Lilian González-Segura

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.

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.

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.

Potential monovalent cation-binding sites in aldehyde dehydrogenases

Potassium ions are non-essential activators of several aldehyde dehydrogenases (ALDHs), whereas a few others require the cation for activity. Two kinds of cation-binding sites, which we named intra-subunit and inter-subunit, have been observed in crystal structures of ALDHs, and based on reported crystallographic data, we here propose the existence of a third kind located in the central cavity of some tetrameric ALDHs. Given the high structural similarity between these enzymes, cation-binding sites may be present in many other members of this superfamily. To explore the prevalence of these sites, we compared 37 known crystal structures from 13 different ALDH families and evaluated the possible existence of a cation on the basis of the number, distance and geometry of its potential interactions, as well as of B-factor values of modeled cations obtained in new refinements of some reported crystal structures. Also, by performing multiple alignments of 855 non-redundant amino acid sequences, we assessed the degree of conservation in their respective families of the amino acid residues putatively relevant for cation binding. Among the ALDH enzymes studied, and according to our analyses, potential intra-subunit cation-binding sites seem to be present in most members of ALDH2, ALDH1L, ALDH4, ALDH5, ALDH7, ALDH10, and ALDH25 families, as well as in the bacterial and fungal members of the ALDH9 family and in a few ALDH1, ALDH6, ALDH11 and ALDH26 enzymes; potential inter-subunit sites in members of ALDH1L, ALDH3, ALDH4 from bacillales, ALDH5, ALDH7, ALDH9, ALDH10, ALDH11 and ALDH25 families; and potential central-cavity sites only in some bacterial and animal ALDH9s and in most members of the ALDH1L family. Because potassium is the most abundant intracellular cation, we propose that these are potassium-binding sites, but the specific structural and/or functional roles of the cation bound to these different sites remain to be investigated.

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.

Identification of the allosteric site for neutral amino acids in the maize C4isozyme of phosphoenolpyruvate carboxylase: The critical role of Ser-100.

The isozymes of photosynthetic phosphoenolpyruvate carboxylase from C4 plants (PEPC-C4) play a critical role in their atmospheric CO2 assimilation and productivity. They are allosterically activated by phosphorylated trioses or hexoses, such as d-glucose 6-phosphate, and inhibited by l-malate or l-aspartate. Additionally, PEPC-C4 isozymes from grasses are activated by glycine, serine, or alanine, but the allosteric site for these compounds remains unknown. Here, we report a new crystal structure of the isozyme from Zea mays (ZmPEPC-C4) with glycine bound at the monomer–monomer interfaces of the two dimers of the tetramer, making interactions with residues of both monomers. This binding site is close to, but different from, the one proposed to bind glucose 6-phosphate. Docking experiments indicated that d/l-serine or d/l-alanine could also bind to this site, which does not exist in the PEPC-C4 isozyme from the eudicot plant Flaveria, mainly because of a lysyl residue at the equivalent position of Ser-100 in ZmPEPC-C4. Accordingly, the ZmPEPC-C4 S100K mutant is not activated by glycine, serine, or alanine. Amino acid sequence alignments showed that PEPC-C4 isozymes from the monocot family Poaceae have either serine or glycine at this position, whereas those from Cyperaceae and eudicot families have lysine. The size and charge of the residue equivalent to Ser-100 are not only crucial for the activation of PEPC-C4 isozymes by neutral amino acids but also affect their affinity for the substrate phosphoenolpyruvate and their allosteric regulation by glucose 6-phosphate and malate, accounting for the reported kinetic differences between PEPC-C4 isozymes from monocot and eudicot plants.