A comparative study of two aldehyde dehydrogenases from Sphingobium sp.: the substrate spectrum and catalytic mechanism.

Biocatalytic oxidation is one of the most important and indispensable organic reactions for the development of green and sustainable biomanufacturing processes. NAD(P)+-dependent aldehyde dehydrogenase (ALDH) catalyzes the oxidation of aldehydes to carboxylic acids. Here, two ALDHs, SpALDH1 and SpALDH2, were identified from Sphingobium sp. SYK-6. They belong to different ALDH families and share only 32.30% amino acid identity. Interestingly, SpALDH1 and SpALDH2 exhibit significantly different enzymatic properties and substrate profiles. SpALDH2 has better thermostability than SpALDH1. SpALDH1 is a metalloenzyme and is activated by potassium ions, while SpALDH2 is not metallic-dependent. Compared with SpALDH1, SpALDH2 has a relatively broad substrate spectrum toward aromatic aldehydes. Based on homology modeling and molecular docking analysis, mechanisms underlying the substrate specificity of ALDHs were elucidated. For both ALDHs, hydrophobicity of substrate binding pockets is important for the catalytic properties, especially substrate specificity. Notably, optimization of the flexible loop 444-457 reforms a hydrogen bond between pyridine substrates and SpALDH1, contributing to the high catalytic activity. Finally, a coupling reaction catalyzed by ALDHs and NOX was constructed for efficient production of aromatic carboxylic acids.

Crystal structure of aldehyde dehydrogenase 1A1 from mouse.

Aldehyde dehydrogenase 1A1 (ALDH1A1) is an enzyme that catalyzes the NAD+-dependent oxidation of aldehydes to carboxylic acids, participating in various metabolic processes. Currently, only structures from human and Ovis aries have been reported. Here we show a 2.89 Å resolution structure of ALDH1A1 from mice using X-ray crystallography. We performed a detailed analysis of the structure and compared it with ALDH1A1 structures from two other species, highlighting the significance of the differences. Structural superimposition reveals that the tetrameric molecule is asymmetrical, and the NAD+-binding domain exhibits a certain rotation. In addition, the noticeable structural differences were detected, including the unique contact between Ser461 and Asp148, as well as the side chain orientations of three amino acids residues, Asn474, Met471 and Phe466. This study helps to expand the structural diversity of the ALDH family.

Curcumin-based-fluorescent probes targeting ALDH1A3 as a promising tool for glioblastoma precision surgery and early diagnosis.

Glioblastoma (GBM) is the most aggressive primary brain tumour for which both effective treatments and efficient tools for an early-stage diagnosis are lacking. Herein, we present curcumin-based fluorescent probes that are able to bind to aldehyde dehydrogenase 1A3 (ALDH1A3), an enzyme overexpressed in glioma stem cells (GSCs) and associated with stemness and invasiveness of GBM. Two compounds are selective versus ALDH1A3, without showing any appreciable interaction with other ALDH1A isoenzymes. Indeed, their fluorescent signal is detectable only in our positive controls in vitro and absent in cells that lack ALDH1A3. Remarkably, in vivo, our Probe selectively accumulate in glioblastoma cells, allowing the identification of the growing tumour mass. The significant specificity of our compounds is the necessary premise for their further development into glioblastoma cells detecting probes to be possibly used during neurosurgical operations.

Targeting colorectal cancer with small-molecule inhibitors of ALDH1B1.

Aldehyde dehydrogenases (ALDHs) are promising cancer drug targets, as certain isoforms are required for the survival of stem-like tumor cells. We have discovered selective inhibitors of ALDH1B1, a mitochondrial enzyme that promotes colorectal and pancreatic cancer. We describe bicyclic imidazoliums and guanidines that target the ALDH1B1 active site with comparable molecular interactions and potencies. Both pharmacophores abrogate ALDH1B1 function in cells; however, the guanidines circumvent an off-target mitochondrial toxicity exhibited by the imidazoliums. Our lead isoform-selective guanidinyl antagonists of ALDHs exhibit proteome-wide target specificity, and they selectively block the growth of colon cancer spheroids and organoids. Finally, we have used genetic and chemical perturbations to elucidate the ALDH1B1-dependent transcriptome, which includes genes that regulate mitochondrial metabolism and ribosomal function. Our findings support an essential role for ALDH1B1 in colorectal cancer, provide molecular probes for studying ALDH1B1 functions and yield leads for developing ALDH1B1-targeting therapies.

Overexpression of the Aldehyde Dehydrogenase Gene ZmALDH Confers Aluminum Tolerance in Arabidopsis thaliana.

Aluminum (Al) toxicity is the main factor limiting plant growth and the yield of cereal crops in acidic soils. Al-induced oxidative stress could lead to the excessive accumulation of reactive oxygen species (ROS) and aldehydes in plants. Aldehyde dehydrogenase (ALDH) genes, which play an important role in detoxification of aldehydes when exposed to abiotic stress, have been identified in most species. However, little is known about the function of this gene family in the response to Al stress. Here, we identified an ALDH gene in maize, ZmALDH, involved in protection against Al-induced oxidative stress. Al stress up-regulated ZmALDH expression in both the roots and leaves. The expression of ZmALDH only responded to Al toxicity but not to other stresses including low pH and other metals. The heterologous overexpression of ZmALDH in Arabidopsis increased Al tolerance by promoting the ascorbate-glutathione cycle, increasing the transcript levels of antioxidant enzyme genes as well as the activities of their products, reducing MDA, and increasing free proline synthesis. The overexpression of ZmALDH also reduced Al accumulation in roots. Taken together, these findings suggest that ZmALDH participates in Al-induced oxidative stress and Al accumulation in roots, conferring Al tolerance in transgenic Arabidopsis.

Evolution of aldehyde dehydrogenase genes and proteins in diploid and allotetraploid Xenopus frog species.

At least 19 human aldehyde dehydrogenase (ALDH) genes and enzymes have been studied among vertebrate organisms. BLAT and BLAST analyses were undertaken of Xenopus tropicalis (western clawed frog) and Xenopus laevis (African clawed frog) genomes which are related diploid (N = 20) and allotetraploid (N = 36) species, respectively. The corresponding ALDH genes and proteins within these Xenopus genomes were identified and studied. Evidence is presented for tetraploid copies of 10 Xenopus laevis ALDH genes, whereas another 7 identified ALDH genes were diploid in nature. Xenopus laevis and Xenopus tropicalis ALDH amino acid sequences were highly homologous with the human enzymes, with the exception of the mitochondrial signal peptide sequences. Amino acids performing catalytic and structural roles were conserved and identified based on previous reports of 3D structures for the corresponding mammalian enzymes.

Study of ALDH from Thermus thermophilus-Expression, Purification and Characterisation of the Non-Substrate Specific, Thermophilic Enzyme Displaying Both Dehydrogenase and Esterase Activity.

Aldehyde dehydrogenases (ALDH), found in all kingdoms of life, form a superfamily of enzymes that primarily catalyse the oxidation of aldehydes to form carboxylic acid products, while utilising the cofactor NAD(P)+. Some superfamily members can also act as esterases using p-nitrophenyl esters as substrates. The ALDHTt from Thermus thermophilus was recombinantly expressed in E. coli and purified to obtain high yields (approximately 15-20 mg/L) and purity utilising an efficient heat treatment step coupled with IMAC and gel filtration chromatography. The use of the heat treatment step proved critical, in its absence decreased yield of 40% was observed. Characterisation of the thermophilic ALDHTt led to optimum enzymatic working conditions of 50 °C, and a pH of 8. ALDHTt possesses dual enzymatic activity, with the ability to act as a dehydrogenase and an esterase. ALDHTt possesses broad substrate specificity, displaying activity for a range of aldehydes, most notably hexanal and the synthetic dialdehyde, terephthalaldehyde. Interestingly, para-substituted benzaldehydes could be processed efficiently, but ortho-substitution resulted in no catalytic activity. Similarly, ALDHTt displayed activity for two different esterase substrates, p-nitrophenyl acetate and p-nitrophenyl butyrate, but with activities of 22.9% and 8.9%, respectively, compared to the activity towards hexanal.

Nuclear receptors NHR-49 and NHR-79 promote peroxisome proliferation to compensate for aldehyde dehydrogenase deficiency in C. elegans.

The intracellular level of fatty aldehydes is tightly regulated by aldehyde dehydrogenases to minimize the formation of toxic lipid and protein adducts. Importantly, the dysregulation of aldehyde dehydrogenases has been implicated in neurologic disorder and cancer in humans. However, cellular responses to unresolved, elevated fatty aldehyde levels are poorly understood. Here, we report that ALH-4 is a C. elegans aldehyde dehydrogenase that specifically associates with the endoplasmic reticulum, mitochondria and peroxisomes. Based on lipidomic and imaging analysis, we show that the loss of ALH-4 increases fatty aldehyde levels and reduces fat storage. ALH-4 deficiency in the intestine, cell-nonautonomously induces NHR-49/NHR-79-dependent hypodermal peroxisome proliferation. This is accompanied by the upregulation of catalases and fatty acid catabolic enzymes, as indicated by RNA sequencing. Such a response is required to counteract ALH-4 deficiency since alh-4; nhr-49 double mutant animals are sterile. Our work reveals unexpected inter-tissue communication of fatty aldehyde levels and suggests pharmacological modulation of peroxisome proliferation as a therapeutic strategy to tackle pathology related to excess fatty aldehydes.

Mining of two novel aldehyde dehydrogenases (DHY-SC-VUT5 and DHY-G-VUT7) from metagenome of hydrocarbon contaminated soils.

BACKGROUND: Aldehyde dehydrogenases are vital for aerobic hydrocarbon degradation and is involved in the last step of catalysing the oxidation of aldehydes to carboxylic acids. With the global increase in hydrocarbon pollution of different environments, these enzymes have the potential to be used in enzymatic bioremediation applications. RESULTS: Fifteen fosmid clones with hydrocarbon degrading potential were functionally screened to identify dehydrogenase enzymes. Accordingly, the fosmid insert of the positive clones were sequenced using PacBio next generation sequencing platform and de novo assembled using CLC Genomic Work Bench. The 1233 bp long open reading frame (ORF) for DHY-SC-VUT5 was found to share a protein sequence similarity of 97.7% to short-chain dehydrogenase from E. coli. The 1470 bp long ORF for DHY-G-VUT7 was found to share a protein sequence similarity of 23.9% to glycine dehydrogenase (decarboxylating) (EC 1.4.4.2) from Caulobacter vibrioides (strain NA1000 / CB15N) (Caulobacter crescentus). The in silico analyses and blast against UNIPROT protein database with the stated similarity show that the two dehydrogenases are novel. Biochemical characterization revealed, that the highest relative activity was observed at substrate concentrations of 150 mM and 50 mM for DHY-SC-VUT5 and DHY-G-VUT7, respectively. The Km values were found to be 13.77 mM with a Vmax of 0.009135 µmol.min- 1 and 2.832 mM with a Vmax of 0.005886 µmol.min- 1 for DHY-SC-VUT5 and DHY-G-VUT7, respectively. Thus, a potent and efficient enzyme for alkyl aldehyde conversion to carboxylic acid. CONCLUSION: The microorganisms overexpressing the novel aldehyde dehydrogenases could be used to make up microbial cocktails for biodegradation of alkanes. Moreover, since the discovered enzymes are novel it would be interesting to solve their structures by crystallography and explore the downstream applications.

Impact of missense mutations in the ALDH7A1 gene on enzyme structure and catalytic function.

Certain mutations in the ALDH7A1 gene cause pyridoxine-dependent epilepsy (PDE), an autosomal recessive metabolic disease characterized by seizures, and in some cases, intellectual disability. The mutational spectrum of PDE is vast and includes over 70 missense mutations. This review summarizes the current state of biochemical and biophysical research on the impact of PDE missense mutations on the structure and catalytic activity of ALDH7A1. Paradoxically, some mutations that target active site residues have a relatively modest impact on structure and function, while those remote from the active site can have profound effects. For example, missense mutations targeting remote residues in oligomer interfaces tend to strongly impact catalytic function by inhibiting formation of the active tetramer. These results shows that it remains very difficult to predict the impact of missense mutations, even when the structure of the wild-type enzyme is known. Additional biophysical analyses of many more disease-causing mutations are needed to develop the rules for predicting the impact of genetic mutations on enzyme structure and catalytic function.

The plant pathogen enzyme AldC is a long-chain aliphatic aldehyde dehydrogenase.

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.

Purification and characterization of molybdenum-containing aldehyde dehydrogenase that oxidizes benzyl maltol derivative from Pseudomonas nitroreducens SB32154.

Maltol derivatives are used in a variety of fields due to their metal-chelating abilities. In the previous study, it was found that cytochrome P450 monooxygenase, P450nov, which has the ability to effectively convert the 2-methyl group in a maltol derivative, transformed 3-benzyloxy-2-methyl-4-pyrone (BMAL) to 2-(hydroxymethyl)-3-(phenylmethoxy)-4H-pyran-4-one (BMAL-OH) and slightly to 3-benzyloxy-4-oxo-4 H-pyran-2-carboxaldehyde (BMAL-CHO). We isolated Pseudomonas nitroreducens SB32154 with the ability to convert BMAL-CHO to BMAL-COOH from soil. The enzyme responsible for aldehyde oxidation, a BMAL-CHO dehydrogenase, was purified from P. nitroreducens SB32154 and characterized. The purified BMAL-CHO dehydrogenase was found to be a xanthine oxidase family enzyme with unique structure of heterodimer composed of 75 and 15 kDa subunits containing a molybdenum cofactor and [Fe-S] clusters, respectively. The enzyme showed broad substrate specificity toward benzaldehyde derivatives. Furthermore, one-pot conversion of BMAL to BMAL-COOH via BMAL-CHO by the combination of the BMAL-CHO dehydrogenase with P450nov was achieved.

Inhibition, crystal structures, and in-solution oligomeric structure of aldehyde dehydrogenase 9A1.

Aldehyde dehydrogenase 9A1 (ALDH9A1) is a human enzyme that catalyzes the NAD+-dependent oxidation of the carnitine precursor 4-trimethylaminobutyraldehyde to 4-N-trimethylaminobutyrate. Here we show that the broad-spectrum ALDH inhibitor diethylaminobenzaldehyde (DEAB) reversibly inhibits ALDH9A1 in a time-dependent manner. Possible mechanisms of inhibition include covalent reversible inactivation involving the thiohemiacetal intermediate and slow, tight-binding inhibition. Two crystal structures of ALDH9A1 are reported, including the first of the enzyme complexed with NAD+. One of the structures reveals the active conformation of the enzyme, in which the Rossmann dinucleotide-binding domain is fully ordered and the inter-domain linker adopts the canonical ß-hairpin observed in other ALDH structures. The oligomeric structure of ALDH9A1 was investigated using analytical ultracentrifugation, small-angle X-ray scattering, and negative stain electron microscopy. These data show that ALDH9A1 forms the classic ALDH superfamily dimer-of-dimers tetramer in solution. Our results suggest that the presence of an aldehyde substrate and NAD+ promotes isomerization of the enzyme into the active conformation.

Coupled regulations of enzymatic activity and structure formation of aldehyde dehydrogenase Ald4p.

Previously, we have developed an extramitochondrial assembly system, where mitochondrial targeting signal (MTS) can be removed from a given mitochondrial enzyme, which could be used to characterize the regulatory factors involved in enzyme assembly/disassembly in vivo Here, we demonstrate that addition of exogenous acetaldehyde can quickly induce the supramolecular assembly of MTS-deleted aldehyde dehydrogenase Ald4p in yeast cytoplasm. Also, by using PCR-based modification of the yeast genome, cytoplasmically targeted Ald4p cannot polymerize into long filaments when key functional amino acid residues are substituted, as shown by N192D, S269A, E290K and C324A mutations. This study has confirmed that extramitochondrial assembly could be a powerful external system for studying mitochondrial enzyme assembly, and its regulatory factors outside the mitochondria. In addition, we propose that mitochondrial enzyme assembly/disassembly is coupled to the regulation of a given mitochondrial enzyme activity.

Molecular Dynamics Analysis of a Rationally Designed Aldehyde Dehydrogenase Gives Insights into Improved Activity for the Non-Native Cofactor NAD.

The aldehyde dehydrogenase from Thermoplasma acidophilum was previously implemented as a key enzyme in a synthetic cell-free reaction cascade for the production of alcohols. In order to engineer the enzyme's cofactor specificity from NADP+ to NAD+, we identified selectivity-determining residues with the CSR-SALAD tool and investigated further positions based on the crystal structure. Stepwise combination of the initially discovered six point mutations allowed us to monitor the cross effects of each mutation, resulting in a final variant with reduced KM for the non-native cofactor NAD+ (from 18 to 0.6 mM) and an increased activity for the desired substrate d-glyceraldehyde (from 0.4 to 1.5 U/mg). Saturation mutagenesis of the residues at the entrance of the substrate pocket could eliminate substrate inhibition. Molecular dynamics simulations showed a significant gain of flexibility at the cofactor binding site for the final variant. The concomitant increase in stability against isobutanol and only a minor reduction in its temperature stability render the final variant a promising candidate for future optimization of our synthetic cell-free enzymatic cascade.

Functional models of nonheme diiron enzymes: reactivity of the µ-oxo-µ-1,2-peroxo-diiron(iii) intermediate in electrophilic and nucleophilic reactions.

The reactivity of the previously reported peroxo-adduct [FeIII2(µ-O)(µ-1,2-O2)(IndH)2(solv)2]2+ (1) (IndH = 1,3-bis(2-pyridyl-imino)isoindoline) has been investigated in nucleophilic (e.g., deformylation of alkyl and aryl alkyl aldehydes) and electrophilic (e.g. oxidation of phenols) stoichiometric reactions as biomimics of ribonucleotide reductase (RNR-R2) and aldehyde deformylating oxygenase (ADO) enzymes. Based on detailed kinetic and mechanistic studies, we have found further evidence for the ambiphilic behaviour of the peroxo intermediates proposed for diferric oxidoreductase enzymes.

Structural analysis of pathogenic mutations targeting Glu427 of ALDH7A1, the hot spot residue of pyridoxine-dependent epilepsy.

Certain loss-of-function mutations in the gene encoding the lysine catabolic enzyme aldehyde dehydrogenase 7A1 (ALDH7A1) cause pyridoxine-dependent epilepsy (PDE). Missense mutations of Glu427, especially Glu427Gln, account for ~30% of the mutated alleles in PDE patients, and thus Glu427 has been referred to as a mutation hot spot of PDE. Glu427 is invariant in the ALDH superfamily and forms ionic hydrogen bonds with the nicotinamide ribose of the NAD+ cofactor. Here we report the first crystal structures of ALDH7A1 containing pathogenic mutations targeting Glu427. The mutant enzymes E427Q, Glu427Asp, and Glu427Gly were expressed in Escherichia coli and purified. The recombinant enzymes displayed negligible catalytic activity compared to the wild-type enzyme. The crystal structures of the mutant enzymes complexed with NAD+ were determined to understand how the mutations impact NAD+ binding. In the E427Q and E427G structures, the nicotinamide mononucleotide is highly flexible and lacks a defined binding pose. In E427D, the bound NAD+ adopts a "retracted" conformation in which the nicotinamide ring is too far from the catalytic Cys residue for hydride transfer. Thus, the structures revealed a shared mechanism for loss of function: none of the variants are able to stabilise the nicotinamide of NAD+ in the pose required for catalysis. We also show that these mutations reduce the amount of active tetrameric ALDH7A1 at the concentration of NAD+ tested. Altogether, our results provide the three-dimensional molecular structural basis of the most common pathogenic variants of PDE and implicate strong (ionic) hydrogen bonds in the aetiology of a human disease.

Structural and biochemical consequences of pyridoxine-dependent epilepsy mutations that target the aldehyde binding site of aldehyde dehydrogenase ALDH7A1.

In humans, certain mutations in the gene encoding aldehyde dehydrogenase 7A1 are associated with pyridoxine-dependent epilepsy (PDE). Understanding the impact of PDE-causing mutations on the structure and activity of ALDH7A1 could allow for the prediction of symptom-severity and aid the development of patient-specific medical treatments. Herein, we investigate the biochemical and structural consequences of PDE missense mutations targeting residues in the aldehyde substrate binding site: N167S, P169S, A171V, G174V, and W175G. All but G174V could be purified for biochemical and X-ray crystallographic analysis. W175G has a relatively mild kinetic defect, exhibiting a fivefold decrease in kcat with no change in Km . P169S and N167S have moderate defects, characterized by catalytic efficiencies of 20- and 100-times lower than wild-type, respectively. A171V has a profound functional defect, with catalytic efficiency 2000-times lower than wild-type. The crystal structures of the variants are the first for any PDE-associated mutant of ALDH7A1. The structures show that missense mutations that decrease the steric bulk of the side chain tend to create a cavity in the active site. The protein responds by relaxing into the vacant space, and this structural perturbation appears to cause misalignment of the aldehyde substrate in W175G and N167S. The P169S structure is nearly identical to that of the wild-type enzyme; however, analysis of B-factors suggests the catalytic defect may result from altered protein dynamics. The A171V structure suggests that the potential for steric clash with Val171 prevents Glu121 from ion pairing with the amino group of the aldehyde substrate. ENZYMES: Aldehyde dehydrogenase 7A1 (EC1.2.1.31). DATABASES: Coordinates have been deposited in the Protein Data Bank under the following accession codes: 6O4B, 6O4C, 6O4D, 6O4E, 6O4F, 6O4G, 6O4H.

Enhancement in the Catalytic Activity of Human Salivary Aldehyde Dehydrogenase by Alliin from Garlic: Implications in Aldehyde Toxicity and Oral Health.

BACKGROUND: Lower human salivary aldehyde dehydrogenase (hsALDH) activity increases the risk of aldehyde mediated pathogenesis including oral cancer. Alliin, the bioactive compound of garlic, exhibits many beneficial health effects. OBJECTIVE: To study the effect of alliin on hsALDH activity. METHODS: Enzyme kinetics was performed to study the effect of alliin on the activity of hsALDH. Different biophysical techniques were employed for structural and binding studies. Docking analysis was done to predict the binding region and the type of binding forces. RESULTS: Alliin enhanced the dehydrogenase activity of the enzyme. It slightly reduced the Km and significantly enhanced the Vmax value. At 1 µM alliin concentration, the initial reaction rate increased by about two times. Further, it enhanced the hsALDH esterase activity. Biophysical studies indicated a strong complex formation between the enzyme and alliin (binding constant, Kb: 2.35 ± 0.14 x 103 M-1). It changes the secondary structure of hsALDH. Molecular docking study indicated that alliin interacts to the enzyme near the substrate binding region involving some active site residues that are evolutionary conserved. There was a slight increase in the nucleophilicity of active site cysteine in the presence of alliin. Ligand efficiency metrics values indicate that alliin is an efficient ligand for the enzyme. CONCLUSION: Alliin activates the catalytic activity of the enzyme. Hence, consumption of alliincontaining garlic preparations or alliin supplements and use of alliin in pure form may lower aldehyde related pathogenesis including oral carcinogenesis.

Expression, purification and crystallization of the novel Xenopus tropicalis ALDH16B1, a homologue of human ALDH16A1.

ALDH16 is a novel family of the aldehyde dehydrogenase (ALDH) superfamily with unique structural characteristics that distinguish it from the other ALDH superfamily members. In addition to structural characteristics, there is an evolutionary-related grouping within the ALDH 16 genes. The ALDH16 isozymes in frog, lower animals, and bacteria possess a critical Cys residue in their active site, which is absent from ALDH16 in mammals and fish. Genomic analysis and plasma metabolomic studies have associated ALDH16A1 with the pathogenesis of gout in humans, although its actual involvement in this disease is poorly understood. Insight into the structure of ALDH16A1 is an important step in deciphering its function in gout. Herein, we report our efforts towards the structural characterization of Xenopus tropicalis ALDH16B1 (the homolog of human ALDH16A1) that was predicted to be catalytically-active. Recombinant ALDH16B1 was expressed in Sf9 cells and purified using affinity and size exclusion chromatography. Crystallization of ALDH16B1 was achieved by vapor diffusion. A data set was collected at 2.5 Šand preliminary crystallographic analysis showed that the frog ALDH16B1 crystals belong to the P 212 121 space group with unit cell parameters a = 80.48 Å, b = 89.73 Å, c = 190.92 Å, α = ß = γ = 90.00°. Structure determination is currently in progress.