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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.

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The Bacillus subtilis strain 168 genome contains the chr3N-chr3C genes encoding the Chr3N/Chr3C protein pair of the chromate ion transporter (CHR) superfamily. Chr3N/Chr3C confers chromate resistance in Escherichia coli only when both proteins are expressed. Upstream of chr3N is the chrS gene encoding ChrS, a protein with homology to the Lrp/AsnC family of transcriptional regulators. When the chrS-chr3N-chr3C gene cluster was transferred to E. coli, a diminished level of chromate resistance was observed, as compared with E. coli transformants bearing only the chromate resistance genes, which displayed full resistance. These data suggested that the chrS gene product acts as negative regulator. RT-PCR assays demonstrated that expression of chrS diminishes transcription of the chromate resistance genes in E. coli, and that this repression was overcome by chromate. Electrophoretic mobility shift assays showed that purified ChrS protein specifically binds to the 5' region of chrS. These results indicate that the chr gene cluster forms an operon regulated negatively by ChrS binding to its own gene's regulatory region, and positively by chromate ions. Sequence analysis revealed similar operons in many Bacillales strains, suggesting some adaptive advantage. This is the first example of a bacterial heavy-metal resistance system controlled by an Lrp-type transcriptional regulator.

Assuntos

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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.

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Within the aldehyde dehydrogenase (ALDH) superfamily, proteins belonging to the ALDH9, ALDH10, ALDH25, ALDH26 and ALDH27 families display activity as ω-aminoaldehyde dehydrogenases (AMADHs). These enzymes participate in polyamine, choline and arginine catabolism, as well as in synthesis of several osmoprotectants and carnitine. Active site aromatic and acidic residues are involved in binding the ω-aminoaldehydes in plant ALDH10 enzymes. In order to ascertain the degree of conservation of these residues among AMADHs and to evaluate their possible relevance in determining the aminoaldehyde specificity, we compared the known amino acid sequences of every ALDH family that have at least one member with known crystal structure, as well as the electrostatic potential surface of the aldehyde binding sites of these structures. Our analyses showed that four or three aromatic residues form a similar "aromatic box" in the active site of the AMADH enzymes, being the equivalents to Phe170 and Trp177 (human ALDH2 numbering) strictly conserved in all of them, which supports their relevance in binding the aminoaldehyde by cation-π interactions. In addition, all AMADHs exhibit a negative electrostatic potential surface in the aldehyde-entrance tunnel, due to side-chain carboxyl and hydroxyl groups or main-chain carbonyl groups. In contrast, ALDHs that have non-polar or negatively charged substrates exhibit neutral or positive electrostatic potential surfaces, respectively. Finally, our comparative sequence analyses revealed that the residues equivalent to Asp121 and Phe170 are highly conserved in many ALDH families irrespective of their substrate specificity-suggesting that they perform a role in catalysis additional or different to binding of the substrate-and that the positions Met124, Cys301, and Cys303 are hot spots changed during evolution to confer aldehyde specificity to several ALDH families.

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Alcohol dehydrogenase (ADH) activity is widely distributed in all phyla. In animals, three non-homologous NAD(P)(+)-dependent ADH protein families are reported. These arose independently throughout evolution and possess different structures and mechanisms of reaction: type I (medium-chain) ADHs are zinc-containing enzymes and comprise the most studied group in vertebrates; type II (short-chain) ADHs lack metal cofactor and have been extensively studied in Drosophila; and type III ADHs are iron-dependent/-activated enzymes that were initially identified only in microorganisms. The presence of these different ADHs in animals has been assumed to be a consequence of chronic exposure to ethanol. By far the most common natural source of ethanol is fermentation of fruit sugars by yeast, and available data support that this fruit trait evolved in concert with the characteristics of their frugivorous seed dispersers. Therefore, if the presence of ADHs in animals evolved as an adaptive response to dietary ethanol exposure, then it can be expected that the enzymogenesis of these enzymes began after the appearance of angiosperms with fleshy fruits, because substrate availability must precede enzyme selection. In this work, available evidence supporting this possibility is discussed. Phylogenetic analyses reveal that type II ADHs suffered several duplications, all of these restricted to flies (order Diptera). Induction of type II Adh by ethanol exposure, a positive correlation between ADH activity and ethanol resistance, and the fact that flies and type II Adh diversification occurred in concert with angiosperm diversification, strongly suggest that type II ADHs were recruited to allow larval flies to exploit new restricted niches with high ethanol content. In contrast, phyletic distribution of types I and III ADHs in animals showed that these appeared before angiosperms and land plants, independently of ethanol availability. Because these enzymes are not induced by ethanol exposure and possess a high affinity and/or catalytic efficiency for non-ethanol endogenous substrates, it can be concluded that the participation of types I and III ADHs in ethanol metabolism can be considered as incidental, and not adaptive.

Assuntos

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The relative apportionment of hydrocarbons (HCs) coming from mobile, fixed, and other sources (not correlated either to carbon monoxide [CO] or sulfur dioxide [SO2] emissions as solvent evaporation and biogenic sources) is calculated as previously proposed by Riveros et al.1 through the linear approximation [HC]tol = [HC]0 + m1 [CO] + m2 [SO2], where m1 and m2 are fitted constants. The obtained apportionment with air samples taken in 1993 is consistent with the earlier published apportionment with air samples taken in 1992, validating the previous procedure. This analysis suggests that 75% of HC originate from mobile sources, 5-18% from fixed sources, and 7-20% from other sources (mainly solvents and bio-genic sources). A similar analysis was employed to estimate the relative contribution of HCs and nitric oxides (NO2) to ozone (O3) formation, the most important air pollutant in Mexico City. In this way, through a local lineation of O3 isopleths, simultaneous measurements of HC and NO2 in the atmosphere were fitted to the equation-[O3]peak = [O3]0 + ma [HC] + mb [NO2]-to predict O3 peak. With these data, the adjusted parameters show that NO2, not HC as was proposed previously, is the most important contributor to O3 formation.

RESUMO

Ehtanol or wthyl alcohol is a molecule that, in mammals, is naturally present at low concentrations due to its production by gastrointestinal flora fermentation activity. However, it is remarkable that this metabolite, with a clearly minor role in regular vertebrate metabolism, can be oxidized into acetaldehyde through several ensymatic and non-enzymatic mechanisms, which comprise the activity of more than ten ensymes and isozymes, many of them broadly distributed in different specie and tissues. In correspondence, acetaldehyde can also be oxidized into acetate through several enzymatic pathways that involve about ten enzymes and isozymes which also have a broad distribution In this article, a complete review of the aforementioned metabolic pathways is elaborated. From this group, the participation and wide distribution of alcohol dehydrogenase and aldehyde dehydrogenase systems are emphasized. The mechanism of reaction, kinetic characteristics and physiological relevance are described, and finally, the possible physiological role of these enzymatic systems as responsible to synthesize or catabolize several endogenous metabolites that regulate growth, metabolism, differentiation and neuroendocrine function in mammals are discussed

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