrin
authors.initials: K
authors.lastname: Castiglione
mesh_terms: D000117:Acetylglucosamine / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism; D002238:Carbohydrate Epimerases / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D003001:Cloning, Molecular; D000458:Cyanobacteria / Q000145:classification / Q000201:enzymology* / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D006595:Hexosamines / Q000378:metabolism; D019289:Pyruvic Acid / Q000494:pharmacology*; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D004269:DNA, Bacterial; D006595:Hexosamines; D019289:Pyruvic Acid; D000255:Adenosine Triphosphate; D002238:Carbohydrate Epimerases; C024519:N-acyl-D-glucosamine 2-epimerase; D000117:Acetylglucosamine; C002022:N-acetylmannosamine
keywords: AGE; Epimerization; GlcNAc; ManNAc; N-Acetyl-d-mannosamine; N-Acetylneuraminic acid; N-Acyl-d-glucosamine 2-epimerase; N-acetyl-d-glucosamine; N-acetyl-d-mannosamine; N-acetylneuraminate lyase; N-acetylneuraminic acid; N-acyl-d-glucosamine 2-epimerase; NAL; Neu5Ac; Sialic acid; TCA; trichloroacetic acid