Rapid purification and properties of betaine aldehyde dehydrogenase from Pseudomonas aeruginosa

Betaine aldehyde dehydrogenase (BADH) (EC 1.2.1.8) catalyzes the last, irre versible step in the synthesis of the osmoprotectant glycine betaine from c holine, In Pseudomonas aeruginosa this reaction is also an obligatory step in the assimilation of carbon and nitrogen when bacteria are growing in cho line or choline precursors. We present here a method for the rapid purifica tion to homogeneity of this enzyme by the use of ion-exchange and affinity chromatographies on 2',5'-ADP-Sepharose, which results in a high yield of p ure enzyme with a specific activity at 30 degrees C and pH 7.4 of 74.5 U/mg of protein. Analytical ultracentrifugation, gel filtration, chemical cross -linking, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis sug gest that BADH from P. aeruginosa is a homodimer with 61-kDa subunits, The amino acid composition and the N-terminal sequence of 21 amino acid residue s showed significant similarity with those of the enzymes from Xanthomonas translucens and Escherichia coli. Neither BADH activity nor BADH protein wa s found in cell extracts from bacteria grown in the absence of choline, In contrast to other BADHs studied to date, the Pseudomonas enzyme cannot use positively charged aldehydes other than betaine aldehyde as substrates, The oxidation reaction has an activation energy of 39.8 kJ mol(-1). The pH dep endence of the velocity indicated an optimum at PH 8.0 to 8.5 and the exist ence of two ionizable groups with macroscopic pK values of 7.0 +/- 0.1 and 9.7 +/- 0.1 involved in catalysis and/or binding of substrates. The enzyme is inactivated at 40 degrees C, but activity is regained when the heated en zyme is cooled to 30 degrees C or lower. At the optimum pH of 8.0, the enzy me is inactivated by dilution, but it is stable at pH 6.5 even at very low concentrations. Also, P. aeruginosa BADH activity is rapidly lost on remova l of K+, In all cases studied, inactivation involves a biphasic process, wh ich was dependent on the enzyme concentration only in the case of inactivat ion by dilution. NADP(+) considerably protected the enzyme against these in activating conditions.