title: Gene cloning and biochemical characterization of 4-N-trimethylaminobutyraldehyde dehydrogenase II from Pseudomonas sp. 13CM.
abstract: The gene encoding 4-N-trimethylaminobutyraldehyde dehydrogenase (TMABaldehyde-DH) from Pseudomonas sp. 13CM, responsible for the conversion of 4-N-trimethylaminobutyraldehyde (TMABaldehyde) to γ-butyrobetaine in the carnitine biosynthesis pathway, isolated by shotgun cloning and expressed in Escherichia coli DH5α. The recombinant TMABaldehyde-DH was purified 19.5 fold to apparent homogeneity by hydrophobic and affinity chromatography and biochemically characterized. The enzyme was found to be a trimer with identical 52 kDa subunits. The isoelectric point was found to be 4.5. Optimum pH and temperature were found respectively as pH 9.5 and 40 °C. The Km values for TMABaldehyde, 4-dimethylaminobutyraldehyde, and NAD+ were respectively, 0.31, 0.62, and 1.16 mM. The molecular and catalytic properties differed from those of TMABaldehyde-DH I, which was discovered initially in Pseudomonas sp. 13CM. The new enzyme, designated TMABaldehyde-DH II, structural gene was inserted into an expression vector pET24b (+) and over-expressed in E. coli BL21 (DE3) under the control of a T7 promoter. The recombinant TMABaldehyde-DH from Pseudomonas sp. 13CM can now be obtained in large quantity necessary for further biochemical characterization and applications.
authors.affiliation: United Graduate School of Agricultural Sciences, Tottori University, Tottori 680-8550, Japan.
authors.forename: Md Rezaul
authors.initials: MR
authors.lastname: Bari
authors.forename: Maizom
authors.initials: M
authors.lastname: Hassan
authors.forename: Naoki
authors.initials: N
authors.lastname: Akai
authors.forename: Jiro
authors.initials: J
authors.lastname: Arima
authors.forename: Nobuhiro
authors.initials: N
authors.lastname: Mori
mesh_terms: D000445:Aldehyde Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001622:Betaine / Q000031:analogs & derivatives / Q000378:metabolism; D002331:Carnitine / Q000378:metabolism; D002853:Chromatography, Liquid; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D055503:Protein Multimerization; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013696:Temperature
chemical_list: D004269:DNA, Bacterial; D011994:Recombinant Proteins; D009243:NAD; D001622:Betaine; C002889:gamma-butyrobetaine; C023095:4-N-trimethylaminobutyraldehyde dehydrogenase; D000445:Aldehyde Oxidoreductases; D002331:Carnitine