Journal of Molecular Biology
Volume 385, Issue 2, 16 January 2009, Pages 542-557
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The Crystal Structure of A Ternary Complex of Betaine Aldehyde Dehydrogenase from Pseudomonas aeruginosa Provides New Insight into the Reaction Mechanism and Shows A Novel Binding Mode of the 2′-Phosphate of NADP+ and A Novel Cation Binding Site

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Summary

In the human pathogen Pseudomonas aeruginosa, the NAD(P)+-dependent betaine aldehyde dehydrogenase (PaBADH) may play the dual role of assimilating carbon and nitrogen from choline or choline precursors—abundant at infection sites—and producing glycine betaine and NADPH, potentially protective against the high-osmolarity and oxidative stresses prevalent in the infected tissues. Disruption of the PaBADH gene negatively affects the growth of bacteria, suggesting that this enzyme could be a target for antibiotic design. PaBADH is one of the few ALDHs that efficiently use NADP+ and one of the even fewer that require K+ ions for stability. Crystals of PaBADH were obtained under aerobic conditions in the presence of 2-mercaptoethanol, glycerol, NADP+ and K+ ions. The three-dimensional structure was determined at 2.1-Å resolution. The catalytic cysteine (C286, corresponding to C302 of ALDH2) is oxidized to sulfenic acid or forms a mixed disulfide with 2-mercaptoethanol. The glutamyl residue involved in the deacylation step (E252, corresponding to E268 of ALDH2) is in two conformations, suggesting a proton relay system formed by two well-conserved residues (E464 and K162, corresponding to E476 and K178, respectively, of ALDH2) that connects E252 with the bulk water. In some active sites, a bound glycerol molecule mimics the thiohemiacetal intermediate; its hydroxyl oxygen is hydrogen bonded to the nitrogen of the amide groups of the side chain of the conserved N153 (N169 of ALDH2) and those of the main chain of C286, which form the “oxyanion hole.” The nicotinamide moiety of the nucleotide is not observed in the crystal, and the adenine moiety binds in the usual way. A salt bridge between E179 (E195 of ALDH2) and R40 (E53 of ALDH2) moves the carboxylate group of the former away from the 2′-phosphate of the NADP+, thus avoiding steric clashes and/or electrostatic repulsion between the two groups. Finally, the crystal shows two K+ binding sites per subunit. One is in an intrasubunit cavity that we found to be present in all known ALDH structures. The othersingle bondnot described before for any ALDH but most likely present in most of themsingle bondis located in between the dimeric unit, helping structure a region involved in coenzyme binding and catalysis. This may explain the effects of K+ ions on the activity and stability of PaBADH.

Introduction

In a wide variety of prokaryotic and eukaryotic organisms, the enzyme betaine aldehyde dehydrogenase (ALDH) [betaine aldehyde: NAD(P)+ oxidoreductase, E.C. 1.2.1.8, BADH] catalyzes the irreversible NAD(P)+-dependent oxidation of betaine aldehyde, producing the osmoprotectant glycine betaine.1, 2, 3 The reaction catalyzed by BADH constitutes the second step of the choline catabolic pathway in certain bacteria that are able to grow in choline or choline precursors as their only carbon, nitrogen and energy sources, such as the human pathogen Pseudomonas aeruginosa.4, 5 Inhibition of the enzyme from P. aeruginosa (PaBADH) would not only block or reduce choline catabolism or the supply of glycine betaine but also lead to the accumulation of betaine aldehyde, a highly toxic aldehyde.6 This enzyme might therefore be a target for a much needed antibiotic, given the high prevalence of antibiotic-resistant strains of P. aeruginosa and the susceptibility to infection of a growing immunodepressed population. Validation of PaBADH as a suitable target for antimicrobial agents comes from the finding that disruption of the PaBADH gene severely affects the growth of the bacterium on glucose if osmotic stress conditions and choline are also present in the medium.6 Given that the nucleophilic attack of an essential cysteine residue on the aldehyde substrate is the first step in the catalytic mechanism of every ALDH, thiol-specific reagents have been tested as potential inhibitors of PaBADH activity and P. aeruginosa growth.7, 8 For rational development and/or optimization of clinically useful inhibitors, however, we require knowledge of the enzyme structure and its reaction mechanism.

The chemical mechanism of the reaction catalyzed by ALDHs involves two nucleophilic attacks, one occurring in the acylation step (i.e., formation of the thiohemiacetal intermediate, carried out by the thiol group of the catalytic cysteine) and the other in the deacylation step (i.e., hydrolysis of the thioester intermediate, carried out by a water molecule). Both nucleophiles need to be “activated” by lowering their high pKa so they can lose a proton producing the reactive thiolate and hydroxyl ion, respectively, at physiological pH. In the vicinity of the catalytic cysteine, there are two highly conserved glutamate residues (ALDH2 numbering, E268 and E398; PaBADH numbering, E252 and E387), each of which has been proposed to be the general base in the reaction catalyzed by different ALDHs.9, 10, 11 In the case of PaBADH, it is not known which of these two glutamyl residues participates in the activation of the reaction nucleophiles.

Unlike most ALDHs that prefer NAD+, PaBADH uses NADP+ with the same efficiency as NAD+,12 even though sequence alignments13 have shown that it has the glutamyl residue (ALDH2 numbering, E195; PaBADH numbering, E179) for long thought to be incompatible with the binding of the 2′-phosphate of NADP+ and therefore characteristic of NAD+-dependent ALDHs.14 The only three-dimensional structure of a BADH so far determined, that of the NAD+-dependent cod liver enzyme,15 suggests that a proline at a position equivalent to that of E179(E195)‡ would produce a steric clash with the 2′-phosphate, thus accounting for its low affinity for NADP+. The three-dimensional structures of ALDHs that can bind NADP+ with similar or higher affinity than NAD+ have shown different strategies to accommodate the 2′-phosphate group.16, 17, 18, 19, 20 From analysis of the PaBADH amino acid sequence, however, as well as that of the amino acids known to be involved in binding of NADP+, it is not clear which of these described modes, if any, applies in the case of PaBADH.

We also do not know the structural bases of the stabilizing effects that monovalent cations, particularly K+ ions, have on PaBADH.21, 22, 23 In the absence of K+ ions, PaBADH loses activity in a time-dependent manner, which is caused by enzyme dissociation. Other BADHs—such as those from Escherichia coli,24 amaranth leaves,25 Bacillus subtilis,26 porcine kidney27 and horseshoe crab28—were found to be activated by K+ ions to some extent, but of these, only the stability of the kidney BADH has been reported to be affected by monovalent cations.22 Regarding the ALDH superfamily, the possible role of monovalent cations on the activity and stability of these enzymes has not been extensively studied, and this may be the reason that the only examples to date of K+-activated ALDHs are an ethanol-inducible enzyme from P. aeruginosa29 and two mitochondrial ALDH isoenzymes from Saccharomyces cerevisiae.30, 31 The structure of any of these K+-dependent ALDHs has so far not been determined, nor have the ligands of the K+ binding sites been identified. The human ALDH2 has a Na+ ion bound per subunit,32 but its functional or structural relevance is still unknown.

Here, we present the crystal structure of PaBADHsingle bondcomplexed with glycerol, NADP+ and K+ ionssingle bonddetermined at a resolution of 2.1 Å, revealing a novel mode of binding of the 2′-phosphate group of the NADP+ and a novel intersubunit K+ ion binding site, as well as an intrasubunit K+ ion binding site similar to that found in ALDH2.32 These cation binding sites appear to be a general feature of ALDHs. In addition, the structure shows the catalytic cysteine modified to sulfenic acid or forming a mixed disulfide, a glycerol molecule bound to some active sites mimicking the thiohemiacetal intermediate and a likely proton relay system that participates in catalysis.

Section snippets

Structure determination and main characteristics

The structure of PaBADH complexed with NADP+ has been determined at 2.1-Å resolution. The crystal belongs to the C121 space group and contains eight subunits in the asymmetric unit describing two tetramers, each of which corresponds to the biological unit of the enzyme.22 The solution of the molecular replacement showed that seven of the eight subunits clearly localized. The eighth subunit (labeled as subunit H)single bondwhose electron density was remarkably weaker compared with the other subunitssingle bondwas

Expression and purification of PaBADH

Plasmid pCALbetB containing the full sequence of the gene betB that encodes PaBADH was used for the expression of the enzyme in E. coli cells as previously described.4 The recombinant enzyme was purified to homogeneity by a two-step procedure previously described.50 Protein concentrations were determined by the Coomassie G dye binding technique of Bradford51 using bovine serum albumin as a standard or spectrophotometrically using a molar absorptivity at 280 nm of 52,060 M 1 cm 1, deduced from

Acknowledgements

This research was financially supported by Dirección General de Asuntos del Personal Académico de la Universidad Nacional Autónoma de México (IN228106) and Consejo Nacional de Ciencia y Tecnología de México (50581). We thank Dr. Adela Rodríguez-Romero from the Laboratorio Universitario de Estructura de Proteínas, Universidad Nacional Autónoma de México, and Instituto de Física de San Carlos, Universidade de São Paulo, Brazil, for the initial BADH data sets and the staff at beam line X6a of the

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    Present address: L. González-Segura, Departamento de Bioquímica, Facultad de Química, Universidad Nacional Autónoma de México, México DF 04510, Mexico.

    L. González-Segura and E. Rudiño-Piñera contributed equally to this work.

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