Carlos Mújica-Jiménez

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.

Studies of the allosteric properties of maize leaf phosphoenolpyruvate carboxylase with the phosphoenolpyruvate analog phosphomycin as activator

The antibiotic phosphomycin (1,2-epoxypropylphosphonic acid), an analog of phosphoenolpyruvate (PEP), behaved not as an inhibitor, but as an activator, of the enzyme phosphoenolpyruvate carboxylase (PEPC) from maize leaves. Multiple activation studies indicated that the analog binds to the Glc6P-allosteric site producing a more activated enzyme than Glc6P itself. Because of this, we used phosphomycin as a tool to further extend our understanding of the mechanisms of allosteric regulation of C4-PEPC. Initial velocity data from detailed kinetic studies, in which the concentrations of free and Mg-complexed PEP and phosphomycin were controlled, are consistent with: (1) the true activator is free phosphomycin, which competes with free PEP for the Glc6P-allosteric site; and (2) the Mg-phosphomycin complex caused inhibition by binding to the active site in competition with MgPEP. Therefore, although the Glc6P-allosteric site and the active site are able to bind the same ligands, they differ in the form of substrate and activator they bind. This important difference allows the full expression of the potential of activation and prevents inhibition by the activators, including the physiological ones, which are mostly uncomplexed at physiological free Mg2+ concentrations. At fixed low substrate concentrations, the saturation kinetics of the enzyme by phosphomycin showed positive cooperativity at pH 7.3 and 8.3, although at the latter pH, the kinetics of saturation by the substrate was hyperbolic. The cosolute glycerol greatly increased the affinity of the enzyme for phosphomycin and abolished the cooperativity in its binding, but did not eliminate the heterotropic effects of the activator. Therefore, the heterotropic and homotropic effects of the activator are not always coupled to the homotropic effects of the substrate, which argues against the two-state model previously proposed to explain the allosteric properties of maize-leaf PEPC.

Exploring the evolutionary route of the acquisition of betaine aldehyde dehydrogenase activity by plant ALDH10 enzymes: implications for the synthesis of the osmoprotectant glycine betaine

BackgroundPlant ALDH10 enzymes are aminoaldehyde dehydrogenases (AMADHs) that oxidize different ω-amino or trimethylammonium aldehydes, but only some of them have betaine aldehyde dehydrogenase (BADH) activity and produce the osmoprotectant glycine betaine (GB). The latter enzymes possess alanine or cysteine at position 441 (numbering of the spinach enzyme, SoBADH), while those ALDH10s that cannot oxidize betaine aldehyde (BAL) have isoleucine at this position. Only the plants that contain A441- or C441-type ALDH10 isoenzymes accumulate GB in response to osmotic stress. In this work we explored the evolutionary history of the acquisition of BAL specificity by plant ALDH10s.ResultsWe performed extensive phylogenetic analyses and constructed and characterized, kinetically and structurally, four SoBADH variants that simulate the parsimonious intermediates in the evolutionary pathway from I441-type to A441- or C441-type enzymes. All mutants had a correct folding, average thermal stabilities and similar activity with aminopropionaldehyde, but whereas A441S and A441T exhibited significant activity with BAL, A441V and A441F did not. The kinetics of the mutants were consistent with their predicted structural features obtained by modeling, and confirmed the importance of position 441 for BAL specificity. The acquisition of BADH activity could have happened through any of these intermediates without detriment of the original function or protein stability. Phylogenetic studies showed that this event occurred independently several times during angiosperms evolution when an ALDH10 gene duplicate changed the critical Ile residue for Ala or Cys in two consecutive single mutations. ALDH10 isoenzymes frequently group in two clades within a plant family: one includes peroxisomal I441-type, the other peroxisomal and non-peroxisomal I441-, A441- or C441-type. Interestingly, high GB-accumulators plants have non-peroxisomal A441- or C441-type isoenzymes, while low-GB accumulators have the peroxisomal C441-type, suggesting some limitations in the peroxisomal GB synthesis.ConclusionOur findings shed light on the evolution of the synthesis of GB in plants, a metabolic trait of most ecological and physiological relevance for their tolerance to drought, hypersaline soils and cold. Together, our results are consistent with smooth evolutionary pathways for the acquisition of the BADH function from ancestral I441-type AMADHs, thus explaining the relatively high occurrence of this event.

Ligand-induced conformational changes of betaine aldehyde dehydrogenase from Pseudomonas aeruginosa and Amaranthus hypochondriacus L. leaves affecting the reactivity of the catalytic thiol.

The reaction catalyzed by betaine aldehyde dehydrogenase (BADH) involves the nucleophilic attack of a catalytic cysteinyl residue on the aldehyde substrate. As a possible mechanism of regulation, we have studied the modulation by ligands of the reactivity and/or accessibility of the essential thiol of the enzyme from the human pathogen Pseudomonas aeruginosa and the leaves of the plant Amaranthus hypochondriacus (amaranth). In the absence of ligands, the kinetics of inactivation by thiol modifying reagents of both enzymes were biphasic, suggesting the existence of two enzyme conformers differing in the reactivity of their catalytic thiolate. Preincubation of P. aeruginosa BADH with the coenzymes or the aldehyde prior to the chemical modification brought about active site rearrangements that resulted in an important decrease in the inactivation rate. Amaranth BADH responded similarly to the preincubation with NADH or betaine aldehyde but NAD(+) elicited opposite changes, increasing the rate of inactivation after prolonged preincubation. In amaranth BADH, the different behavior of both coenzymes, and the observed biphasic inactivation kinetics are consistent with the previously proposed iso kinetic mechanism, characterized by the existence of two interconvertible apoenzyme forms, one able to bind NAD(+) and the other NADH. Taken together, our results suggest that ligand-induced conformational changes in BADH from the two sources studied might be important for both proper enzyme function and protection against oxidation.

Reversible, partial inactivation of plant betaine aldehyde dehydrogenase by betaine aldehyde: mechanism and possible physiological implications

In plants, the last step in the biosynthesis of the osmoprotectant glycine betaine (GB) is the NAD(+)-dependent oxidation of betaine aldehyde (BAL) catalysed by some aldehyde dehydrogenase (ALDH) 10 enzymes that exhibit betaine aldehyde dehydrogenase (BADH) activity. Given the irreversibility of the reaction, the short-term regulation of these enzymes is of great physiological relevance to avoid adverse decreases in the NAD(+):NADH ratio. In the present study, we report that the Spinacia oleracea BADH (SoBADH) is reversibly and partially inactivated by BAL in the absence of NAD(+)in a time- and concentration-dependent mode. Crystallographic evidence indicates that the non-essential Cys(450)(SoBADH numbering) forms a thiohemiacetal with BAL, totally blocking the productive binding of the aldehyde. It is of interest that, in contrast to Cys(450), the catalytic cysteine (Cys(291)) did not react with BAL in the absence of NAD(+) The trimethylammonium group of BAL binds in the same position in the inactivating or productive modes. Accordingly, BAL does not inactivate the C(450)SSoBADH mutant and the degree of inactivation of the A(441)I and A(441)C mutants corresponds to their very different abilities to bind the trimethylammonium group. Cys(450)and the neighbouring residues that participate in stabilizing the thiohemiacetal are strictly conserved in plant ALDH10 enzymes with proven or predicted BADH activity, suggesting that inactivation by BAL is their common feature. Under osmotic stress conditions, this novel partial and reversible covalent regulatory mechanism may contribute to preventing NAD(+)exhaustion, while still permitting the synthesis of high amounts of GB and avoiding the accumulation of the toxic BAL.

Reaction of the catalytic cysteine of betaine aldehyde dehydrogenase from Pseudomonas aeruginosa with arsenite-BAL and phenylarsine oxide

Betaine aldehyde dehydrogenase (BADH) catalyses the irreversible oxidation of betaine aldehyde to glycine betaine with the concomitant reduction of NAD(P)(+) to NAD(P)H. In the opportunistic pathogen Pseudomonas aeruginosa, this enzyme (PaBADH) could be an antimicrobial target. Several aldehyde dehydrogenases (ALDHs) are inactivated by arsenite in the presence of a low molecular thiol, a finding that was interpreted as a demonstration of the existence of vicinal thiols in these enzymes. As part of our studies on the susceptibility to chemical modification of the catalytic cysteine (C286) of PaBADH, we treated the enzyme with two arsenical reagents widely used to inhibit enzymes that have vicinal thiols: sodium m-arsenite plus 2,3-dimercaptopropanol (arsenite-BAL) and phenylarsine oxide (PAO). Here we report that they readily and reversibly inactivate PaBADH, even though the four cysteine residues of this enzyme (C286, C353, C377, and C439) are far from each other in the three-dimensional structure. Modification of PaBADH by both reagents was reversible by an excess of a dithiol (dithiothreitol), but only the PAO-modified enzyme could be reactivated by a monothiol (2-mercaptoethanol). C286 is the reactive residue as indicated by the following findings: (i) betaine aldehyde and NADP(+) afforded full protection against enzyme inactivation; (ii) the mutant proteins C353A, C377A, and C439A showed similar inactivation kinetics that the wild-type enzyme, and (iii) pretreatment of PaBADH with arsenite-BAL prevented irreversible inactivation by N-ethylmaleimide. Our results confirm previous findings on other ALDHs, and indicate that these vicinal thiol-specific reagents readily react with certain monothiols, such as the one of the catalytic cysteinyl residue of ALDHs. As arsenicals are being recently used to treat certain cancers, human ALDHs, even those not having conformationally vicinal thiols, may be unsuspected targets in these treatments.

Complexes of NADH with betaine aldehyde dehydrogenase from leaves of the plant Amaranthus hypochondriacus L.

The kinetic mechanism of betaine aldehyde dehydrogenase from leaves of the plant Amaranthus hypochondriacus is ordered with NAD(+) adding first. NADH is a noncompetitive inhibitor against NAD(+), which was interpreted before as evidence of an iso mechanism, in which NAD(+) and NADH binds to different forms of free enzyme. With the aim of testing the proposed kinetic mechanism, we have now investigated the ability of NADH to form different complexes with the enzyme. By initial velocity and equilibrium binding studies, we found that the steady-state levels of E.glycine betaine are negligible, ruling out binding of NADH to this complex. However, NADH readily bind to E.betaine aldehyde, whose levels most likely are kinetically significant given its low dissociation constant. Also, NADH combined with E.NADH and E.NAD(+). Finally, NADH was not able to revert the hydride transfer step, what suggest that there is no acyl-enzyme intermediate, i.e. the release of the reduced dinucleotide takes place after the deacylation step. Although formation of the complex E.NAD(+).NADH would produce an uncompetitive effect in the inhibition of NADH against NAD(+), the iso mechanism cannot be conclusively discarded.

Phosphoenolpyruvate Carboxylase and Malic Enzyme in Leaves of two Populations of Maize Differing in Grain Yield

Summary We investigated the relationship between grain yield and the activities of the C 4 metabolism enzymes, phosphoenolpyruvate carboxylase (PEPC) and NADP-malic enzyme (NADP-ME), in two genetically related maize populations differing in grain yield. In both populations, specific activity of PEPC and NADP-ME and the amount of PEPC protein in the leaf immediately above the ear increased during the first weeks after anthesis and declined afterwards, while total protein content remained almost constant from an thesis through senescence. We found that catalytic properties of the two enzymes were the same in both populations and throughout the period studied. These results suggest that changes in the amount of these enzymes, rather than activation-deactivation processes, take place during this period. In the second leaf of seedlings, PEPC protein, and PEPC and NADP-ME activities increased as the leaf developed, reaching their maxima in mature leaves in both populations. NADP-ME did not show any qualitative change during the development period, while PEPC changed from a non-photosynthetic isozyme, prevalent during the first days of development, to a photosynthetic isozyme prevalent afterwards. We did not find significant differences between the original Zacatecas 58 population and the high-grain-yield MS20 population, either in the activity of these two enzymes, or in the amount of PEPC protein. Therefore, we conclude that, in the populations we studied, increases in grain yield do not result from increases in the activity levels of PEPC nor of NADP-ME.