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 synthe sis of the osmoprotectant glycine betaine from choline. Although betaine al dehyde 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, ma ture, E103Q, E103K, and chimera BADHs were constructed. These enzymes toget her with Escherichia coli BADH were expressed in E. coli and purified. The affinities for betaine aldehyde were similar in the spinach and E. coli BAD Hs, whereas those for omega -aminoaldehydes were higher in spinach BADH tha n in E. coli BADH. A chimera BADH in which part of the Rossmann type fold i n the spinach BADH was replaced with that of E. coli BADH, showed propertie s which resembled spinach BADH more than E. coli BADH. The spinach E103K mu tant was almost inactive, whereas the E103Q mutant showed a similar activit y for the oxidation of betaine aldehyde to that of wild type BADH, but a lo wer affinity for omega -aminoaldehydes. All spinach BADHs were dimers where as E. coli BADH was a tetramer. E. coli BADH was more stable at high temper ature than spinach BADHs. The E103Q mutant was most labile to high temperat ure. These properties are discussed in relation to the structure of spinach BADH.