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• W3048821712 abstract "Aldehyde dehydrogenases are versatile enzymes that serve a range of biochemical functions. Although traditionally considered metabolic housekeeping enzymes because of their ability to detoxify reactive aldehydes, like those generated from lipid peroxidation damage, the contributions of these enzymes to other biological processes are widespread. For example, the plant pathogen Pseudomonas syringae strain PtoDC3000 uses an indole-3-acetaldehyde dehydrogenase to synthesize the phytohormone indole-3-acetic acid to elude host responses. Here we investigate the biochemical function of AldC from PtoDC3000. Analysis of the substrate profile of AldC suggests that this enzyme functions as a long-chain aliphatic aldehyde dehydrogenase. The 2.5 Å resolution X-ray crystal of the AldC C291A mutant in a dead-end complex with octanal and NAD+ reveals an apolar binding site primed for aliphatic aldehyde substrate recognition. Functional characterization of site-directed mutants targeting the substrate- and NAD(H)-binding sites identifies key residues in the active site for ligand interactions, including those in the “aromatic box” that define the aldehyde-binding site. Overall, this study provides molecular insight for understanding the evolution of the prokaryotic aldehyde dehydrogenase superfamily and their diversity of function. Aldehyde dehydrogenases are versatile enzymes that serve a range of biochemical functions. Although traditionally considered metabolic housekeeping enzymes because of their ability to detoxify reactive aldehydes, like those generated from lipid peroxidation damage, the contributions of these enzymes to other biological processes are widespread. For example, the plant pathogen Pseudomonas syringae strain PtoDC3000 uses an indole-3-acetaldehyde dehydrogenase to synthesize the phytohormone indole-3-acetic acid to elude host responses. Here we investigate the biochemical function of AldC from PtoDC3000. Analysis of the substrate profile of AldC suggests that this enzyme functions as a long-chain aliphatic aldehyde dehydrogenase. The 2.5 Å resolution X-ray crystal of the AldC C291A mutant in a dead-end complex with octanal and NAD+ reveals an apolar binding site primed for aliphatic aldehyde substrate recognition. Functional characterization of site-directed mutants targeting the substrate- and NAD(H)-binding sites identifies key residues in the active site for ligand interactions, including those in the “aromatic box” that define the aldehyde-binding site. Overall, this study provides molecular insight for understanding the evolution of the prokaryotic aldehyde dehydrogenase superfamily and their diversity of function. In all organisms, the diversification of large superfamilies of enzymes provides a foundation for the evolution of biochemical capacity and the ability to metabolize varied small molecules (1Michael A.J. Evolution of biosynthetic diversity.Biochem. J. 2017; 474 (28655863): 2277-229910.1042/BCJ20160823Crossref PubMed Scopus (26) Google Scholar). Typically, across a superfamily, the core chemical mechanism is retained with substrate profiles becoming either specialized or promiscuous, depending on evolution and the metabolic needs of the organism (2Newton M.S. Arcus V.L. Gerth M.L. Patrick W.M. Enzyme evolution: innovation is easy, optimization is complicated.Curr. Opin. Struct. Biol. 2018; 48 (29207314): 110-11610.1016/j.sbi.2017.11.007Crossref PubMed Scopus (52) Google Scholar). Classic examples of enzyme superfamilies found across all kingdoms include the cytochromes P450, alcohol dehydrogenases, aldo-keto reductases, and aldehyde dehydrogenases (3Guengerich F.P. Cytochrome P450 and chemical toxicology.Chem. Res. Toxicol. 2008; 21 (18052394): 70-8310.1021/tx700079zCrossref PubMed Scopus (1174) Google Scholar, 4Nelson D.R. Cytochrome P450 and the individuality of species.Arch. Biochem. Biophys. 1999; 369 (10462435): 1-1010.1006/abbi.1999.1352Crossref PubMed Scopus (467) Google Scholar, 5Persson B. Hedlund J. Jörnvall H. Medium- and short-chain dehydrogenase/reductase gene and protein families: the MDR superfamily.Cell. Mol. Life Sci. 2008; 65 (19011751): 3879-389410.1007/s00018-008-8587-zCrossref PubMed Scopus (145) Google Scholar, 6Jörnvall H. Persson B. Krook M. Atrian S. Gonzàlez-Duarte R. Jeffery J. Ghosh D. Short-chain dehydrogenases/reductases (SDR).Biochemistry. 1995; 34 (7742302): 6003-601310.1021/bi00018a001Crossref PubMed Scopus (1158) Google Scholar, 7Penning T.M. The aldo-keto reductases (AKRs): overview.Chem.-Biol. Interact. 2015; 234 (25304492): 236-24610.1016/j.cbi.2014.09.024Crossref PubMed Scopus (254) Google Scholar, 8Jez J.M. Bennett M.J. Schlegel B.P. Lewis M. Penning T.M. Comparative anatomy of the aldo-keto reductase superfamily.Biochem. J. 1997; 326 (9307009): 625-63610.1042/bj3260625Crossref PubMed Scopus (534) Google Scholar, 9Yoshida A. Rzhetsky A. Hsu L.C. Chang C. Human aldehyde dehydrogenase gene family.Eur. J. Biochem. 1998; 251 (9490025): 549-55710.1046/j.1432-1327.1998.2510549.xCrossref PubMed Scopus (389) Google Scholar, 10Brocker C. Vasiliou M. Carpenter S. Carpenter C. Zhang Y. Wang X. Kotchoni S.O. Wood A.J. Kirch H.H. Kopečný D. Nebert D.W. Vasiliou V. Aldehyde dehydrogenase (ALDH) superfamily in plants: gene nomenclature and comparative genomics.Planta. 2013; 237 (23007552): 189-21010.1007/s00425-012-1749-0Crossref PubMed Scopus (112) Google Scholar). For example, aldehyde dehydrogenases are NAD(P)(H)-dependent enzymes that metabolize a wide range of aldehydes to their corresponding carboxylic acids in prokaryotes and eukaryotes and tend to be encoded by multiple genes in the genome of a given species (9Yoshida A. Rzhetsky A. Hsu L.C. Chang C. Human aldehyde dehydrogenase gene family.Eur. J. Biochem. 1998; 251 (9490025): 549-55710.1046/j.1432-1327.1998.2510549.xCrossref PubMed Scopus (389) Google Scholar, 10Brocker C. Vasiliou M. Carpenter S. Carpenter C. Zhang Y. Wang X. Kotchoni S.O. Wood A.J. Kirch H.H. Kopečný D. Nebert D.W. Vasiliou V. Aldehyde dehydrogenase (ALDH) superfamily in plants: gene nomenclature and comparative genomics.Planta. 2013; 237 (23007552): 189-21010.1007/s00425-012-1749-0Crossref PubMed Scopus (112) Google Scholar) (Fig. 1). Aldehyde dehydrogenases are generally associated with the detoxification of aldehydes, which are highly reactive compounds generated through cellular metabolism (11O'Brien P.J. Siraki A.G. Shangari N. Aldehyde sources, metabolism, molecular toxicity mechanisms, and possible effects on human health.Crit. Rev. Toxicol. 2005; 35 (16417045): 609-66210.1080/10408440591002183Crossref PubMed Scopus (509) Google Scholar). For example, these enzymes can scavenge aldehydes, such as malondialdehyde resulting from lipid peroxidation, and convert them to a less chemically reactive carboxylic acid (12Vasiliou V. Pappa A. Estey T. Role of human aldehyde dehydrogenases in endobiotic and xenobiotic metabolism.Drug Metab. Rev. 2004; 36 (15237855): 279-29910.1081/dmr-120034001Crossref PubMed Scopus (246) Google Scholar). Aldehyde dehydrogenases and their biochemical functions are also linked to a wide variety of biochemical processes ranging from ethanol metabolism via oxidation of acetaldehyde into acetate (13Klyosov A.A. Kinetics and specificity of human liver aldehyde dehydrogenases toward aliphatic, aromatic, and fused polycyclic aldehydes.Biochemistry. 1996; 35 (8605195): 4457-446710.1021/bi9521102Crossref PubMed Scopus (128) Google Scholar, 14Steinmetz C.G. Xie P. Weiner H. Hurley T.D. Structure of mitochondrial aldehyde dehydrogenase: the genetic component of ethanol aversion.Structure. 1997; 5 (9195888): 701-71110.1016/S0969-2126(97)00224-4Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar, 15Ni L. Zhou J. Hurley T.D. Weiner H. Human liver mitochondrial aldehyde dehydrogenase: three-dimensional structure and the restoration of solubility and activity of chimeric forms.Protein Sci. 1999; 8 (10631996): 2784-279010.1110/ps.8.12.2784Crossref PubMed Scopus (73) Google Scholar), polyamine metabolism (16Brocker C. Lassen N. Estey T. Pappa A. Cantore M. Orlova V. Chavakis T. Kavanagh K.L. Oppermann U. Vasiliou V. Aldehyde dehydrogenase 7A1 (ALDH7A1) is a novel enzyme involved in cellular defense against hyperosmotic stress.J. Biol. Chem. 2010; 285 (20207735): 18452-1846310.1074/jbc.M109.077925Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar), and plant cell wall ester biogenesis (17Nair R.B. Bastress K.L. Ruegger M.O. Denault J.W. Chapple C. The Arabidopsis thaliana REDUCED EPIDERMAL FLUORESCENCE1 gene encodes an aldehyde dehydrogenase involved in ferulic acid and sinapic acid biosynthesis.Plant Cell. 2004; 16 (14729911): 544-55410.1105/tpc.017509Crossref PubMed Scopus (193) Google Scholar, 18Bosch M. Mayer C.D. Cookson A. Donnison I.S. Identification of genes involved in cell wall biogenesis in grasses by differential gene expression profiling of elongating and non-elongating maize internodes.J. Exp. Bot. 2011; 62 (21402660): 3545-356110.1093/jxb/err045Crossref PubMed Scopus (95) Google Scholar) to protective cellular responses to stresses such as dehydration, osmotic shock, and temperature changes (19Kotchoni S.O. Kuhns C. Ditzer A. Kirch H.H. Bartels D. Over-expression of different aldehyde dehydrogenase genes in Arabidopsis thaliana confers tolerance to abiotic stress and protects plants against lipid peroxidation and oxidative stress.Plant Cell Environ. 2006; 29 (17080931): 1033-104810.1111/j.1365-3040.2005.01458.xCrossref PubMed Scopus (268) Google Scholar, 20Rodrigues S.M. Andrade M.O. Gomes A.P. Damatta F.M. Baracat-Pereira M.C. Fontes E.P. Arabidopsis and tobacco plants ectopically expressing the soybean antiquitin-like ALDH7 gene display enhanced tolerance to drought, salinity, and oxidative stress.J. Exp. Bot. 2006; 57 (16595581): 1909-191810.1093/jxb/erj132Crossref PubMed Scopus (137) Google Scholar). Recent work has also linked aldehyde dehydrogenase activity to the synthesis of indole-3-acetic acid, the primary plant hormone auxin, in the plant pathogenic microbe Pseudomonas syringae strain PtoDC3000 (21McClerklin S.A. Lee S.G. Harper C.P. Nwumeh R. Jez J.M. Kunkel B.N. Indole-3-acetaldehyde dehydrogenase-dependent auxin synthesis contributes to virulence of Pseudomonas syringae strain DC 3000.PLoS Pathog. 2018; 14 (29293681): e100681110.1371/journal.ppat.1006811Crossref PubMed Scopus (74) Google Scholar). To suppress host defenses and promote diseases development, Pseudomonas syringae produces a variety of virulence factors, including phytohormones or chemical mimics of hormones, to manipulate hormone signaling in its host plants (22Xin X.F. He S.Y. Pseudomonas syringae pv. tomato DC3000: a model pathogen for probing disease susceptibility and hormone signaling in plants.Annu. Rev. Phytopathol. 2013; 51 (23725467): 473-49810.1146/annurev-phyto-082712-102321Crossref PubMed Scopus (378) Google Scholar, 23Jones J.D.G. Dangl J.L. The plant immune system.Nature. 2006; 444 (17108957): 323-32910.1038/nature05286Crossref PubMed Scopus (8272) Google Scholar, 24Chisholm S.T. Coaker G. Day B. Staskawicz B.J. Host–microbe interactions: shaping the evolution of the plant immune response.Cell. 2006; 124 (16497589): 803-81410.1016/j.cell.2006.02.008Abstract Full Text Full Text PDF PubMed Scopus (2067) Google Scholar). P. syringae and many other plant-associated microbial pathogens can synthesize the major auxin indole-3-acetic acid (IAA), whose production is implicated in pathogen virulence (21McClerklin S.A. Lee S.G. Harper C.P. Nwumeh R. Jez J.M. Kunkel B.N. Indole-3-acetaldehyde dehydrogenase-dependent auxin synthesis contributes to virulence of Pseudomonas syringae strain DC 3000.PLoS Pathog. 2018; 14 (29293681): e100681110.1371/journal.ppat.1006811Crossref PubMed Scopus (74) Google Scholar, 25Djami-Tchatchou A.T. Harrison G.A. Harper C.P. Wang R. Prigge M. Estelle M. Kunkel B.N. Dual role of auxin in regulating plant defense and bacterial virulence gene expression during Pseudomonas syringae Pto DC3000 pathogenesis.Mol. Plant Microbe Interact. 2020; 33 (32407150): 1059-107110.1094/MPMI-02-20-0047-RCrossref PubMed Scopus (22) Google Scholar, 26Kunkel B.N. Harper C.P. The roles of auxin during interactions between bacterial plant pathogens and their hosts.J. Exp. Bot. 2018; 69 (29272462): 245-25410.1093/jxb/erx447Crossref PubMed Scopus (92) Google Scholar, 27Aragón I.M. Pérez-Martínez I. Moreno-Pérez A. Cerezo M. Ramos C. New insights into the role of indole-3-acetic acid in the virulence of Pseudomonas savastanoi pv. savastanoi.FEMS Microbiol. Lett. 2014; 356 (24606017): 184-19210.1111/1574-6968.12413Crossref PubMed Scopus (41) Google Scholar, 28Mutka A.M. Fawley S. Tsao T. Kunkel B.N. Auxin promotes susceptibility to Pseudomonas syringae via a mechanism independent of suppression of salicylic acid-mediated defenses.Plant J. 2013; 74 (23521356): 746-75410.1111/tpj.12157Crossref PubMed Scopus (70) Google Scholar, 29Manulis S. Haviv-Chesner A. Brandl M.T. Lindow S.E. Barash I. Differential involvement of indole-3-acetic acid biosynthetic pathways in pathogenicity and epiphytic fitness of Erwinia herbicola pv. gypsophilae.Mol. Plant Microbe. Interact. 1998; 11 (9650296): 634-64210.1094/MPMI.1998.11.7.634Crossref PubMed Scopus (129) Google Scholar). PtoDC3000 was shown to synthesize IAA using an uncharacterized pathway requiring indole-3-acetaldehyde dehydrogenase activity (21McClerklin S.A. Lee S.G. Harper C.P. Nwumeh R. Jez J.M. Kunkel B.N. Indole-3-acetaldehyde dehydrogenase-dependent auxin synthesis contributes to virulence of Pseudomonas syringae strain DC 3000.PLoS Pathog. 2018; 14 (29293681): e100681110.1371/journal.ppat.1006811Crossref PubMed Scopus (74) Google Scholar). Previously, a mutation in Azospirilum brasilense (aldA) was identified that decreased IAA production and was linked to a gene and annotated as encoding an aldehyde dehydrogenase (30Xie B. Xu K. Zhao H.X. Chen S.F. Isolation of transposon mutants from Azospirillum brasilense Yu62 and characterization of genes involved in indole-3-acetic acid biosynthesis.FEMS Microbiol. Lett. 2005; 248 (15961260): 57-6310.1016/j.femsle.2005.05.020Crossref PubMed Scopus (19) Google Scholar). Bioinformatic analysis of PtoDC3000 identified a set of aldehyde dehydrogenases (AldA–C) sharing 30–40% amino acid identity with each other. Subsequent metabolic, biochemical, and in planta analyses of AldA (UniProt: PSPTO_0092), AldB (UniProt: PSPTO_2673), and AldC (UniProt: PSPTO_3644) demonstrated that AldA functions as an indole-3-acetaldehyde dehydrogenase, is essential for IAA synthesis, and contributes to virulence of PtoDC3000 (21McClerklin S.A. Lee S.G. Harper C.P. Nwumeh R. Jez J.M. Kunkel B.N. Indole-3-acetaldehyde dehydrogenase-dependent auxin synthesis contributes to virulence of Pseudomonas syringae strain DC 3000.PLoS Pathog. 2018; 14 (29293681): e100681110.1371/journal.ppat.1006811Crossref PubMed Scopus (74) Google Scholar). AldB may also contribute to IAA synthesis but is not as metabolically or kinetically efficient as AldA for IAA production (21McClerklin S.A. Lee S.G. Harper C.P. Nwumeh R. Jez J.M. Kunkel B.N. Indole-3-acetaldehyde dehydrogenase-dependent auxin synthesis contributes to virulence of Pseudomonas syringae strain DC 3000.PLoS Pathog. 2018; 14 (29293681): e100681110.1371/journal.ppat.1006811Crossref PubMed Scopus (74) Google Scholar). Analysis of AldC indicates that it lacks a role in IAA synthesis (21McClerklin S.A. Lee S.G. Harper C.P. Nwumeh R. Jez J.M. Kunkel B.N. Indole-3-acetaldehyde dehydrogenase-dependent auxin synthesis contributes to virulence of Pseudomonas syringae strain DC 3000.PLoS Pathog. 2018; 14 (29293681): e100681110.1371/journal.ppat.1006811Crossref PubMed Scopus (74) Google Scholar); however, its potential biochemical role in PtoDC3000 was not fully examined. Here we investigate the substrate profile of AldC from PtoDC3000, which suggests that this enzyme functions as a long-chain aliphatic aldehyde dehydrogenase. The 2.5 Å resolution X-ray crystal of the AldC C291A mutant in complex with octanal and NAD+ and biochemical analysis of a set of site-directed mutants provide insight on substrate recognition in this enzyme. Comparison of the three-dimensional structures of AldA and AldC from PtoDC3000 reveals the sequence and structural changes that lead to distinct substrate profiles of these aldehyde dehydrogenases. Previously, three aldehyde dehydrogenases (AldA–C) from the plant pathogen P. syringae strain PtoDC3000 were identified and examined for their contribution to the synthesis of IAA and virulence (21McClerklin S.A. Lee S.G. Harper C.P. Nwumeh R. Jez J.M. Kunkel B.N. Indole-3-acetaldehyde dehydrogenase-dependent auxin synthesis contributes to virulence of Pseudomonas syringae strain DC 3000.PLoS Pathog. 2018; 14 (29293681): e100681110.1371/journal.ppat.1006811Crossref PubMed Scopus (74) Google Scholar). Each protein was expressed and purified to homogeneity for enzyme assays using indole-3-acetaldehyde as a substrate (21McClerklin S.A. Lee S.G. Harper C.P. Nwumeh R. Jez J.M. Kunkel B.N. Indole-3-acetaldehyde dehydrogenase-dependent auxin synthesis contributes to virulence of Pseudomonas syringae strain DC 3000.PLoS Pathog. 2018; 14 (29293681): e100681110.1371/journal.ppat.1006811Crossref PubMed Scopus (74) Google Scholar). Unlike AldA and AldB, which functioned as physiological tetramers, AldC was shown to be a homodimer (21McClerklin S.A. Lee S.G. Harper C.P. Nwumeh R. Jez J.M. Kunkel B.N. Indole-3-acetaldehyde dehydrogenase-dependent auxin synthesis contributes to virulence of Pseudomonas syringae strain DC 3000.PLoS Pathog. 2018; 14 (29293681): e100681110.1371/journal.ppat.1006811Crossref PubMed Scopus (74) Google Scholar). Steady-state kinetic parameters for AldA using indole-3-acetaldehyde showed this enzyme to be 130- and 710-fold more efficient than AldB and AldC, respectively (21McClerklin S.A. Lee S.G. Harper C.P. Nwumeh R. Jez J.M. Kunkel B.N. Indole-3-acetaldehyde dehydrogenase-dependent auxin synthesis contributes to virulence of Pseudomonas syringae strain DC 3000.PLoS Pathog. 2018; 14 (29293681): e100681110.1371/journal.ppat.1006811Crossref PubMed Scopus (74) Google Scholar). Bioinformatic analysis predicted that AldC contains the PF00171 domain, a signature domain of the aldehyde dehydrogenase superfamily, ranging from Ser33 to Ile482 (Fig. 2A). Specifically, the 4 catalytic residues (Asn159, Glu257, Gly288, and Cys291) and 19 residues in the NAD+-binding site (Ile155–Asn159, Lys182, Gly219, Ile233–Ser236, Ala239, Leu242, Glu257, Leu258, Gly259, Cys291, Glu391, and Phe393) define the aldehyde dehydrogenase consensus sequence motifs of AldC. The NCBI Conserved Protein Domain Family server places AldC in the cd01738/ALDH_CddD_SSP0762 family, 1 of 42 aldehyde dehydrogenase families found in the Pseudomonas (31Sophos N.A. Vasiliou V. Aldehyde dehydrogenase gene superfamily: the 2002 update.Chem. Biol. Interact. 2003; 143–144 (12604184): 5-2210.1016/S0009-2797(02)00163-1Crossref PubMed Scopus (284) Google Scholar, 32Riveros-Rosas H. Julián-Sánchez A. Moreno-Hagelsieb G. Muñoz-Clares R.A. Aldehyde dehydrogenase diversity in bacteria of the Pseudomonas genus.Chem. Biol. Interact. 2019; 304 (30862475): 83-8710.1016/j.cbi.2019.03.006Crossref PubMed Scopus (19) Google Scholar). The Pseudomonas orthologous groups classification system in the Pseudomonas Genome Database (RRID:SCR_006590) found orthologs of AldC (group ID POG018413) from 93 Pseudomonas species and strains, including P. aeruginosa, P. putida, P. fluorescens, and P. savastanoi (Fig. 2B and Table S1). AldC belongs to a clade consisting of aldehyde dehydrogenases, which share ∼90% sequence identity, from various plant pathogenic Pseudomonas spp.: P. syringae, P. viridiflava, P. savastanoi, and P. amygdali in the P. syringae phylogenetic group. None of AldC-related enzymes from Pseudomonas had previously been experimentally characterized, and their substrates and functions had yet to be described. To determine the substrate preference of AldC, a panel of 23 molecules (Table S2), including short- to long-chain aliphatic aldehydes (acetaldehyde, propionaldehyde, butyraldehyde, valeraldehyde (pentanal), isovaleraldehyde, 3-methylcrotonaldehyde, hexanal, trans-2-hexen-1-al, heptanal, octanal, nonanal, trans-2-nonenal), betaine aldehyde, and aromatic aldehydes (benzaldehyde, m-anisaldehyde, p-anisaldehyde, phenylacetaldehyde, cinnamaldehyde, hydrocinnamaldehyde, coniferylaldehyde, sinapaldehyde, 4-pyridinecarboxaldehyde, and indole-3-acetaldehyde) was used to screen for enzymatic activity. Spectrophotometric assays of AldC identified aliphatic aldehydes of 5–9-carbon length, as well as hydrocinnamaldehyde and 4-pyridinecarboxyaldehyde, as substrates (Fig. 3A) with octanal having the highest specific activity (Fig. 3B). These data suggest that short 2–4-carbon aldehydes, branched aliphatic aldehydes, and larger aromatic aldehydes are poor substrates for AldC. To evaluate the nicotinamide cofactor preference of AldC, the activity of the enzyme was tested with octanal and either NAD+ or NADP+, which showed a distinct preference for NAD+ (Fig. 3C). Steady-state kinetic analysis of AldC with valeraldehyde, hexanal, heptanal, octanal, and nonanal indicates that the 8-carbon substrate (i.e. octanal) is the preferred aliphatic aldehyde substrate (Table 1). Although the kcat values of AldC vary less than 3-fold between these substrates, the Km value for octanal is the lowest (1.2 mm) with that of valeraldehyde as the highest (48.8 mm). The overall effect is a marked difference in catalytic efficiency (kcat/Km) between octanal and the other aliphatic substrates ranging from use of nonanal exhibiting a 10-fold reduction in kcat/Km to ∼80-fold less efficient use of the 5-carbon substrate valeraldehyde. Generally, the aromatic aldehyde substrates hydrocinnamaldehyde and indole-3-acetaldehyde were poorly used by AldC with catalytic efficiencies comparable with valeraldehyde and 4-pyridinecarboxaldehyde used with a kcat/Km value 6-fold lower than octanal (Table 1). Overall, the biochemical analysis of AldC suggests that this enzyme functions primarily as a long-chain aliphatic aldehyde dehydrogenase (Fig. 3D). Moreover, conservation of related homologs in 93 different Pseudomonas species and strains (Fig. 2B) indicates a likely conserved function across these organisms.Table 1Steady-state kinetic parameters of AldC with aldehyde substratesSubstrateKcatKmKcat/Kmmin−1mmm−1 s−1Valeraldehyde/pentanal34.3 ± 4.248.8 ± 17.112Hexanal22.9 ± 1.016.4 ± 2.323Heptanal22.1 ± 1.57.4 ± 2.049Octanal67.4 ± 1.41.2 ± 0.1924Nonanal27.7 ± 5.04.9 ± 2.394Hydrocinnamaldehyde16.8 ± 1.614.2 ± 3.1204-Pyridinecarboxaldehyde38.7 ± 1.94.4 ± 0.6147Indole-3-acetaldehydeaSteady-state kinetic parameters of AldC with indole-3-acetaldehyde were previously reported (21).3.6 ± 0.21.3 ± 0.314NAD+ (octanal)30.9 ± 0.40.5 ± 0.11,050a Steady-state kinetic parameters of AldC with indole-3-acetaldehyde were previously reported (21McClerklin S.A. Lee S.G. Harper C.P. Nwumeh R. Jez J.M. Kunkel B.N. Indole-3-acetaldehyde dehydrogenase-dependent auxin synthesis contributes to virulence of Pseudomonas syringae strain DC 3000.PLoS Pathog. 2018; 14 (29293681): e100681110.1371/journal.ppat.1006811Crossref PubMed Scopus (74) Google Scholar). Open table in a new tab To understand how AldC recognizes aliphatic aldehyde substrates, crystals of the AldC C291A mutant were grown in the presence of octanal and NAD+. The analogous cysteine to Cys291 of AldC in aldehyde dehydrogenases, including AldA from P. syringae, is the essential catalytic residue (9Yoshida A. Rzhetsky A. Hsu L.C. Chang C. Human aldehyde dehydrogenase gene family.Eur. J. Biochem. 1998; 251 (9490025): 549-55710.1046/j.1432-1327.1998.2510549.xCrossref PubMed Scopus (389) Google Scholar, 10Brocker C. Vasiliou M. Carpenter S. Carpenter C. Zhang Y. Wang X. Kotchoni S.O. Wood A.J. Kirch H.H. Kopečný D. Nebert D.W. Vasiliou V. Aldehyde dehydrogenase (ALDH) superfamily in plants: gene nomenclature and comparative genomics.Planta. 2013; 237 (23007552): 189-21010.1007/s00425-012-1749-0Crossref PubMed Scopus (112) Google Scholar, 14Steinmetz C.G. Xie P. Weiner H. Hurley T.D. Structure of mitochondrial aldehyde dehydrogenase: the genetic component of ethanol aversion.Structure. 1997; 5 (9195888): 701-71110.1016/S0969-2126(97)00224-4Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar, 15Ni L. Zhou J. Hurley T.D. Weiner H. Human liver mitochondrial aldehyde dehydrogenase: three-dimensional structure and the restoration of solubility and activity of chimeric forms.Protein Sci. 1999; 8 (10631996): 2784-279010.1110/ps.8.12.2784Crossref PubMed Scopus (73) Google Scholar, 16Brocker C. Lassen N. Estey T. Pappa A. Cantore M. Orlova V. Chavakis T. Kavanagh K.L. Oppermann U. Vasiliou V. Aldehyde dehydrogenase 7A1 (ALDH7A1) is a novel enzyme involved in cellular defense against hyperosmotic stress.J. Biol. Chem. 2010; 285 (20207735): 18452-1846310.1074/jbc.M109.077925Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar, 21McClerklin S.A. Lee S.G. Harper C.P. Nwumeh R. Jez J.M. Kunkel B.N. Indole-3-acetaldehyde dehydrogenase-dependent auxin synthesis contributes to virulence of Pseudomonas syringae strain DC 3000.PLoS Pathog. 2018; 14 (29293681): e100681110.1371/journal.ppat.1006811Crossref PubMed Scopus (74) Google Scholar). The AldC C291A point mutant was generated by PCR mutagenesis, and the resulting protein was expressed, purified, and crystallized with the goal of obtaining a dead-end complex of the enzyme with octanal and NAD+ bound in the active site. The 2.5 Å resolution X-ray crystal structure of the AldC(C291A)·octanal·NAD+ complex was solved by molecular replacement using the three-dimensional structure of P. syringae AldA as a search model (21McClerklin S.A. Lee S.G. Harper C.P. Nwumeh R. Jez J.M. Kunkel B.N. Indole-3-acetaldehyde dehydrogenase-dependent auxin synthesis contributes to virulence of Pseudomonas syringae strain DC 3000.PLoS Pathog. 2018; 14 (29293681): e100681110.1371/journal.ppat.1006811Crossref PubMed Scopus (74) Google Scholar) (Table 2). Each of the two unique protein chains in the asymmetric unit form a corresponding physiological dimer through crystallographic symmetry (Fig. 4A). The secondary structure features and domains of the AldC monomer are similar to those of other aldehyde dehydrogenase family members (Fig. 4, B and C). The N-terminal Rossmann-fold domain contains a central β-sheet (β9-β8-β7-β10-β11) surrounded by α-helices to form the NAD(H)-binding site (Fig. 4, B and C, blue). The C-terminal region consists of a mixed α/β domain, which includes the catalytic cysteine residue and forms the aldehyde-binding site (Fig. 4, B and C, red). An interdomain linker region (Fig. 4, B and C, green) connects the N- and C-terminal domains of AldC. A small three-stranded β-sheet domain (Fig. 4, B and C, gold) facilitates oligomerization.Table 2Summary of crystallographic statistics for AldC structuresCrystalAldC (C291A)·octanal·NAD+Space groupC2221Cell dimensions (Å) a92.19 b92.17 c231.4Data collection Wavelength0.979 Å Resolution range (highest shell)39.6–2.52 Å (2.54–2.52 Å) Reflections (total/unique)116,137/32,956 Completeness (highest shell)97.8% (99.1%) <I/σ> (highest shell)12.8 (1.8) Rsymasym = Σ|Ih − <Ih>|/ΣIh, where <Ih> is the average intensity over symmetry. (highest shell)6.0% (52.9%)Refinement Rcrystbcryst = Σ|Fo − <Fc>|/ΣFo, where summation is over the data used for refinement./Rfreecfree is defined the same as Rcryst but was calculated using 5% of data excluded from refinement.17.1%/21.9% No. of protein atoms7,180 No. of waters197 No. of ligand atoms106 RMSD Bond lengths (Å)0.008 Bond angles (°)0.93 Average B-factor (Å2) Protein45.9 Water43.7 Ligand55.9 Stereochemistry (%) Favored95.7 Allowed4.3 Outliers0aR sym = Σ|Ih − <Ih>|/ΣIh, where <Ih> is the average intensity over symmetry.bR cryst = Σ|Fo − <Fc>|/ΣFo, where summation is over the data used for refinement.cR free is defined the same as Rcryst but was calculated using 5% of data excluded from refinement. Open table in a new tab The overall fold of AldC shares structural similarity with multiple aldehyde dehydrogenases, including P. syringae AldA, from a variety of microbes and eukaryotes (Fig. S1 and Table S3). Overlays of the AldC structure with twelve aldehyde, retinal, betaine aldehyde, and indole-3-acetaldehyde (i.e. AldA) dehydrogenases (14Steinmetz C.G. Xie P. Weiner H. Hurley T.D. Structure of mitochondrial aldehyde dehydrogenase: the genetic component of ethanol aversion.Structure. 1997; 5 (9195888): 701-71110.1016/S0969-2126(97)00224-4Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar, 15Ni L. Zhou J. Hurley T.D. Weiner H. Human liver mitochondrial aldehyde dehydrogenase: three-dimensional structure and the restoration of solubility and activity of chimeric forms.Protein Sci. 1999; 8 (10631996): 2784-279010.1110/ps.8.12.2784Crossref PubMed Scopus (73) Google Scholar, 21McClerklin S.A. Lee S.G. Harper C.P. Nwumeh R. Jez J.M. Kunkel B.N. Indole-3-acetaldehyde dehydrogenase-dependent auxin synthesis contributes to virulence of Pseudomonas syringae strain DC 3000.PLoS Pathog. 2018; 14 (29293681): e100681110.1371/journal.ppat.1006811Crossref PubMed Scopus (74) Google Scholar, 33Lamb A.L. Newcomer M.E. The structure of retinal dehydrogenase type II at 2.7 A resolution: implications for retinal specificity.Biochemistry. 1999; 38 (10320326): 6003-601110.1021/bi9900471Crossref PubMed Scopus (84) Google Scholar, 34Bateman O.A. Purkiss A.G. van Montfort R. Slingsby C. Graham C. Wistow G. Crystal structure of eta-crysta" @default.
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• W3048821712 title "The plant pathogen enzyme AldC is a long-chain aliphatic aldehyde dehydrogenase" @default.
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