Rosario A. Muñoz-Clares

Purification and Properties of Betaine Aldehyde Dehydrogenase Extracted from Detached Leaves of Amaranthus hypochondriacus L. Subjected to Water Deficit

Summary The activity of the enzyme betaine aldehyde dehydrogenase (BADH, EC 1.2.1.8) from leaves of Amaranthus hypochondriacus L. rises from undetectable levels to 10-3 units/ mg protein after 4 h of treatment with 17% (w/v) polyethyleneglycol to produce a water deficit. This enzyme was purified to apparent homogeneity in only three consecutive steps: fractional precipitation with ammonium sulfate, ion exchange, affinity chromatography on 5′-AMP Sepharose. A specific activity of 2.6 mol/min kg (protein) was obtained. The enzyme has a native molecular mass of 125 kDa, estimated by gel filtration chromatography, a subunit molecular mass of 63 kDa, determined by SDS-PAGE. The reaction is highly specific for betaine aldehyde, which is an inhibitor at high concentrations, but can use NAD+ or NADP+ as nucleotide. The estimated Km values at pH 8.0 and 30 °C for NAD+, NADP+, betaine aldehyde were 80 μM, 2.5 mM, 69 μM respectively. The reaction could not be reversed even at very high glycine betaine concentrations. The optimum pH and temperature were 8.0 and 50 °C, respectively. The pH dependence of the velocity indicated the existence of two ionizable groups of macroscopic pK values of 6.78 ± 0.02 and 9.38 ± 0.01 involved in catalysis and/or binding of the substrates. Chemical modification studies suggested the presence of essential cisteine(s), histidine(s), arginine(s) residues. The enzyme was activated by relatively low concentrations of K+, sucrose, proline, while it was inhibited by NH+4, Na+, high concentrations of glycine betaine. Mg++ up to 150 mM and Ca++ up to 50 mM did not have any effect on the activity.

Kinetic and structural features of betaine aldehyde dehydrogenases: mechanistic and regulatory implications.

The betaine aldehyde dehydrogenases (BADH; EC 1.2.1.8) are so-called because they catalyze the irreversible NAD(P)(+)-dependent oxidation of betaine aldehyde to glycine betaine, which may function as (i) a very efficient osmoprotectant accumulated by both prokaryotic and eukaryotic organisms to cope with osmotic stress, (ii) a metabolic intermediate in the catabolism of choline in some bacteria such as the pathogen Pseudomonas aeruginosa, or (iii) a methyl donor for methionine synthesis. BADH enzymes can also use as substrates aminoaldehydes and other quaternary ammonium and tertiary sulfonium compounds, thereby participating in polyamine catabolism and in the synthesis of gamma-aminobutyrate, carnitine, and 3-dimethylsulfoniopropionate. This review deals with what is known about the kinetics and structural properties of these enzymes, stressing those properties that have only been found in them and not in other aldehyde dehydrogenases, and discussing their mechanistic and regulatory implications.

Amino Acid Residues Critical for the Specificity for Betaine Aldehyde of the Plant ALDH10 Isoenzyme Involved in the Synthesis of Glycine Betaine

Plant Aldehyde Dehydrogenase10 (ALDH10) enzymes catalyze the oxidation of ω-primary or ω-quaternary aminoaldehydes, but, intriguingly, only some of them, such as the spinach (Spinacia oleracea) betaine aldehyde dehydrogenase (SoBADH), efficiently oxidize betaine aldehyde (BAL) forming the osmoprotectant glycine betaine (GB), which confers tolerance to osmotic stress. The crystal structure of SoBADH reported here shows tyrosine (Tyr)-160, tryptophan (Trp)-167, Trp-285, and Trp-456 in an arrangement suitable for cation-π interactions with the trimethylammonium group of BAL. Mutation of these residues to alanine (Ala) resulted in significant Km(BAL) increases and Vmax/Km(BAL) decreases, particularly in the Y160A mutant. Tyr-160 and Trp-456, strictly conserved in plant ALDH10s, form a pocket where the bulky trimethylammonium group binds. This space is reduced in ALDH10s with low BADH activity, because an isoleucine (Ile) pushes the Trp against the Tyr. Those with high BADH activity instead have Ala (Ala-441 in SoBADH) or cysteine, which allow enough room for binding of BAL. Accordingly, the mutation A441I decreased the Vmax/Km(BAL) of SoBADH approximately 200 times, while the mutation A441C had no effect. The kinetics with other ω-aminoaldehydes were not affected in the A441I or A441C mutant, demonstrating that the existence of an Ile in the second sphere of interaction of the aldehyde is critical for discriminating against BAL in some plant ALDH10s. A survey of the known sequences indicates that plants have two ALDH10 isoenzymes: those known to be GB accumulators have a high-BAL-affinity isoenzyme with Ala or cysteine in this critical position, while non GB accumulators have low-BAL-affinity isoenzymes containing Ile. Therefore, BADH activity appears to restrict GB synthesis in non-GB-accumulator plants.

Betaine aldehyde dehydrogenase from Pseudomonas aeruginosa: cloning, over-expression in Escherichia coli, and regulation by choline and salt

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.

Monovalent cations requirements for the stability of betaine aldehyde dehydrogenase from Pseudomonas aeruginosa, porcine kidney and amaranth leaves

Betaine aldehyde dehydrogenase from the human pathogen Pseudomonas aeruginosa requires K(+) ions for maintenance of its active conformation. In order to explore if this property is shared by other BADHs of different origins and to further understand the mechanism underlying the effects of these ions, we carried out a comparative study on the stability and quaternary structure of P. aeruginosa, porcine kidney and amaranth leaves BADHs in the absence of K(+) ions. At low enzyme concentrations, the bacterial and porcine enzymes were totally inactivated upon removal of K(+) following biphasic and monophasic kinetics, respectively, whereas the amaranth enzyme retained its activity. Inactivation of P. aeruginosa BADH was much faster than that of the porcine enzyme. The oxidized coenzyme protected both enzymes against inactivation by the absence of K(+), whereas betaine aldehyde afforded partial protection to the bacterial BADH and increased the inactivation rate of the porcine. Reactivation of the inactive enzymes, by adding back to the incubation medium K(+) ions, was dependent on enzyme concentration, suggesting that enzyme dissociation takes place in the absence of K(+). In the bacterial enzyme, NH(4)(+) but not Na(+) ions could mimic the effects of K(+), whereas the three cations tested reactivated porcine BADH, indicating a requirement of this enzyme for high ionic strength rather than for a specific monovalent cation. Size exclusion chromatography of the inactivated enzymes confirmed that K(+) ions or other monovalent cations are required for the maintenance of the quaternary structure of these two BADHs. At pH 7.0, in the absence of K(+) in a buffer of low ionic strength, the active tetrameric form of P. aeruginosa BADH dissociated into inactive monomers and that of porcine kidney BADH into inactive dimers. Once reactivated, both enzymes reassociated into active tetramers.

Kinetic evidence of the existence of a regulatory phosphoenolpyruvate binding site in maize leaf phosphoenolpyruvate carboxylase

Phenylphosphate, a structural analog of phosphoenolpyruvate (PEP), was found to be an activator of phosphoenolpyruvate carboxylase (PEP carboxylase) purified from maize leaves. This finding suggested the presence in the enzyme of a regulatory site, to which PEP could bind. We carried out kinetic studies on this enzyme using controlled concentrations of free PEP and of Mg-PEP complex and developed a theoretical kinetic model of the reaction. In summary, the main conclusions drawn from our results, and taken as assumptions of the model, were the following: (i) The affinity of the active site for the complex Mg-PEP is much higher than that for free PEP and Mg2+ ions, and therefore it can be considered that the preferential substrate of the PEP-catalyzed reaction is Mg-PEP. (ii) The enzyme has a regulatory site specific for free PEP, to which Mg2+ ions can not bind. (iii) The binding of free PEP, or an analog molecule, to this regulatory site yields a modified enzyme that has much lower apparent Km values and apparent Vmax values than the unmodified enzyme. So, free PEP behaves as an excellent activator of the reaction at subsaturating substrate concentrations, and as an inhibitor at saturating substrate concentrations. These findings may have important physiological implications on the regulation of the PEP carboxylase in vivo activity and, consequently, of the C4 pathway, since increased reaction rates would be obtained when the concentration of PEP rises, even at limiting Mg2+ concentrations.

Thermal inactivation of the plasma membrane H+-ATPase from Kluyveromyces lactis. Protection by trehalose.

The activity of the isolated plasma membrane H+-ATPase from Kluyveromyces lactis was measured during incubation at 35-45 degrees C and in the absence or in the presence of 0-0.6 M trehalose. As the temperature of incubation was raised from 35 to 45 degrees C, increasing enzyme inactivation rates were observed. Thermal inactivation kinetics of the H+-ATPase were biphasic exhibiting a first rapid phase and then a second slow phase. The transition from the native state occurred through a temperature-mediated increase in the inactivation rate constants of both phases. A model is proposed where the native H+-ATPase yields a partially active intermediary during the first phase of inactivation and then the intermediary is slowly converted into a totally inactive enzyme in the second phase. At each of these temperatures trehalose protected the enzymatic activity in a concentration dependent manner. Full protection was observed at 0.6 M trehalose in the range of 35-40 degrees C. Whereas, at 42 and 45 degrees C, the trehalose-mediated thermoprotection of the H+-ATPase was only partial. Trehalose stabilized the enzyme mainly by preventing the temperature dependent increase of the first and second inactivation rate constants.

Crystallographic evidence for active-site dynamics in the hydrolytic aldehyde dehydrogenases. Implications for the deacylation step of the catalyzed reaction

The overall chemical mechanism of the reaction catalyzed by the hydrolytic aldehyde dehydrogenases (ALDHs) involves three main steps: (1) nucleophilic attack of the thiol group of the catalytic cysteine on the carbonyl carbon of the aldehyde substrate; (2) hydride transfer from the tetrahedral thiohemiacetal intermediate to the pyridine ring of NAD(P)(+); and (3) hydrolysis of the resulting thioester intermediate (deacylation). Crystal structures of different ALDHs from several organisms-determined in the absence and presence of bound NAD(P)(+), NAD(P)H, aldehydes, or acid products-showed specific details at the atomic level about the catalytic residues involved in each of the catalytic steps. These structures also showed the conformational flexibility of the nicotinamide half of the cofactor, and of the catalytic cysteinyl and glutamyl residues, the latter being the general base that activates the hydrolytic water molecule in the deacylation step. The architecture of the ALDH active site allows for this conformational flexibility, which, undoubtedly, is crucial for catalysis in these enzymes. Focusing in the deacylation step of the ALDH-catalyzed reaction, here we review and systematize the crystallographic evidence of the structural features responsible for the conformational flexibility of the catalytic glutamyl residue, and for the positioning of the hydrolytic water molecule inside the ALDH active site. Based on the analysis of the available crystallographic data and of energy-minimized models of the thioester reaction intermediate, as well as on the results of theoretical calculations of the pK(a) of the carboxyl group of the catalytic glutamic acid in its three different conformations, we discuss the role that the conformational flexibility of this residue plays in the activation of the hydrolytic water. We also propose a critical participation in the water activation process of the peptide bond to which the catalytic glutamic acid in the intermediate conformation is hydrogen bonded.

Inactivation of betaine aldehyde dehydrogenase from Pseudomonas aeruginosa and Amaranthus hypochondriacus L. leaves by disulfiram.

Betaine aldehyde dehydrogenase (BADH) activity might be crucial for the growth of the human pathogen Pseudomonas aeruginosa under conditions of infection and therefore appears to be a suitable target for antimicrobial agents. As a first step in the search for BADH inhibitors, we have tested the effects of the known aldehyde dehydrogenase inhibitor disulfiram (DSF) on the activity of P. aeruginosa and Amaranthus hypochondriacus (amaranth) leaf BADHs. DSF totally inactivated both enzymes in a time- and dose-dependent manner. In the case of the Pseudomonas enzyme, inactivation kinetics were monophasic with a second-order inactivation rate constant at pH 6.9 of 4.9+/-0.4 M(-1) s(-1), whereas the plant enzyme was inactivated in a biphasic process with second-order inactivation rate constants at pH 7.5 of 6.8+/-0.6 and 0.33+/-0.04 M(-1) s(-1). At pH 8.8, the second-order rate constants for inactivation of the bacterial enzyme was 1 x 10(3) M(-1) s(-1), which compare well with that reported for human liver mitochondrial aldehyde dehydrogenase (ALDH2), the target of DSF inhibition in the aversion therapy of alcoholism. Both BADHs were inactivated faster in the presence of NAD(P)(+) than in its absence, whereas NAD(P)H and betaine aldehyde protected the bacterial, but increased the inactivation rate of the plant enzyme. The inactivated enzymes were reactivated by dithiothreitol, but not by a high concentration of the physiological reductant glutathione. The high in vitro sensitivity of the Pseudomonas BADH to DSF, particularly in the presence of NAD(P)(+), together with the lack of reversibility of DSF modification by glutathione, makes this inhibitor a potential antimicrobial agent and suggests that it might be worth testing its effects and those of its metabolites in vivo, under culture conditions in which the activity of BADH is required for growth of the bacteria.

Trehalose-Mediated Inhibition of the Plasma Membrane H+-ATPase from Kluyveromyces lactis: Dependence on Viscosity and Temperature

The effect of increasing trehalose concentrations on the kinetics of the plasma membrane H+-ATPase from Kluyveromyces lactis was studied at different temperatures. At 20 degrees C, increasing concentrations of trehalose (0.2 to 0.8 M) decreased V(max) and increased S(0.5) (substrate concentration when initial velocity equals 0.5 V(max)), mainly at high trehalose concentrations (0.6 to 0.8 M). The quotient V(max)/S(0.5) decreased from 5.76 micromol of ATP mg of protein(-1) x min(-1) x mM(-1) in the absence of trehalose to 1.63 micromol of ATP mg of protein(-1) x min(-1) x mM(-1) in the presence of 0.8 M trehalose. The decrease in V(max) was linearly dependent on solution viscosity (eta), suggesting that inhibition was due to hindering of protein domain diffusional motion during catalysis and in accordance with Kramers theory for reactions in solution. In this regard, two other viscosity-increasing agents, sucrose and glycerol, behaved similarly, exhibiting the same viscosity-enzyme inhibition correlation predicted. In the absence of trehalose, increasing the temperature up to 40 degrees C resulted in an exponential increase in V(max) and a decrease in enzyme cooperativity (n), while S(0.5) was not modified. As temperature increased, the effect of trehalose on V(max) decreased to become negligible at 40 degrees C, in good correlation with the temperature-mediated decrease in viscosity. The trehalose-mediated increase in S(0.5) was similar at all temperatures tested, and thus, trehalose effects on V(max)/S(0.5) were always observed. Trehalose increased the activation energy for ATP hydrolysis. Trehalose-mediated inhibition of enzymes may explain why yeast rapidly hydrolyzes trehalose when exiting heat shock.