No abstract

In the human pathogen Pseudomonas aeruginosa, betaine aldehyde dehydrogenase (BADH) may play a dual role assimilating carbon and nitrogen from choline or choline precursors--abundant at infection sites--and producing glycine betaine, which protects the bacteria against the high-osmolarity stress prevalent in the infected tissues. We cloned the P. aeruginosa BADH gene and expressed the BADH protein in Escherichia coli. The recombinant protein appears identical to its native counterpart, as judged by Western blot, N-terminal amino acid sequence, tryptophan-fluorescence emission spectra, circular-dichroism spectroscopy, size-exclusion chromatography, and kinetic properties. Computational analysis indicated that the promoter sequence of the putative operon that includes the BADH gene has a consensus-binding site for the choline-sensing transcription repressor BetI, and putative boxes for ArcA and Lrp transcription factors but no known elements of response to osmotic stress. This is consistent with the strong induction of BADH expression by choline and with the lack of effect of NaCl. As there were significant amounts of BADH protein and activity in P. aeruginosa cells grown on glucose plus choline, as well as the BADH activity exhibiting tolerance to salt, it is likely that glycine betaine is synthesized in vivo and could play an important osmoprotectant role under conditions of infection.

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Betaine aldehyde dehydrogenase (BADH) (EC 1.2.1.8 ) catalyzes the last, irreversible step in the synthesis of the osmoprotectant glycine betaine from choline. In Pseudomonas aeruginosa this reaction is also an obligatory step in the assimilation of carbon and nitrogen when bacteria are growing in choline or choline precursors. We present here a method for the rapid purification 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 pure enzyme with a specific activity at 30°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 suggest 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 residues showed significant similarity with those of the enzymes from Xanthomonas translucens andEscherichia coli. Neither BADH activity nor BADH protein was found in cell extracts from bacteria grown in the absence of choline. In contrast to other BADHs studied to date, thePseudomonas 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 dependence of the velocity indicated an optimum at pH 8.0 to 8.5 and the existence 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°C, but activity is regained when the heated enzyme is cooled to 30°C or lower. At the optimum pH of 8.0, the enzyme 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 removal of K+. In all cases studied, inactivation involves a biphasic process, which was dependent on the enzyme concentration only in the case of inactivation by dilution. NADP+ considerably protected the enzyme against these inactivating conditions.