Structural and Functional Analysis of Human SIRT1

SIRT1 is a NAD⁺-dependent deacetylase that plays important roles in many cellular processes. SIRT1 activity is uniquely controlled by a C-terminal regulatory segment (CTR). Here we present crystal structures of the catalytic domain of human SIRT1 in complex with the CTR in an open apo form and a closed conformation in complex with a cofactor and a pseudo-substrate peptide. The catalytic domain adopts the canonical sirtuin fold. The CTR forms a β hairpin structure that complements the β sheet of the NAD⁺-binding domain, covering an essentially invariant hydrophobic surface. The apo form adopts a distinct open conformation, in which the smaller subdomain of SIRT1 undergoes a rotation with respect to the larger NAD⁺-binding subdomain. A biochemical analysis identifies key residues in the active site, an inhibitory role for the CTR, and distinct structural features of the CTR that mediate binding and inhibition of the SIRT1 catalytic domain.     

Structural and Functional Characterization of Plant Aminoaldehyde Dehydrogenase from Pisum sativum with a Broad Specificity for Natural and Synthetic Aminoaldehydes

Aminoaldehyde dehydrogenases (AMADHs, EC 1.2.1.19) belong to the large aldehyde dehydrogenase (ALDH) superfamily, namely, the ALDH9 family. They oxidize polyamine-derived ω-aminoaldehydes to the corresponding ω-amino acids. Here, we report the first X-ray structures of plant AMADHs: two isoenzymes, PsAMADH1 and PsAMADH2, from Pisum sativum in complex with β-nicotinamide adenine dinucleotide (NAD⁺) at 2.4 and 2.15 Å resolution, respectively. Both recombinant proteins are dimeric and, similarly to other ALDHs, each monomer is composed of an oligomerization domain, a coenzyme binding domain and a catalytic domain. Each subunit binds NAD⁺ as a coenzyme, contains a solvent-accessible C-terminal peroxisomal targeting signal (type 1) and a cation bound in the cavity close to the NAD⁺ binding site. While the NAD⁺ binding mode is classical for PsAMADH2, that for PsAMADH1 is unusual among ALDHs. A glycerol molecule occupies the substrate binding site and mimics a bound substrate. Structural analysis and substrate specificity study of both isoenzymes in combination with data published previously on other ALDH9 family members show that the established categorization of such enzymes into distinct groups based on substrate specificity is no more appropriate, because many of them seem capable of oxidizing a large spectrum of aminoaldehyde substrates. PsAMADH1 and PsAMADH2 can oxidize N,N,N-trimethyl-4-aminobutyraldehyde into γ-butyrobetaine, which is the carnitine precursor in animal cells. This activity highly suggests that in addition to their contribution to the formation of compatible osmolytes such as glycine betaine, β-alanine betaine and γ-aminobutyric acid, AMADHs might participate in carnitine biosynthesis in plants.     

The tetrameric assembly of 2-aminomuconic 6-semialdehyde dehydrogenase is a functional requirement of cofactor NAD+ binding

The bacterium Pseudomonas sp. AP-3 is able to use the environmental pollutant 2-aminophenol as its sole source of carbon, nitrogen, and energy. Eight genes (amnA, B, C, D, E, F, G, and H) encoding 2-aminophenol metabolizing enzymes are clustered into a single operon. 2-Aminomuconic 6-semialdehyde dehydrogenase (AmnC), a member of the aldehyde dehydrogenase (ALDH) superfamily, is responsible for oxidizing 2-aminomuconic 6-semialdehyde to 2-aminomuconate. In contrast to many other members of the ALDH superfamily, the structural basis of the catalytic activity of AmnC remains elusive. Here, we present the crystal structure of AmnC, which displays a homotetrameric quaternary assembly that is directly involved in its enzymatic activity. The tetrameric state of AmnC in solution was also presented using small-angle X-ray scattering. The tetramerization of AmnC is mediated by the assembly of a protruding hydrophobic beta-strand motif and residues V121 and S123 located in the NAD⁺-binding domain of each subunit. Dimeric mutants of AmnC dramatically lose NAD⁺ binding affinity and failed to oxidize the substrate analogue 2-hydroxymuconate-6-semialdehyde to α-hydroxymuconic acid, indicating that tetrameric assembly of AmnC is functional requirement.     

Enhanced aldehyde dehydrogenase activity by regenerating NAD+ in Klebsiella pneumoniae and implications for the glycerol dissimilation pathways

In Klebsiella pneumoniae, 3-hydroxypropaldehyde is converted to 3-hydroxypropionic acid (3-HP) by aldehyde dehydrogenase (ALDH) with NAD⁺ as a cofactor. Although ALDH overexpression stimulates the formation of 3-HP, it ceases to accumulate when NAD⁺ is exhausted. Here we show that NAD⁺ regeneration, together with ALDH overexpression, facilitates 3-HP production and benefits cell growth. Three distinct NAD⁺-regenerating enzymes: NADH oxidase and NADH dehydrogenase from K. pneumoniae, and glycerol-3-phosphate dehydrogenase (GPD1) from Saccharomyces cerevisiae, were individually expressed in K. pneumoniae. In vitro assay showed their higher activities than that of the control, indicating their capacities to regenerate NAD⁺. When they were respectively co-expressed with ALD4, an ALDH from S. cerevisiae, the activities of ALD4 were significantly elevated compared with that expressing ALD4 alone, suggesting that the regenerated NAD⁺ enhanced the activity of ALD4. More interestingly, the growth rates of all NAD⁺-regenerating strains were prolonged in comparison with the control, indicating that NAD⁺ regeneration stimulated cell proliferation. This study not only reveals the reliance of ALD4 activity on NAD⁺ availability but also provides a method for regulating the dha regulon.     

Aldehyde dehydrogenase enzyme ALDH3H1 from Arabidopsis thaliana: Identification of amino acid residues critical for cofactor specificity

The cofactor-binding site of the NAD⁺-dependent Arabidopsis thaliana aldehyde dehydrogenase ALDH3H1 was analyzed to understand structural features determining cofactor-specificity. Homology modeling and mutant analysis elucidated important amino acid residues. Glu149 occupies a central position in the cofactor-binding cleft, and its carboxylate group coordinates the 2′- and 3′-hydroxyl groups of the adenosyl ribose ring of NAD⁺ and repels the 2′-phosphate moiety of NADP⁺. If Glu149 is mutated to Gln, Asp, Asn or Thr the binding of NAD⁺ is altered and rendered the enzyme capable of using NADP⁺. This change is attributed to a weaker steric hindrance and elimination of the electrostatic repulsion force of the 2′-phosphate of NADP⁺. Simultaneous mutations of Glu149 and Ile200, which is situated opposite of the cofactor binding cleft, improved the enzyme efficiency with NADP⁺. The double mutant ALDH3H1_{Glu149Thr/Ile200Val} showed a good catalysis with NADP⁺. Subsequently a triple mutation was generated by replacing Val178 by Arg in order to create a “closed” cofactor binding site. The cofactor specificity was shifted even further in favor of NADP⁺, as the mutant ALDH3H1_{E149T/V178R/I200V} uses NADP⁺ with almost 7-fold higher catalytic efficiency compared to NAD⁺. Our experiments suggest that residues occupying positions equivalent to 149, 178 and 200 constitute a group of amino acids in the ALDH3H1 protein determining cofactor affinity.     

Investigating the reaction and substrate preference of indole-3-acetaldehyde dehydrogenase from the plant pathogen Pseudomonas syringae PtoDC3000

Aldehyde dehydrogenases (ALDHs) catalyze the conversion of various aliphatic and aromatic aldehydes into corresponding carboxylic acids. Traditionally considered as housekeeping enzymes, new biochemical roles are being identified for members of ALDH family. Recent work showed that AldA from the plant pathogen Pseudomonas syringae strain PtoDC3000 (PtoDC3000) functions as an indole-3-acetaldehyde dehydrogenase for the synthesis of indole-3-acetic acid (IAA). IAA produced by AldA allows the pathogen to suppress salicylic acid-mediated defenses in the model plant Arabidopsis thaliana. Here we present a biochemical and structural analysis of the AldA indole-3-acetaldehyde dehydrogenase from PtoDC3000. Site-directed mutants targeting the catalytic residues Cys³⁰² and Glu²⁶⁷ resulted in a loss of enzymatic activity. The X-ray crystal structure of the catalytically inactive AldA C302A mutant in complex with IAA and NAD⁺ showed the cofactor adopting a conformation that differs from the previously reported structure of AldA. These structures suggest that NAD⁺ undergoes a conformational change during the AldA reaction mechanism similar to that reported for human ALDH. Site-directed mutagenesis of the IAA binding site indicates that changes in the active site surface reduces AldA activity; however, substitution of Phe¹⁶⁹ with a tryptophan altered the substrate selectivity of the mutant to prefer octanal. The present study highlights the inherent biochemical versatility of members of the ALDH enzyme superfamily in P. syringae.     

Structural and Biochemical Characterization of Aldehyde Dehydrogenase 12, the Last Enzyme of Proline Catabolism in Plants

Heterokonts, Alveolata protists, green algae from Charophyta and Chlorophyta divisions, and all Embryophyta plants possess an aldehyde dehydrogenase (ALDH) gene named ALDH12. Here, we provide a biochemical characterization of two ALDH12 family members from the lower plant Physcomitrella patens and higher plant Zea mays. We show that ALDH12 encodes an NAD⁺-dependent glutamate γ-semialdehyde dehydrogenase (GSALDH), which irreversibly converts glutamate γ-semialdehyde (GSAL), a mitochondrial intermediate of the proline and arginine catabolism, to glutamate. Sedimentation equilibrium and small-angle X-ray scattering analyses reveal that in solution both plant GSALDHs exist as equilibrium between a domain-swapped dimer and the dimer-of-dimers tetramer. Plant GSALDHs share very low-sequence identity with bacterial, fungal, and animal GSALDHs (classified as ALDH4), which are the closest related ALDH superfamily members. Nevertheless, the crystal structure of ZmALDH12 at 2.2-Å resolution shows that nearly all key residues involved in the recognition of GSAL are identical to those in ALDH4, indicating a close functional relationship with ALDH4. Phylogenetic analysis suggests that the transition from ALDH4 to ALDH12 occurred during the evolution of the endosymbiotic plant ancestor, prior to the evolution of green algae and land plants. Finally, ALDH12 expression in maize and moss is downregulated in response to salt and drought stresses, possibly to maintain proline levels. Taken together, these results provide molecular insight into the biological roles of the plant ALDH12 family.     

Insight into the Mechanism of Intramolecular Inhibition of the Catalytic Activity of Sirtuin 2 (SIRT2)

Sirtuin 2 (SIRT2) is a NAD⁺-dependent deacetylase that has been associated with neurodegeneration and cancer. SIRT2 is composed of a central catalytic domain, the structure of which has been solved, and N- and C-terminal extensions that are thought to control SIRT2 function. However structural information of these N- and C-terminal regions is missing. Here, we provide the first full-length molecular models of SIRT2 in the absence and presence of NAD⁺. We also predict the structural alterations associated with phosphorylation of SIRT2 at S331, a modification that inhibits catalytic activity. Bioinformatics tools and molecular dynamics simulations, complemented by in vitro deacetylation assays, provide a consistent picture based on which the C-terminal region of SIRT2 is suggested to function as an autoinhibitory region. This has the capacity to partially occlude the NAD⁺ binding pocket or stabilize the NAD⁺ in a non-productive state. Furthermore, our simulations suggest that the phosphorylation at S331 causes large conformational changes in the C-terminal region that enhance the autoinhibitory activity, consistent with our previous findings that phosphorylation of S331 by cyclin-dependent kinases inhibits SIRT2 catalytic activity. The molecular insight into the role of the C-terminal region in controlling SIRT2 function described in this study may be useful for future design of selective inhibitors targeting SIRT2 for therapeutic applications.     

The three-dimensional structural basis of type II hyperprolinemia

Type II hyperprolinemia is an autosomal recessive disorder caused by a deficiency in Δ¹-pyrroline-5-carboxylate dehydrogenase (P5CDH; also known as ALDH4A1), the aldehyde dehydrogenase that catalyzes the oxidation of glutamate semialdehyde to glutamate. Here, we report the first structure of human P5CDH (HsP5CDH) and investigate the impact of the hyperprolinemia-associated mutation of Ser352 to Leu on the structure and catalytic properties of the enzyme. The 2. 5-Å-resolution crystal structure of HsP5CDH was determined using experimental phasing. Structures of the mutant enzymes S352A (2.4 Å) and S352L (2.85 Å) were determined to elucidate the structural consequences of altering Ser352. Structures of the 93% identical mouse P5CDH complexed with sulfate ion (1.3 Å resolution), glutamate (1.5 Å), and NAD⁺ (1.5 Å) were determined to obtain high-resolution views of the active site. Together, the structures show that Ser352 occupies a hydrophilic pocket and is connected via water-mediated hydrogen bonds to catalytic Cys348. Mutation of Ser352 to Leu is shown to abolish catalytic activity and eliminate NAD⁺ binding. Analysis of the S352A mutant shows that these functional defects are caused by the introduction of the nonpolar Leu352 side chain rather than the removal of the Ser352 hydroxyl. The S352L structure shows that the mutation induces a dramatic 8-Å rearrangement of the catalytic loop. Because of this conformational change, Ser349 is not positioned to interact with the aldehyde substrate, conserved Glu447 is no longer poised to bind NAD⁺, and Cys348 faces the wrong direction for nucleophilic attack. These structural alterations render the enzyme inactive.     

Rossmann-fold motifs can confer multiple functions to metabolic enzymes: RNA binding and ribonuclease activity of a UDP-glucose dehydrogenase

Metabolic enzymes are usually characterized to have one specific function, and this is the case of UDP-glucose dehydrogenase that catalyzes the twofold NAD⁺-dependent oxidation of UDP-glucose into UDP-glucuronic acid. We have determined that this enzyme is also capable of participating in other cellular processes. Here, we report that the bacterial UDP-glucose dehydrogenase (UgdG) from Sphingomonas elodea ATCC 31461, which provides UDP-glucuronic acid for the synthesis of the exopolysaccharide gellan, is not only able to bind RNA but also acts as a ribonuclease. The ribonucleolytic activity occurs independently of the presence of NAD⁺ and the RNA binding site does not coincide with the NAD⁺ binding region. We have also performed the kinetics of interaction between UgdG and RNA. Moreover, computer analysis reveals that the N- and C-terminal domains of UgdG share structural features with ancient mitochondrial ribonucleases named MAR. MARs are present in lower eukaryotic microorganisms, have a Rossmannoid-fold and belong to the isochorismatase superfamily. This observation reinforces that the Rossmann structural motifs found in NAD⁺-dependent dehydrogenases can have a dual function working as a nucleotide cofactor binding domain and as a ribonuclease.     

Cloning,expression and characterization of a novel aldehyde dehydrogenase gene from Flammeovirga pacifica

A novel putative aldehyde dehydrogenase (ALDH) gene aldh1413 from Flammeovirga pacifica isolated from deep sea sediment was cloned, expressed, and characterized. The molecular weight of the ALDH1413 (479 amino acids) was estimated by SDS-PAGE to be 53 kDa. The optimum temperature and pH for ALDH1413 were 35°C and 9.0, respectively. In the presence of either NAD⁺ or NADP⁺, the enzyme could oxidize a number of aliphatic aldehydes, particularly C3-and C5-aliphatic aldehydes and aromatic aldehydes such as benzaldehyde, which indicates that the enzyme belongs to broad-specific (ALDH) superfamily. Steady-state kinetic study revealed that ALDH1413 had a K _M value of 0.545 mM and a k _cat value of 7.48 s^?1 when propionaldehyde was used as the substrate. The Na⁺ could enhance ALDH1413 activity, which indicated it might be adapt to its habitat, marine environment.     

Identification and characterization of Klebsiella pneumoniae aldehyde dehydrogenases increasing production of 3-hydroxypropionic acid from glycerol

Klebsiella pneumoniae produces 3-hydroxypropionic acid (3-HP) from glycerol with oxidation of 3-hydroxypropionaldehyde (3-HPA) to 3-HP in a reaction catalyzed by aldehyde dehydrogenase (ALDH). In the present study, two putative ALDHs of K. pneumoniae, YneI and YdcW were identified and characterized. Recombinant YneI was specifically active on 3-HPA and preferred NAD⁺ as a cofactor, whereas YdcW exhibited broad substrate specificity and preferred NADP⁺ as a cofactor. Overexpression of ALDHs in the glycerol oxidative pathway-deficient mutant K. pneumoniae AK resulted in a significant increase in 3-HP production upon shake-flask culture. The final titers of 3-HP were 2.4 and 1.8 g L^?1 by recombinants overexpressing YneI and YdcW, respectively. Deletion of the ALDH gene from K. pneumoniae did not affect the extent of 3-HP synthesis, implying non-specific activity of ALDHs on 3-HPA. The ALDHs might play major roles in detoxifying the aldehyde generated in glycerol metabolism.     

Kinetic evidence for human liver and stomach aldehyde dehydrogenase-3 representing an unique class of isozymes

Substrate and coenzyme specificities of human liver and stomach aldehyde dehydrogenase (ALDH) isozymes were compared by staining with various aldehydes including propionaldehyde, heptaldehyde, decaldehyde, 2-furaldehyde, succinic semialdehyde, and glutamic -semialdehyde and with NAD⁺ or NADP⁺ on agarose isoelectric focusing gels. ALDH3 isozyme was isolated from a liver via carboxymethyl-Sephadex and blue Sepharose chromatographies and its kinetic constants for various substrates and coenzymes were determined. Consistent with the previously proposed genetic model for human ALDH3 isozymes (Yinet al., Biochem. Genet. 26:343, 1988), a single liver form and multiple stomach forms exhibited similar kinetic properties, which were strikingly distinct from those of ALDH1, ALDH2, and ALDH4 (glutamic -semialdehyde dehydrogenase). A set of activity assays using various substrates, coenzymes, and an inhibitor to distinguish ALDH1, ALDH2, ALDH3, and ALDH4 is presented. As previously reported in ALDH1 and ALDH2, a higher catalytic efficiency (V _max/K _m) for oxidation of long-chain aliphatic aldehydes was found in ALDH3, suggesting that these enzymes have a hydrophobic barrel-shape substrate binding pocket. Since theK _m value for acetaldehyde for liver ALDH3, 83 mM, is very much higher than those of ALDH1 and ALDH2, ALDH3 thus represents an unique class of human ALDH isozymes and it appears not to be involved in ethanol metabolism.This work was supported by grants from the National Science Council and the Academia Sinica, Republic of China.     

Aldehyde dehydrogenase (ALDH) superfamily in plants: gene nomenclature and comparative genomics

In recent years, there has been a significant increase in the number of completely sequenced plant genomes. The comparison of fully sequenced genomes allows for identification of new gene family members, as well as comprehensive analysis of gene family evolution. The aldehyde dehydrogenase (ALDH) gene superfamily comprises a group of enzymes involved in the NAD⁺- or NADP⁺-dependent conversion of various aldehydes to their corresponding carboxylic acids. ALDH enzymes are involved in processing many aldehydes that serve as biogenic intermediates in a wide range of metabolic pathways. In addition, many of these enzymes function as ‘aldehyde scavengers’ by removing reactive aldehydes generated during the oxidative degradation of lipid membranes, also known as lipid peroxidation. Plants and animals share many ALDH families, and many genes are highly conserved between these two evolutionarily distinct groups. Conversely, both plants and animals also contain unique ALDH genes and families. Herein we carried out genome-wide identification of ALDH genes in a number of plant species—including Arabidopsis thaliana (thale crest), Chlamydomonas reinhardtii (unicellular algae), Oryza sativa (rice), Physcomitrella patens (moss), Vitis vinifera (grapevine) and Zea mays (maize). These data were then combined with previous analysis of Populus trichocarpa (poplar tree), Selaginella moellindorffii (gemmiferous spikemoss), Sorghum bicolor (sorghum) and Volvox carteri (colonial algae) for a comprehensive evolutionary comparison of the plant ALDH superfamily. As a result, newly identified genes can be more easily analyzed and gene names can be assigned according to current nomenclature guidelines; our goal is to clarify previously confusing and conflicting names and classifications that might confound results and prevent accurate comparisons between studies.     

Interaction of sheep liver cytosolic aldehyde dehydrogenase with quercetin,resveratrol and diethylstilbestrol

The effects of quercetin and resveratrol (substances found in red wine) on the activity of cytosolic aldehyde dehydrogenase in vitro are compared with those of the synthetic hormone diethylstilbestrol. It is proposed that quercetin inhibits the enzyme by binding competitively in both the aldehyde substrate binding-pocket and the NAD⁺-binding site, whereas resveratrol and diethylstilbestrol can only bind in the aldehyde site. When inhibition is overcome by high aldehyde and NAD⁺ concentrations (1 mM of each), the modifiers enhance the activity of the enzyme; we hypothesise that this occurs through binding to the enzyme-NADH complex and consequent acceleration of the rate of dissociation of NADH. The proposed ability of quercetin to bind in both enzyme sites is supported by gel filtration experiments with and without NAD⁺, by studies of the esterase activity of the enzyme, and by modelling the quercetin molecule into the known three-dimensional structure of the enzyme. The possibility that interaction between aldehyde dehydrogenase and quercetin may be of physiological significance is discussed.     

NAD+ binding to the Escherichia coli K+-uptake protein TrkA and sequence similarity between TrkA and domains of a family of dehydrogenases suggest a role for NAD+ in bacterial transport

The nucleotide sequence of trkA, a gene encoding a surface component of the constitutive K⁺-uptake systems TrkG and TrkH from Escherichia coli, was determined. The structure of the TrkA protein deduced from the nucleotide sequence accords with the view that TrkA is peripherally bound to the inner side of the cytoplasmic membrane. Analysis by a dot matrix revealed that TrkA is composed of similar halves. The M-terminal part of each TrkA half (residues 1–130 and 234–355, respectively) is similar to the complete NAD⁺-binding domain of NAD⁺-dependent dehydrogenases. The C-terminal part of each TrkA half (residues 131–233 and 357–458, respectively) aligns with the first 100 residues of the catalytic domain of glyceraldehyde-3-phosphate dehydrogenase. Strong u.v. illumination at 252 nm led to cross-linking of NAD⁺ or NADH, but not of ATP to the isolated TrkA protein.     

Structural and functional characterization of a plant S-nitrosoglutathione reductase from Solanum lycopersicum

S-nitrosoglutathione reductase (GSNOR), also known as S-(hydroxymethyl)glutathione (HMGSH) dehydrogenase, belongs to the large alcohol dehydrogenase superfamily, namely to the class III ADHs. GSNOR catalyses the oxidation of HMGSH to S-formylglutathione using a catalytic zinc and NAD⁺ as a coenzyme. The enzyme also catalyses the NADH-dependent reduction of S-nitrosoglutathione (GSNO). In plants, GSNO has been suggested to serve as a nitric oxide (NO) reservoir locally or possibly as NO donor in distant cells and tissues. NO and NO-related molecules such as S-nitrosothiols (S-NOs) play a central role in the regulation of normal plant physiological processes and host defence. The enzyme thus participates in the cellular homeostasis of S-NOs and in the metabolism of reactive nitrogen species. Although GSNOR has recently been characterized from several organisms, this study represents the first detailed biochemical and structural characterization of a plant GSNOR, that from tomato (Solanum lycopersicum). SlGSNOR gene expression is higher in roots and stems compared to leaves of young plants. It is highly expressed in the pistil and stamens and in fruits during ripening. The enzyme is a dimer and preferentially catalyses reduction of GSNO while glutathione and S-methylglutathione behave as non-competitive inhibitors. Using NAD⁺, the enzyme oxidizes HMGSH and other alcohols such as cinnamylalcohol, geraniol and ω-hydroxyfatty acids. The crystal structures of the apoenzyme, of the enzyme in complex with NAD⁺ and in complex with NADH, solved up to 1.9 Å resolution, represent the first structures of a plant GSNOR. They confirm that the binding of the coenzyme is associated with the active site zinc movement and changes in its coordination. In comparison to the well characterized human GSNOR, plant GSNORs exhibit a difference in the composition of the anion-binding pocket, which negatively influences the affinity for the carboxyl group of ω-hydroxyfatty acids.     

Structural and biochemical characterization of a novel aldehyde dehydrogenase encoded by the benzoate oxidation pathway in Burkholderia xenovorans LB400

The recently identified benzoate oxidation (box) pathway in Burkholderia xenovorans LB400 (LB400 hereinafter) assimilates benzoate through a unique mechanism where each intermediate is processed as a coenzyme A (CoA) thioester. A key step in this process is the conversion of 3,4-dehydroadipyl-CoA semialdehyde into its corresponding CoA acid by a novel aldehyde dehydrogenase (ALDH) (EC 1.2.1.x). The goal of this study is to characterize the biochemical and structural properties of the chromosomally encoded form of this new class of ALDHs from LB400 (ALDH_C) in order to better understand its role in benzoate degradation. To this end, we carried out kinetic studies with six structurally diverse aldehydes and nicotinamide adenine dinucleotide (phosphate) (NAD ⁺ and NADP ⁺). Our data definitively show that ALDH_C is more active in the presence of NADP ⁺ and selective for linear medium-chain to long-chain aldehydes. To elucidate the structural basis for these biochemical observations, we solved the 1.6-Å crystal structure of ALDH_C in complex with NADPH bound in the cofactor-binding pocket and an ordered fragment of a polyethylene glycol molecule bound in the substrate tunnel. These data show that cofactor selectivity is governed by a complex network of hydrogen bonds between the oxygen atoms of the 2′-phosphoryl moiety of NADP ⁺ and a threonine/lysine pair on ALDH_C. The catalytic preference of ALDH_C for linear longer-chain substrates is mediated by a deep narrow configuration of the substrate tunnel. Comparative analysis reveals that reorientation of an extended loop (Asn478-Pro490) in ALDH_C induces the constricted structure of the substrate tunnel, with the side chain of Asn478 imposing steric restrictions on branched-chain and aromatic aldehydes. Furthermore, a key glycine (Gly104) positioned at the mouth of the tunnel allows for maximum tunnel depth required to bind medium-chain to long-chain aldehydes. This study provides the first integrated biochemical and structural characterization of a box-pathway-encoded ALDH from any organism and offers insight into the catalytic role of ALDH_C in benzoate degradation.     

Pocket analysis of the full-length cholix toxin. An assessment of the structure–dynamics of the apo catalytic domain

Cholix toxin from Vibrio cholerae is the third member of the diphtheria toxin (DT) group of mono-ADP-ribosyltransferase (mART) bacterial toxins. It shares structural and functional properties with Pseudomonas aeruginosa exotoxin A and Corynebacterium diphtheriae DT. Cholix toxin is an important model for the development of antivirulence approaches and therapeutics against these toxins from pathogenic bacteria. Herein, we have used the high-resolution X-ray structure of full-length cholix complexed with NAD⁺ to describe the properties of the NAD⁺-binding pocket at the residue level, including the role of crystallographic water molecules in the NAD⁺ substrate interaction. The full-length apo cholix structure is used to describe the putative NAD⁺-binding site(s) and to correlate biochemical with crystallographic data to study the stoichiometry and orientation of bound NAD⁺ molecules. We quantitatively describe the NAD⁺ substrate interactions on a residue basis for the main 22 pocket residues in cholix_f, a glycerol and 5 contact water molecules as part of the recognition surface by the substrate according to the conditions of crystallization. In addition, the dynamic properties of an in silico version of the catalytic domain were investigated in order to understand the lack of electronic density for one of the main flexible loops (R-loop) in the pocket of X-ray complexes. Implications for a rational drug design approach for mART toxins are derived.