The uncharacterized Pseudomonas aeruginosa PA4189 is a novel and efficient aminoacetaldehyde dehydrogenase.

Neither the Pseudomonas aeruginosa aldehyde dehydrogenase encoded by the PA4189 gene nor its ortholog proteins have been biochemically or structurally characterized and their physiological function is unknown. We cloned the PA4189 gene, obtained the PA4189 recombinant protein, and studied its structure-function relationships. PA4189 is an NAD+-dependent aminoaldehyde dehydrogenase highly efficient with protonated aminoacetaldehyde and 3-aminopropionaldehyde, which are much more preferred to the non-protonated species as indicated by pH studies. Based on the higher activity with aminoacetaldehyde than with 3-aminopropionaldehyde, we propose that aminoacetaldehyde might be the PA4189 physiological substrate. Even though at the physiological pH of P. aeruginosa cells the non-protonated aminoacetaldehyde species will be predominant, and despite the competition of these species with the protonated ones, PA4189 would very efficiently oxidize ACTAL in vivo, producing glycine. To our knowledge, PA4189 is the first reported enzyme that might metabolize ACTAL, which is considered a dead-end metabolite because its consuming reactions are unknown. The PA4189 crystal structure reported here suggested that the charge and size of the active-site residue Glu457, which narrows the aldehyde-entrance tunnel, greatly define the specificity for small positively charged aldehydes, as confirmed by the kinetics of the E457G and E457Q variants. Glu457 and the residues that determine Glu457 conformation inside the active site are conserved in the PA4189 orthologs, which we only found in proteobacteria species. Also is conserved the PA4189 genomic neighborhood, which suggests that PA4189 participates in an uncharacterized metabolic pathway. Our results open the door to future efforts to characterize this pathway.

Structural and biochemical evidence of the glucose 6-phosphate-allosteric site of maize C4-phosphoenolpyruvate carboxylase: its importance in the overall enzyme kinetics.

Activation of phosphoenolpyruvate carboxylase (PEPC) enzymes by glucose 6-phosphate (G6P) and other phospho-sugars is of major physiological relevance. Previous kinetic, site-directed mutagenesis and crystallographic results are consistent with allosteric activation, but the existence of a G6P-allosteric site was questioned and competitive activation-in which G6P would bind to the active site eliciting the same positive homotropic effect as the substrate phosphoenolpyruvate (PEP)-was proposed. Here, we report the crystal structure of the PEPC-C4 isozyme from Zea mays with G6P well bound into the previously proposed allosteric site, unambiguously confirming its existence. To test its functionality, Asp239-which participates in a web of interactions of the protein with G6P-was changed to alanine. The D239A variant was not activated by G6P but, on the contrary, inhibited. Inhibition was also observed in the wild-type enzyme at concentrations of G6P higher than those producing activation, and probably arises from G6P binding to the active site in competition with PEP. The lower activity and cooperativity for the substrate PEP, lower activation by glycine and diminished response to malate of the D239A variant suggest that the heterotropic allosteric activation effects of free-PEP are also abolished in this variant. Together, our findings are consistent with both the existence of the G6P-allosteric site and its essentiality for the activation of PEPC enzymes by phosphorylated compounds. Furthermore, our findings suggest a central role of the G6P-allosteric site in the overall kinetics of these enzymes even in the absence of G6P or other phospho-sugars, because of its involvement in activation by free-PEP.

Identification of the allosteric site for neutral amino acids in the maize C4 isozyme 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.

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.

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.

BACKGROUND: Plant 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. RESULTS: We 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. CONCLUSION: Our 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.

Two new T-state crystal structures of maize C4-phosphoenolpyruvate carboxylase reveal and suggest novel structural features of the allosteric regulation and carboxylation step.

To get a deeper understanding of the structural bases of the allosteric transition between T and R states of plant and bacterial phosphoenolpyruvate carboxylases (PEPCs), we obtained the first T-state crystal structures of the maize photosynthetic PEPC (ZmPEPC-C4) and exhaustively compared them with the previously reported R-state ZmPEPC-C4 and other T-state structures. We identified previously unrecognized significant conformational changes in the T state: that of the α8-α9 loop, which connects the two kinds of activator allosteric sites with the active site, the conversion of the α30 helix into a 310 helix, leading to the disorganization of the active site lid and activators allosteric sites, and the closure of the inhibitor allosteric-site lid. Additionally, we identified previously overlooked, highly conserved residues of potential interest in the allosteric transition, including two histidines whose protonation might stabilize the T state. The crystal structures reported here also suggest similar tetrameric quaternary arrangements of PEPC enzymes in the R and T states, and the location of the bicarbonate binding site, as well as the conformational changes required for the carboxylation step. Our findings and working hypothesis advance the understanding of the structural features of the allosteric PEPC enzymes and provide a foundation for future experiments.

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 K(m)(BAL) increases and V(max)/K(m)(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 V(max)/K(m)(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.

Novel NADPH-cysteine covalent adduct found in the active site of an aldehyde dehydrogenase.

PaBADH (Pseudomonas aeruginosa betaine aldehyde dehydrogenase) catalyses the irreversible NAD(P)+-dependent oxidation of betaine aldehyde to its corresponding acid, the osmoprotector glycine betaine. This reaction is involved in the catabolism of choline and in the response of this important pathogen to the osmotic and oxidative stresses prevalent in infection sites. The crystal structure of PaBADH in complex with NADPH showed a novel covalent adduct between the C2N of the pyridine ring and the sulfur atom of the catalytic cysteine residue, Cys286. This kind of adduct has not been reported previously either for a cysteine residue or for a low-molecular-mass thiol. The Michael addition of the cysteine thiolate in the 'resting' conformation to the double bond of the α,ß-unsaturated nicotinamide is facilitated by the particular conformation of NADPH in the active site of PaBADH (also observed in the crystal structure of the Cys286Ala mutant) and by an ordered water molecule hydrogen bonded to the carboxamide group. Reversible formation of NAD(P)H-Cys286 adducts in solution causes reversible enzyme inactivation as well as the loss of Cys286 reactivity towards thiol-specific reagents. This novel covalent modification may provide a physiologically relevant regulatory mechanism of the irreversible PaBADH-catalysed reaction, preventing deleterious decreases in the intracellular NAD(P)+/NAD(P)H ratios.

The critical role of the aldehyde dehydrogenase PauC in spermine, spermidine, and diaminopropane toxicity in Pseudomonas aeruginosa: Its possible use as a drug target.

The opportunistic human pathogen Pseudomonas aeruginosa exhibits great resistance to antibiotics; so, new therapeutic agents are urgently needed. Since polyamines levels are incremented in infected tissues, we explored whether the formation of a toxic aldehyde in polyamines degradation can be exploited in combating infection. We cloned the gene encoding the only aminoaldehyde dehydrogenase involved in P. aeruginosa polyamines-degradation routes, PaPauC, overexpressed this enzyme, and found that it oxidizes 3-aminopropionaldehyde (APAL) and 3-glutamyl-3-aminopropionaldehyde (GluAPAL) - produced in spermine (Spm), spermidine (Spd), and diaminopropane (Dap) degradation, as well as 4-aminobutyraldehyde (ABAL) and 4-glutamyl-4-aminobutyraldehyde (GluABAL) - formed in putrescine (Put) degradation. As the catalytic efficiency of PaPauC with APAL was 30-times lower than with GluAPAL, and GluAPAL is predominantly formed, APAL will be poorly oxidized 'in vivo'. We found polyamines-induced increases in the PaPauC activity of cell crude-extracts and in the expression of the PapauC gene that were diminished by glucose. Spm, Spd, or Dap, but not Put, were toxic to P. aeruginosa even in the presence of other carbon and nitrogen sources, particularly to a strain with the PapauC gene disrupted. APAL, but not GluAPAL, was highly toxic even to wild-type cells, suggesting that its accumulation, particularly in the absence of, or low, PaPauC activity is responsible for the toxicity of Spm, Spd, and Dap. Our results shed light on the toxicity mechanism of these three polyamines and strongly support the critical role of PaPauC in this toxicity. Thus, PaPauC emerges as a novel potential drug target whose inhibition might help in combating infection by this important pathogen.

Multiple conformations in solution of the maize C4-phosphoenolpyruvate carboxylase isozyme.

The photosynthetic phosphoenolpyruvate carboxylase isozyme from C4 plants (PEPC-C4) has a complex allosteric regulation, involving positive cooperativity in binding the substrate phosphoenolpyruvate as well as positive and negative allosteric effectors. Besides the proposed R- and T-states, previous kinetic results suggested functionally relevant different R-states of the maize enzyme (ZmPEPC-C4) elicited by PEP or its two kinds of activators, glucose 6-phosphate or glycine. To detect these different R-state conformations, we used as conformational probes the fluorescence of 8-anilino-1-naphthalene sulfonate (ANS), near-UV circular dichroism (CD) spectroscopy, and limited proteolysis by trypsin. Phosphoenolpyruvate and malate binding caused distinct concentration-dependent fluorescence changes of ZmPEPC-C4/ANS, suggesting that they elicited conformational states different from that of the free enzyme, while glucose 6-phosphate or glycine binding did not produce fluorescence changes. Differences were also observed in the near UV CD spectra of the enzyme, free or complexed with its substrate or allosteric effectors. Additionally, differences in the trypsin-digestion fragmentation patterns, as well as in the susceptibility of the free and complexed enzyme to digestion and digestion-provoked loss of activity, provided evidence of several ZmPEPC-C4 conformations in solution elicited by the substrate and the allosteric effectors. Using the already reported ZmPEPC-C4 crystal structures and bioinformatics methods, we predicted that the most probable trypsin-cleavage sites are located in superficial flexible regions, which seems relevant for the protein dynamics underlying the function and allosteric regulation of this enzyme. Together, our findings agree with previous kinetic results, shed light on this enzyme's complex allosteric regulation, and place ZmPEPC-C4 in the growing list of allosteric enzymes possessing an ensemble of closely related R-state conformations.

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.

The importance of assessing aldehyde substrate inhibition for the correct determination of kinetic parameters and mechanisms: the case of the ALDH enzymes.

Substrate inhibition by the aldehyde has been observed for decades in NAD(P)+-dependent aldehyde dehydrogenase (ALDH) enzymes, which follow a Bi Bi ordered steady-state kinetic mechanism. In this work, by using theoretical simulations of different possible substrate inhibition mechanisms in monosubstrate and Bi Bi ordered steady-state reactions, we explored the kind and extent of errors arising when estimating the kinetic parameters and determining the kinetic mechanisms if substrate inhibition is intentionally or unintentionally ignored. We found that, in every mechanism, fitting the initial velocity data of apparently non-inhibitory substrate concentrations to a rectangular hyperbola produces important errors, not only in the estimation of Vmax values, which were underestimated as expected, but, surprisingly, even more in the estimation of Km values, which led to overestimation of the Vmax/Km values. We show that the greater errors in Km arises from fitting data that do experience substrate inhibition, although it may not be evident, to a Michaelis-Menten equation, which causes overestimation of the data at low substrate concentrations. Similarly, we show that if substrate inhibition is not fully assessed when inhibitors are evaluated, the estimated inhibition constants will have significant errors, and the type of inhibition could be grossly mistaken. We exemplify these errors with experimental results obtained with the betaine aldehyde dehydrogenase from spinach showing the errors predicted by the theoretical simulations and that these errors are increased in the presence of NADH, which in this enzyme favors aldehyde substrate inhibition. Therefore, we strongly recommend assessing substrate inhibition by the aldehyde in every ALDH kinetic study, particularly when inhibitors are evaluated. The common practices of using an apparently non-inhibitory concentration range of the aldehyde or a single high concentration of the aldehyde or the coenzyme when varying the other to determine true kinetic parameters should be abandoned.

Aldehyde dehydrogenase diversity in bacteria of the Pseudomonas genus.

Aldehyde dehydrogenases (ALDHs) comprise one of the most ancient protein superfamilies widely distributed in the three domains of life. Their members have been extensively studied in animals and plants, sorted out in different ALDH protein families and their participation in a broad variety of metabolic pathways has been documented. Paradoxically, no systematic studies comprising ALDHs from bacteria have been performed so far. Among bacteria, the genus Pseudomonas occupies numerous ecological niches, and is one of the most complex bacterial genera with the largest number of known species. For these reasons, we selected Pseudomonas as a paradigm to analyze the diversity of ALDHs in bacteria. With this aim, complete Pseudomonas genome sequences and annotations were retrieved from NCBI's RefSeq genome database. The 258 Pseudomonas strains belong to 46 different species, along with 23 with no species designation. The genomes of these Pseudomonas contain from 3,315 to 6,825 annotated protein coding genes. A total of 6,510 ALDH sequences were found in the selected Pseudomonas, with a median of 24 ALDH-coding genes per strain (by comparison humans possess only 19 different ALDH loci). Pseudomonas saudiphocaensis possesses the lowest number of aldh genes (9), while Pseudomonas pseudoalcaligenes KF707 NBRC110670 possesses the maximum number of aldh genes (49). The ALDHs found in Pseudomonas can be sorted out into 42 protein families, with a predominance of 14 families, which contained 76% of all ALDHs found. In this regard, it is important to note that many Pseudomonas genomes have multiple aldh genes coding for proteins belonging to the same family. Given that all strains contained members of families ALDH4, ALDH5, ALDH6, ALDH14, ALDH18 and ALDH27, we consider these families to be part of the core Pseudomonas genome.

Bona fide choline monoxygenases evolved in Amaranthaceae plants from oxygenases of unknown function: Evidence from phylogenetics, homology modeling and docking studies.

Few land plants can synthesize and accumulate the osmoprotectant glycine betaine (GB) even though this metabolic trait has major adaptive importance given the prevalence of drought, hypersaline soils or cold. GB is synthesized from choline in two reactions catalyzed by choline monooxygenases (CMOs) and enzymes of the family 10 of aldehyde dehydrogenases (ALDH10s) that gained betaine aldehyde dehydrogenase activity (BADH). Homolog genes encoding CMO and ALDH10 enzymes are present in all known land plant genomes, but since GB-non-accumulators plants lack the BADH-type ALDH10 isozyme, they would be expected to also lack the CMO activity to avoid accumulation of the toxic betaine aldehyde. To explore CMOs substrate specificity, we performed amino acid sequence alignments, phylogenetic analysis, homology modeling and docking simulations. We found that plant CMOs form a monophyletic subfamily within the Rieske/mononuclear non-heme oxygenases family with two clades: CMO1 and CMO2, the latter diverging from CMO1 after gene duplication. CMO1 enzymes are present in all plants; CMO2s only in the Amaranthaceae high-GB-accumulators plants. CMO2s, and particularly their mononuclear non-heme iron domain where the active site is located, evolved at a faster rate than CMO1s, which suggests positive selection. The homology model and docking simulations of the spinach CMO2 enzyme showed at the active site three aromatic residues forming a box with which the trimethylammonium group of choline could interact through cation-π interactions, and a glutamate, which also may interact with the trimethylammonium group through a charge-charge interaction. The aromatic box and the carboxylate have been shown to be critical for choline binding in other proteins. Interestingly, these residues are conserved in CMO2 proteins but not in CMO1 proteins, where two of these aromatic residues are leucine and the glutamate is asparagine. These findings reinforce our proposal that the CMO1s physiological substrate is not choline but a still unknown metabolite.

Complex, unusual conformational changes in kidney betaine aldehyde dehydrogenase suggested by chemical modification with disulfiram.

The NAD+-dependent animal betaine aldehyde dehydrogenases participate in the biosynthesis of glycine betaine and carnitine, as well as in polyamines catabolism. We studied the kinetics of inactivation of the porcine kidney enzyme (pkBADH) by the drug disulfiram, a thiol-reagent, with the double aim of exploring the enzyme dynamics and investigating whether it could be an in vivo target of disulfiram. Both inactivation by disulfiram and reactivation by reductants were biphasic processes with equal limiting amplitudes. Under certain conditions half of the enzyme activity became resistant to disulfiram inactivation. NAD+ protected almost 100% at 10 microM but only 50% at 5mM, and vice versa if the enzyme was pre-incubated with NAD+ before the chemical modification. NADH, betaine aldehyde, and glycine betaine also afforded greater protection after pre-incubation with the enzyme than without pre-incubation. Together, these findings suggest two kinds of active sites in this seemingly homotetrameric enzyme, and complex, unusual ligand-induced conformational changes. In addition, they indicate that, in vivo, pkBADH is most likely protected against disulfiram inactivation.

Mechanisms of protection against irreversible oxidation of the catalytic cysteine of ALDH enzymes: Possible role of vicinal cysteines.

The catalytic mechanism of the NAD(P)+-dependent aldehyde dehydrogenases (ALDHs) involves the nucleophilic attack of the essential cysteine (Cys302, mature HsALDH2 numbering) on the aldehyde substrate. Although oxidation of Cys302 will inactivate these enzymes, it is not yet well understood how this oxidation is prevented. In this work we explore possible mechanisms of protection by systematically analyzing the reported three-dimensional structures and amino acid sequences of the enzymes of the ALDH superfamily. Specifically, we considered the Cys302 conformational space, the structure and residues conservation of the catalytic loop where Cys302 is located, the observed oxidation states of Cys302, the ability of physiological reductants to revert its oxidation, and the presence of vicinal Cys in the catalytic loop. Our analyses suggested that: 1) In the apo-enzyme, the thiol group of Cys302 is quite resistant to oxidation by ambient O2 or mild oxidative conditions, because the protein environment promotes its high pKa. 2) NAD(P)+ bound in the "hydride transfer" conformation afforded total protection against Cys302 oxidation by an unknown mechanism. 3) If formed, the Cys302-sulfenic acid is protected against irreversible oxidation. 4) Of the physiological reductant agents, the dithiol lipoic acid could reduce a sulfenic or a disulfide bond in the ALDHs active site; glutathione cannot because its thiol group cannot reach Cys302, and other physiological monothiols may be ineffective in those ALDHs where their active site cannot sterically accommodate two molecules of the monothiols. 5) Formation of the disulfides Cys301-Cys302, Cys302-Cys304, Cys302-Cys305 and Cys-302-Cys306 in those ALDHs that have these Cys residues is not probable, because of the permitted Cys conformers as well as the conserved structure and low flexibility of the catalytic loop. 6) Only in some ALDH2, ALDH9, ALDH16 and ALDH23 enzymes, Cys303, alone or in conjunction with Cys301, allows disulfide formation. Interestingly, several of these enzymes are mitochondrial.

Functional and structural analysis of catalase oxidized by singlet oxygen.

Purified catalase-1 (CAT-1) from Neurospora crassa asexual spores is oxidized by singlet oxygen giving rise to active enzyme forms with different electrophoretic mobility. These enzyme forms are detected in vivo under stress conditions and during development at the start of the asexual morphogenetic transitions. CAT-1 heme b is oxidized to heme d by singlet oxygen. Here, we describe functional and structural comparisons of the non-oxidized enzyme with the fully oxidized one. Using a broad H(2)O(2) concentration range (0.01-3.0 M), non-hyperbolic saturation kinetics was found in both enzymes, indicating that kinetic complexity does not arise from heme oxidation. The kinetics was consistent with the existence of two kinds of active sites differing more than 10-times in substrate affinity. Positive cooperativity for one or both of the saturation curves is possible. Kinetic constants obtained at 22 degrees C varied slightly and apparent activation energies for the reaction of both components are not significantly different. Protein fluorescence and circular dicroism of the two enzymes were nearly identical, indicating no gross conformational change with oxidation. Increased sensitivity to inhibition by cyanide indicated a local change at the active site in the oxidized catalase. Oxidized catalase was less resistant to high temperatures, high guanidinium ion concentration, and digestion with subtilisin. It was also less stable than the non-oxidized enzyme at an acid pH. The overall data show that the oxidized enzyme is structurally different from the non-oxidized one, although it conserves most of the remarkable stability and catalytic efficiency of the non-oxidized enzyme. Because the enzyme in the cell can be oxidized under physiological conditions, preservation of functional and structural properties of catalase could have been selected through evolution to assure an active enzyme under oxidative stress conditions.

Site-directed mutagenesis and homology modeling indicate an important role of cysteine 439 in the stability of betaine aldehyde dehydrogenase from Pseudomonas aeruginosa.

Betaine aldehyde dehydrogenase (BADH) from the human pathogen Pseudomonas aeruginosa is a tetrameric enzyme that contains a catalytic Cys286 and three additional cysteine residues, Cys353, 377, and 439, per subunit. In the present study, we have investigated the role of the three non-essentials in enzyme activity and stability by homology modeling and site-directed mutagenesis. Cys353 and Cys377 are located at the protein surface with their sulfur atoms buried, while Cys439 is at the subunit interface between the monomers forming a dimeric pair. All three residues were individually mutated to alanine and Cys439 also to serine and valine. The five mutant proteins were expressed in Escherichia coli and purified to homogeneity. Their steady-state kinetics was not significantly affected, neither was their structure as indicated by circular dicroism spectropolarimetry, protein intrinsic fluorescence, and size-exclusion chromatography. However, stability was severely reduced in the Cys439 mutants particularly in C439S and C439V, which were inactive when expressed at 37 degrees C. They also exhibited higher sensitivity to thermal and chemical inactivation, and higher propensity to dissociation by dilution or exposure to low ionic strength than the wild-type enzyme. Size-exclusion chromatography indicates that substitution of Cys439 lead to unstable dimers or to stable dimeric conformations not compatible with a stable tetrameric structure. To the best of our knowledge, this is the first study of an aldehyde dehydrogenase revealing a residue at the dimer interface involved in holding the dimer, and consequently the tetramer, together.

Residues that influence coenzyme preference in the aldehyde dehydrogenases.

To find out the residues that influence the coenzyme preference of aldehyde dehydrogenases (ALDHs), we reviewed, analyzed and correlated data from their known crystal structures and amino-acid sequences with their published kinetic parameters for NAD(P)(+). We found that the conformation of the Rossmann-fold loops participating in binding the adenosine ribose is very conserved among ALDHs, so that coenzyme specificity is mainly determined by the nature of the residue at position 195 (human ALDH2 numbering). Enzymes with glutamate or proline at 195 prefer NAD(+) because the side-chains of these residues electrostatically and/or sterically repel the 2'-phosphate group of NADP(+). But contrary to the conformational rigidity of proline, the conformational flexibility of glutamate may allow NADP(+)-binding in some enzymes by moving the carboxyl group away from the 2'-phosphate group, which is possible if a small neutral residue is located at position 224, and favored if the residue at position 53 interacts with Glu195 in a NADP(+)-compatible conformation. Of the residues found at position 195, only glutamate interacts with the NAD(+)-adenosine ribose; glutamine and histidine cannot since their side-chain points are opposite to the ribose, probably because the absence of the electrostatic attraction by the conserved nearby Lys192, or its electrostatic repulsion, respectively. The shorter side-chains of other residues-aspartate, serine, threonine, alanine, valine, leucine, or isoleucine-are distant from the ribose but leave room for binding the 2'-phosphate group. Generally, enzymes having a residue different from Glu bind NAD(+) with less affinity, but they can also bind NADP(+) even sometimes with higher affinity than NAD(+), as do enzymes containing Thr/Ser/Gln195. Coenzyme preference is a variable feature within many ALDH families, consistent with being mainly dependent on a single residue that apparently has no other structural or functional roles, and therefore can easily be changed through evolution and selected in response to physiological needs.

Amino acid residues that affect the basicity of the catalytic glutamate of the hydrolytic aldehyde dehydrogenases.

In the catalytic mechanism of hydrolytic aldehyde dehydrogenases (ALDHs) the role of Glu268 (mature human ALDH2 numbering) as a general base is of major relevance. Since Glu268 basicity depends on its protein environment, here we explore its interactions with other amino acid residues in the three different conformations observed in ALDH crystal-structures: "inside", "intermediate" and "outside". In all of them Glu268 is in a hydrophobic environment. In the "inside" conformation, the theoretical pKa estimated by PROPKA3 is the result of the effects of hydrogen bonds with the protonated thiol of the catalytic Cys302 and/or the main-chain amide nitrogen of the highly conserved Gly270, and of charge-charge interactions with neighboring side-chains-Lys178, Glu/Asp476, His465 or Glu399 depending on the enzyme. In the "intermediate" conformation Glu268-carboxyl pKa is influenced by interactions with Glu/Asp476, Arg/Lys475, Lys/Arg178, His465 or Arg459, also depending on the enzyme. In the "outside" conformation, the effects on Glu268-carboxyl pKa arise from hydrogen bonds with the side chains of the strictly conserved Thr224 and/or of Lys/Arg178, and from charge-charge interactions with Lys/Arg/Asp178, Glu476, or Arg459. The estimated pKas and interactions of Glu268-carboxyl in the "intermediate" and "outside" conformations are consistent with their previously proposed roles in activating the hydrolytic water and in a proton relay mechanism, respectively. Water channels connecting Glu268 with the bulk water were found in all hydrolytic ALDHs. In the "inside" conformation the theoretical pKas of the Glu268-carboxyl and Cys302-thiol groups suggest that the carboxyl cannot receive the proton from the thiol. We propose that a protonated Cys302 might perform the nucleophilic attack on the aldehyde, which can be facilitated by Glu268 in the "intermediate" conformation. Finally, the conservation of the residues influencing Glu268 basicity between and within ALDH families suggests that these residues, not previously studied, are important for the catalytic mechanism of many ALDH enzymes.