title: A sialic acid aldolase from Peptoclostridium difficile NAP08 with 4-hydroxy-2-oxo-pentanoate aldolase activity.
abstract: Sialic acid aldolases (E.C.4.1.3.3) catalyze the reversible aldol cleavage of N-acetyl-d-neuraminic acid (Neu5Ac) to from N-acetyl-d-mannosamine (ManNAc) and pyruvate. In this study, a sialic acid aldolase (PdNAL) from Peptoclostridium difficile NAP08 was expressed in Escherichia coli BL21 (DE3). This homotetrameric enzyme was purified with a specific activity of 18.34U/mg for the cleavage of Neu5Ac. The optimal pH and temperature for aldol addition reaction were 7.4 and 65°C, respectively. PdNAL was quite stable at neutral and alkaline pH (6.0-10.0) and maintained about 89% of the activity after incubation at pH 10.0 for 24h. After incubation at 70°C for 15min, almost no activity loss was observed. The high thermostability simplified the purification of this enzyme. Interestingly, substrate profiling showed that PdNAL not only accepted ManNAc but also short chain aliphatic aldehydes such as acetaldehyde, propionaldehyde and n-butyraldehyde as the substrates. This is the first example that a sialic acid aldolase is active toward aliphatic aldehyde acceptors with two or more carbons. The amino acid sequence analysis indicates that PdNAL belongs to the NAL subfamily rather than 4-hydroxy-2-oxopentanoate (HOPA) aldolase, but it is interesting that the enzyme possesses the activity of HOPA aldolase.
authors.affiliation: National Engineering Laboratory for Industrial Enzymes and Tianjin Engineering Center for Biocatalytic Technology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China; University of Chinese Academy of Sciences, Beijing, China.
authors.forename: Qijia
authors.initials: Q
authors.lastname: Chen
authors.affiliation: National Engineering Laboratory for Industrial Enzymes and Tianjin Engineering Center for Biocatalytic Technology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China; University of Chinese Academy of Sciences, Beijing, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Han
authors.affiliation: National Engineering Laboratory for Industrial Enzymes and Tianjin Engineering Center for Biocatalytic Technology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China.
authors.forename: Xi
authors.initials: X
authors.lastname: Chen
authors.affiliation: National Engineering Laboratory for Industrial Enzymes and Tianjin Engineering Center for Biocatalytic Technology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China.
authors.forename: Yunfeng
authors.initials: Y
authors.lastname: Cui
authors.affiliation: National Engineering Laboratory for Industrial Enzymes and Tianjin Engineering Center for Biocatalytic Technology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China.
authors.forename: Jinhui
authors.initials: J
authors.lastname: Feng
authors.affiliation: National Engineering Laboratory for Industrial Enzymes and Tianjin Engineering Center for Biocatalytic Technology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China.
authors.forename: Qiaqing
authors.initials: Q
authors.lastname: Wu
authors.affiliation: National Engineering Laboratory for Industrial Enzymes and Tianjin Engineering Center for Biocatalytic Technology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China; University of Chinese Academy of Sciences, Beijing, China. Electronic address: zhu_dm@tib.cas.cn.
authors.forename: Dunming
authors.initials: D
authors.lastname: Zhu
mesh_terms: D000446:Aldehyde-Lyases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000068016:Eubacteriales / Q000201:enzymology* / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D007700:Kinetics; D007652:Oxo-Acid-Lyases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; C115453:4-hydroxy-2-ketopentanoic acid aldolase; D000446:Aldehyde-Lyases; D007652:Oxo-Acid-Lyases; C018728:N-acetylneuraminate lyase
keywords: 4-Hydroxy-2-oxopentanoate aldolase; Aldol addition reaction; Peptoclostridium difficile; Sialic acid aldolase