Rapid Purification and Properties of Betaine Aldehyde Dehydrogenase fromPseudomonas aeruginosa

Abstract

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.

Structural and functional analysis of betaine aldehyde dehydrogenase fromStaphylococcus aureus

“…6 shows binding sites for Na + , K + and Mg 2+ at the dimerization interface of the protein and in the vicinity of the NAD-binding site. Binding of cations at similar sites have been reported previously (Muñ oz-Clares et al, 2010) and were shown to activate BADH/ALDH through facilitation of NAD(P) + association and to affect the tertiary and quaternary structures of the enzymes (Falkenberg & Strøm, 1990;Valenzuela-Soto & Muñ oz-Clares, 1994;Muñ oz-Clares et al, 2010;Velasco-García et al, 1999;Valenzuela-Soto et al, 2003;Gonzá lez-Segura et al, 2013;Garza-Ramos et al, 2013). Mg 2+ (Fig.…”

“…SaBADH was strongly inhibited by BA at concentrations of higher than 0.15 mM (Fig. 1d, Table 3), similarly to other BADHs (Velasco-García et al, 1999Falkenberg & Strøm, 1990;Valenzuela-Soto & Muñ oz-Clares, 1994;Boch et al, 1997). Detailed kinetic and inhibition studies of SaBADH have been published elsewhere (Chen et al, 2014).…”

Exploration of intraclonal adaptation mechanisms of Pseudomonas brassicacearum facing cadmium toxicity

“…Induction was noticed for the periplasmic components of ATP-binding proteins of ABC-type transporters involved in the transport of sugar (35B11), spermine/putrescine (7F12) and proline/glycine-betaine (83G4 and 1H11) ( Table 1 ). In general, Gram-negative bacteria achieve high intracellular concentrations of proline and glycine-betaine during osmotic or metal stress through increased transport ( Csonka, 1989 ; Velasco-Garcia et al ., 1999 ; Caldas et al ., 1999 ; Sharma and Dietz, 2006 ). Our results, underlying common pathways between heavy metal and osmotic stresses, are in accordance with a recent study on E. coli 's response to Cd toxicity ( Wang and Crowley, 2005 ).…”

Glycinebetaine Accumulation, Physiological Characterizations and Growth Efficiency in Salt‐tolerant and Salt‐sensitive Lines of Indica Rice (Oryza sativaL. ssp.indica) in Response to Salt Stress

“…Each determination was performed at least twice. One unit of activity is defined as the amount of enzyme that catalyses the formation of 1 μ mol of NADH per minute in standard assay (Velasco‐Garcia et al. 1999).…”