Overproduction of spinach betaine aldehyde dehydrogenase in Escherichia coli. Structural and functional properties of wild-type, mutants and E. coli enzymes.
Betaine aldehyde dehydrogenase (BADH) catalyzes the last step in the synthesis of the osmoprotectant glycine betaine from choline. Although betaine aldehyde has been thought to be a specific substrate for BADH, recent studies have shown that human and sugar beet BADHs also catalyze the oxidation of omega-aminoaldehydes. To characterize the kinetic and stability properties of spinach BADH, five kinds of expression vectors encoding full length, mature, E103Q, E103K, and chimera BADHs were constructed. These enzymes together with Escherichia coli BADH were expressed in E. coli and purified. The affinities for betaine aldehyde were similar in the spinach and E. coli BADHs, whereas those for omega-aminoaldehydes were higher in spinach BADH than in E. coli BADH. A chimera BADH in which part of the Rossmann type fold in the spinach BADH was replaced with that of E. coli BADH, showed properties which resembled spinach BADH more than E. coli BADH. The spinach E103K mutant was almost inactive, whereas the E103Q mutant showed a similar activity for the oxidation of betaine aldehyde to that of wild type BADH, but a lower affinity for omega-aminoaldehydes. All spinach BADHs were dimers whereas E. coli BADH was a tetramer. E. coli BADH was more stable at high temperature than spinach BADHs. The E103Q mutant was most labile to high temperature. These properties are discussed in relation to the structure of spinach BADH.
Incharoensakdi
A
A
Research Institute of Meijo University, Tenpaku-ku, Nagoya, Aichi, Japan.
Matsuda
N
N
Hibino
T
T
Meng
Y L
YL
Ishikawa
H
H
Hara
A
A
Funaguma
T
T
Takabe
T
T
Takabe
T
T
D000445:Aldehyde Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D050728:Betaine-Aldehyde Dehydrogenase; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D006358:Hot Temperature; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D018724:Spinacia oleracea / Q000201:enzymology*
D000445:Aldehyde Oxidoreductases; D050728:Betaine-Aldehyde Dehydrogenase