title: Characterization of a novel N-acetylneuraminic acid lyase favoring industrial N-acetylneuraminic acid synthesis.
abstract: N-Acetylneuraminic acid lyase (NAL, E.C. number 4.1.3.3) is a Class I aldolase that catalyzes the reversible aldol cleavage of N-acetylneuraminic acid (Neu5Ac) from pyruvate and N-acetyl-D-mannosamine (ManNAc). Due to the high Neu5Ac cleavage activity in most isozyme forms, the enzyme catalyzes the rate-limiting step of two biocatalytic reactions producing Neu5Ac in industry. We report the biochemical characterization of a novel NAL from a "GRAS" (General recognized as safe) strain C. glutamicum ATCC 13032 (CgNal). Compared to all previously reported NALs, CgNal exhibited the lowest kcat/Km value for Neu5Ac and highest kcat/Km values for ManNAc and pyruvate, which makes CgNal favor industrial Neu5Ac synthesis process in a non-equilibrium condition. The recombinant CgNal reached the highest expression level (480 mg/L culture), and the highest reported yield of Neu5Ac was achieved (194 g/L, 0.63 M). All these unique properties make CgNal a promising biocatalyst for industrial Neu5Ac biosynthesis. Additionally, although showing the best Neu5Ac synthesis activity among the NAL family, CgNal is more related to dihydrodipicolinate synthase (DHDPS) by phylogenetic analysis. The activities of CgNal towards both NAL's and DHDPS' substrates are fairly high, which indicates CgNal a bi-functional enzyme. The sequence analysis suggests that CgNal might have adopted a unique set of residues for substrates recognition.
authors: 1] State Key Laboratory of Materials-Oriented Chemical Engineering, Nanjing, PR China [2] College of Life Science and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, PR China [3] National Engineering Technique Research Center for Biotechnology, Nanjing, PR China.
authors: Wenyan
authors: W
authors: Ji
authors: Joint Department of Biomedical Engineering, University of North Carolina at Chapel Hill and North Carolina State University, Raleigh, United States.
authors: Wujin
authors: W
authors: Sun
authors: Department of Pathogenic Biology, School of Medicine, Jianghan University, Wuhan, China.
authors: Jinmei
authors: J
authors: Feng
authors: College of Life Science and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, PR China.
authors: Tianshun
authors: T
authors: Song
authors: International Cooperation Division, China National Center for Biotechnology Development, Beijing, PR China.
authors: Dalu
authors: D
authors: Zhang
authors: 1] State Key Laboratory of Materials-Oriented Chemical Engineering, Nanjing, PR China [2] College of Life Science and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, PR China [3] National Engineering Technique Research Center for Biotechnology, Nanjing, PR China.
authors: Pingkai
authors: P
authors: Ouyang
authors: Joint Department of Biomedical Engineering, University of North Carolina at Chapel Hill and North Carolina State University, Raleigh, United States.
authors: Zhen
authors: Z
authors: Gu
authors: 1] State Key Laboratory of Materials-Oriented Chemical Engineering, Nanjing, PR China [2] College of Life Science and Pharmaceutical Engineering, Nanjing Tech University, Nanjing, PR China [3] National Engineering Technique Research Center for Biotechnology, Nanjing, PR China.
authors: Jingjing
authors: J
authors: Xie
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055162:Biocatalysis; D003001:Cloning, Molecular; D048230:Corynebacterium glutamicum / Q000737:chemistry* / Q000145:classification / Q000201:enzymology; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015870:Gene Expression; D006595:Hexosamines / Q000378:metabolism; D006836:Hydro-Lyases / Q000737:chemistry / Q000145:classification / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D019158:N-Acetylneuraminic Acid / Q000096:biosynthesis*; D007652:Oxo-Acid-Lyases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010802:Phylogeny; D019289:Pyruvic Acid / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D006595:Hexosamines; D011994:Recombinant Proteins; D019289:Pyruvic Acid; D007652:Oxo-Acid-Lyases; D006836:Hydro-Lyases; C020040:4-hydroxy-tetrahydrodipicolinate synthase; D019158:N-Acetylneuraminic Acid; C002022:N-acetylmannosamine