Structural insights into the octamerization of glycerol dehydrogenase.

Glycerol dehydrogenase (GDH) catalyzes glycerol oxidation to dihydroxyacetone in a NAD+-dependent manner. As an initiator of the oxidative pathway of glycerol metabolism, a variety of functional and structural studies of GDH have been conducted previously. Structural studies revealed intriguing features of GDH, like the flexible ß-hairpin and its significance. Another commonly reported structural feature is the enzyme's octameric oligomerization, though its structural details and functional significance remained unclear. Here, with a newly reported GDH structure, complexed with both NAD+ and glycerol, we analyzed the octamerization of GDH. Structural analyses revealed that octamerization reduces the structural dynamics of the N-domain, which contributes to more consistently maintaining a distance required for catalysis between the cofactor and substrate. This suggests that octamerization may play a key role in increasing the likelihood of the enzyme reaction by maintaining the ligands in an appropriate configuration for catalysis. These findings expand our understanding of the structure of GDH and its relation to the enzyme's activity.

Structural and functional insights into the flexible ß-hairpin of glycerol dehydrogenase.

During glycerol metabolism, the initial step of glycerol oxidation is catalysed by glycerol dehydrogenase (GDH), which converts glycerol to dihydroxyacetone in a NAD+ -dependent manner via an ordered Bi-Bi kinetic mechanism. Structural studies conducted with GDH from various species have mainly elucidated structural details of the active site and ligand binding. However, the structure of the full GDH complex with both cofactor and substrate bound is not determined, and thus, the structural basis of the kinetic mechanism of GDH remains unclear. Here, we report the crystal structures of Escherichia coli GDH with a substrate analogue bound in the absence or presence of NAD+ . Structural analyses including molecular dynamics simulations revealed that GDH possesses a flexible ß-hairpin, and that during the ordered progression of the kinetic mechanism, the flexibility of the ß-hairpin is reduced after NAD+ binding. It was also observed that this alterable flexibility of the ß-hairpin contributes to the cofactor binding and possibly to the catalytic efficiency of GDH. These findings suggest the importance of the flexible ß-hairpin to GDH enzymatic activity and shed new light on the kinetic mechanism of GDH.

DCXR promotes cell proliferation by promoting the activity of aerobic glycolysis in breast cancer.

The function of human dicarbonyl/L­xylulose reductase (DCXR) in the pathophysiology of breast cancer is yet to be elucidated. The present study aimed to investigate the function of DCXR in glycolysis and the cell cycle of breast cancer cells with respect to cell proliferation. Differential expressed DCXR was identified in The Cancer Genome Atlas (TCGA) database and verified in clinical breast cancer tissue. DCXR silencing and overexpression were induced by RNA interference and lentiviral vectors, respectively. Cell cycle progression, proliferation and glycolytic activity of breast cancer cells were detected by flow cytometry, Cell Counting Kit­8 assay and chemical methods, respectively. Tumorigenicity was detected using nude mice xenograft models. The expression of DCXR was increased in TCGA breast cancer database and the function of DCXR was enriched in 'glycolysis' and 'cell cycle'. Further analysis using clinical breast cancer samples confirmed upregulation of DCXR. The silencing of DCXR suppressed proliferation and cell cycle progression of breast cancer cells and significantly decreased the capacity for glycolysis, thereby demonstrating the effect of DCXR on the function of breast cancer cells. Similar conclusions were obtained in DCXR overexpressing cells; notably, DCXR overexpression promoted proliferation, cell cycle progression at S phase and glycolysis. 2­Deoxy­D­glucose inhibited the effect of DCXR on the proliferation and cell cycle progression of breast cancer cells. The present study revealed that DCXR regulated breast cancer cell cycle progression and proliferation by increasing glycolysis activity and thus may serve as an oncogene for breast cancer.

Unidirectional mannitol synthesis of Acinetobacter baumannii MtlD is facilitated by the helix-loop-helix-mediated dimer formation.

Persistence of Acinetobacter baumannii in environments with low water activity is largely attributed to the biosynthesis of compatible solutes. Mannitol is one of the key compatible solutes in A. baumannii, and it is synthesized by a bifunctional mannitol-1-phosphate dehydrogenase/phosphatase (AbMtlD). AbMtlD catalyzes the conversion of fructose-6-phosphate to mannitol in two consecutive steps. Here, we report the crystal structure of dimeric AbMtlD, constituting two protomers each with a dehydrogenase and phosphatase domain. A proper assembly of AbMtlD dimer is facilitated by an intersection comprising a unique helix­loop­helix (HLH) domain. Reduction and dephosphorylation catalysis of fructose-6-phosphate to mannitol is dependent on the transient dimerization of AbMtlD. AbMtlD presents as a monomer under lower ionic strength conditions and was found to be mainly dimeric under high-salt conditions. The AbMtlD catalytic efficiency was markedly increased by cross-linking the protomers at the intersected HLH domain via engineered disulfide bonds. Inactivation of the AbMtlD phosphatase domain results in an intracellular accumulation of mannitol-1-phosphate in A. baumannii, leading to bacterial growth impairment upon salt stress. Taken together, our findings demonstrate that salt-induced dimerization of the bifunctional AbMtlD increases catalytic dehydrogenase and phosphatase efficiency, resulting in unidirectional catalysis of mannitol production.

Milestones in treatments for inborn errors of metabolism: Reflections on Where chemistry and medicine meet.

From Sir Archibald Garrod's initial description of the tetrad of albinism, alkaptonuria, cystinuria, and pentosuria to today, the field of medicine dedicated to inborn errors of metabolism has evolved from disease identification and mechanistic discovery to the development of therapies designed to subvert biochemical defects. In this review, we highlight major milestones in the treatment and diagnosis of inborn errors of metabolism, starting with dietary therapy for phenylketonuria in the 1950s and 1960s, and ending with current approaches in genetic manipulation.

Using galactitol dehydrogenase coupled with water-forming NADH oxidase for efficient enzymatic synthesis of L-tagatose.

L-Tagatose, a promising building block in the production of many value-added chemicals, is generally produced by chemical routes with a low yield, which may not meet the increasing demands. Synthesis of l-tagatose by enzymatic oxidation of d-galactitol has not been applied on an industrial scale because of the high cofactor costs and the lack of efficient cofactor regeneration methods. In this work, an efficient and environmentally friendly enzymatic method containing a galactitol dehydrogenase for d-galactitol oxidation and a water-forming NADH oxidase for regeneration of NAD+ was first designed and used for l-tagatose production. Supplied with only 3 mM NAD+, subsequent reaction optimization facilitated the efficient transformation of 100 mM of d-galactitol into l-tagatose with a yield of 90.2 % after 12 h (obtained productivity: 7.61 mM.h-1). Compared with the current chemical and biocatalytic methods, the strategy developed avoids by-product formation and achieves the highest yield of l-tagatose with low costs. It is expected to become a cleaner and more promising route for industrial biosynthesis of l-tagatose.

Conversion of levoglucosan into glucose by the coordination of four enzymes through oxidation, elimination, hydration, and reduction.

Levoglucosan (LG) is an anhydrosugar produced through glucan pyrolysis and is widely found in nature. We previously isolated an LG-utilizing thermophile, Bacillus smithii S-2701M, and suggested that this bacterium may have a metabolic pathway from LG to glucose, initiated by LG dehydrogenase (LGDH). Here, we completely elucidated the metabolic pathway of LG involving three novel enzymes in addition to LGDH. In the S-2701M genome, three genes expected to be involved in the LG metabolism were found in the vicinity of the LGDH gene locus. These four genes including LGDH gene (lgdA, lgdB1, lgdB2, and lgdC) were expressed in Escherichia coli and purified to obtain functional recombinant proteins. Thin layer chromatography analyses of the reactions with the combination of the four enzymes elucidated the following metabolic pathway: LgdA (LGDH) catalyzes 3-dehydrogenation of LG to produce 3-keto-LG, which undergoes ß-elimination of 3-keto-LG by LgdB1, followed by hydration to produce 3-keto-D-glucose by LgdB2; next, LgdC reduces 3-keto-D-glucose to glucose. This sequential reaction mechanism resembles that proposed for an enzyme belonging to glycoside hydrolase family 4, and results in the observational hydrolysis of LG into glucose with coordination of the four enzymes.

Identification and functional characterization of glycerol dehydrogenase reveal the role in kojic acid synthesis in Aspergillus oryzae.

Glycerol dehydrogenase has been identified and characterized functionally in many species. However, little is known about glycerol dehydrogenase genes and their functions in Aspergillus oryzae. Here, a total of 45 glycerol dehydrogenase genes in Aspergillus oryzae were identified and renamed from AoGld1 to AoGld45 according to their chromosome distribution. They were classified into three groups based on phylogenetic analysis. Synteny analysis revealed that thirteen AoGld genes are conserved among Aspergillus species. Promoter analysis displayed that AoGld3 and AoGld13 harbored multiple binding elements of GATA-type transcription factors and zinc-finger protein msnA that were involved in nitrogen and kojic acid metabolism, respectively. Moreover, the AoGld3 deletion strain Δgld3 was generated by the CRISPR/Cas9 system, which had no visible growth defects compared with the control wild-type strain under the control and osmotic stress treatments. However, disruption of AoGld3 led to the inhibition of kojic acid production, and the expression of kojA, kojR was down-regulated in the Δgld3 strain. Furthermore, when kojA or kojR was overexpressed in the Δgld3 strain, the yield of kojic acid was restored, suggesting that AoGld3 is involved in kojic acid production through affecting the expression of kojR and kojA. Taken together, these findings provide new insights into our understanding of glycerol dehydrogenase and establish foundation for further study of their roles in Aspergillus oryzae.

Selective and high yield transformation of glycerol to lactic acid using NNN pincer ruthenium catalysts.

The conversion of glycerol selectively to lactic acid has been accomplished in high yields (ca. 90%) by using a NNN pincer-Ru catalyst. DFT explains the role of the Ru-P bond and sterics in favoring the catalysis.

Purification and characterization of a novel medium-chain ribitol dehydrogenase from a lichen-associated bacterium Sphingomonas sp.

Sugar alcohols (polyols) are abundant carbohydrates in lichen-forming algae and transported to other lichen symbionts, fungi, and bacteria. Particularly, ribitol is an abundant polyol in the lichen Cetraria sp. Polyols have important physiological roles in lichen symbiosis, but polyol utilization in lichen-associated bacteria has been largely unreported. Herein, we purified and characterized a novel ribitol dehydrogenase (RDH) from a Cetraria sp.-associated bacterium Sphingomonas sp. PAMC 26621 grown on a minimal medium containing D-ribitol (the RDH hereafter referred to as SpRDH). SpRDH is present as a trimer in its native form, and the molecular weight of SpRDH was estimated to be 39 kDa by SDS-PAGE and 117 kDa by gel filtration chromatography. SpRDH converted D-ribitol to D-ribulose using NAD+ as a cofactor. As far as we know, SpRDH is the first RDH belonging to the medium-chain dehydrogenase/reductase family. Multiple sequence alignments indicated that the catalytic amino acid residues of SpRDH consist of Cys37, His65, Glu66, and Glu157, whereas those of short-chain RDHs consist of Ser, Tyr, and Lys. Furthermore, unlike other short-chain RDHs, SpRDH did not require divalent metal ions for its catalytic activity. Despite SpRDH originating from a psychrophilic Arctic bacterium, Sphingomonas sp., it had maximum activity at 60°C and exhibited high thermal stability within the 4-50°C range. Further studies on the structure/function relationship and catalytic mechanism of SpRDH will expand our understanding of its role in lichen symbiosis.

Dimeric dihydrodiol dehydrogenase is an efficient primate 1,5-anhydro-D-fructose reductase.

1,5-Anhydro-D-fructose (AF), a metabolite of the anhydrofructose pathway of glycogen metabolism, has recently been shown to react with intracellular proteins and form advanced glycation end-products. The reactive AF is metabolized to non-reactive 1,5-anhydro-D-glucitol by AF reductase in animal tissues and human cells. Pig and mouse AF reductases were characterized, but primate AF reductase remains unknown. Here, we examined the AF-reducing activity of eleven primate NADPH-dependent reductases with broad substrate specificity for carbonyl compounds. AF was reduced by monkey dimeric dihydrodiol dehydrogenase (DHDH), human aldehyde reductase (AKR1A1) and human dicarbonyl/L-xylulose reductase (DCXR). DHDH showed the lowest KM (21 µM) for AF, and its kcat/KM value (1208 s-1mM-1) was much higher than those of AKR1A1 (1.3 s-1mM-1), DCXR (1.1 s-1mM-1) and the pig and mouse AF reductases. AF is a novel substrate with higher affinity and catalytic efficiency than known substrates of DHDH. Docking simulation study suggested that Lys156 in the substrate-binding site of DHDH contributes to the high affinity for AF. Gene database searches identified DHDH homologues (with >95% amino acid sequence identity) in humans and apes. Thus, DHDH acts as an efficient AF reductase in primates.

Permeabilized Escherichia coli Whole Cells Containing Co-Expressed Two Thermophilic Enzymes Facilitate the Synthesis of scyllo-Inositol from myo-Inositol.

Scyllo-inositol (SI), a stereoisomer of inositol, is regarded as a promising therapeutic agent for Alzheimer's disease. Here, an in vitro cofactor-balance biotransformation for the production of SI from myo-inositol (MI) by thermophilic myo-inositol 2-dehydrogenase (IDH) and scyllo-inositol 2-dehydrogenase (SIDH) is presented. These two enzymes (i.e., IDH and SIDH from Geobacillus kaustophilus) are co-expressed in Escherichia coli BL21(DE3), and E. coli cells containing the two enzymes are permeabilized by heat treatment as whole-cell catalysts to convert MI to SI. After condition optimizations about permeabilized temperature, reaction temperature, and initial MI concentration, about 82 g L-1 of SI is produced from 250 g L-1 of MI within 24 h without any cofactor supplementation. This final titer of SI produced is the highest to the authors' limited knowledge. This study provides a promising method for the large-scale industrial production of SI.

Combined cross-linked enzyme aggregates of glycerol dehydrogenase and NADH oxidase for high efficiency in situ NAD+ regeneration.

Cofactor regeneration is an important method to avoid the consumption of large quantities of oxidized cofactor NAD+ in enzyme-catalyzed reactions. Herein, glycerol dehydrogenase (GDH) and NADH oxidase preparations by aggregating enzymes with ammonium sulphate followed by cross-linking formed aggregates for effective regeneration of NAD+. After optimization, the activity of combi-CLEAs and separate CLEAs mixtures were 950 and 580 U/g, respectively. And the catalytic stability of combi-CLEAs against pH and temperature was superior to the free enzyme mixture. After ten cycles of reuse, the catalytic efficiency could still retain 63.3% of its initial activity, indicating that the constructed combi-CLEAs system had excellent reusability. Also, the conversion of glycerol to 1,3-dihydroxyacetone (DHA) was improved by the constructed NAD+ regeneration system, resulting in 4.6%, which was 2.5 times of the free enzyme system. Thus, wide applications of this co-immobilization method in the production of various chiral chemicals could be expected in the industry for its high efficiency at a low cost.

Biochemical and Molecular Characterization of Glycerol Dehydrogenas from Klebsiella pneumoniae.

Glycerol dehydrogenase (GlyDH) catalyzes the oxidation of glycerol to dihydroxyacetone (DHA), which is the first step in the glycerol metabolism pathway. GlyDH has attracted great interest for its potential industrial applications, since DHA is a precursor for the synthesis of many commercially valuable chemicals and various drugs. In this study, GlyDH from Klebsiella pneumoniae (KpGlyDH) was overexpressed in E. coli and purified to homogeneity for biochemical and molecular characterization. KpGlyDH exhibits an exclusive preference for NAD+ over NADP+. The enzymatic activity of KpGlyDH is maximal at pH 8.6 and pH 10.0. Of the three common polyol substrates, KpGlyDH showed the highest kcat/Km value for glycerol, which is three times higher than for racemic 2,3-butanediol and 32 times higher than for ethylene glycol. The kcat value for glycerol oxidation is notably high at 87.1 ± 11.3 sec-1. KpGlyDH was shown to exist in an equilibrium between two different oligomeric states, octamer and hexadecamer, by size-exclusion chromatography analysis. KpGlyDH is structurally thermostable, with a Tm of 83.4°C, in thermal denaturation experiment using circular dichroism spectroscopy. The biochemical and biophysical characteristics of KpGlyDH revealed in this study should provide the basis for future research on its glycerol metabolism and possible use in industrial applications.

An enzymatic assay for quantification of inositol in human term placental tissue.

A modified sensitive, cheap and simple enzymatic assay method is described for the quantitation of inositol (6-carbon polyol) in human placental tissue. Water-soluble and total (water-soluble and lipid-bound) inositol isomers were extracted and quantified using a 96-well adaptation of the Megazyme® assay. This assay specifically recognized myo-inositol (predominant isomer), d-chiro-, epi-, and allo-inositols, but not scyllo-inositol, glucose or fucose. In term placenta, water-soluble and total inositol contents were high [489 (±58) and 635 (±69) µg/g respectively], and reliably quantified with good reproducibility. This modified assay could facilitate placental inositol biology research, particularly pertinent now with interest in myo-inositol supplementation for gestational diabetes (GDM) prevention.

Targeting Mannitol Metabolism as an Alternative Antimicrobial Strategy Based on the Structure-Function Study of Mannitol-1-Phosphate Dehydrogenase in Staphylococcus aureus.

Mannitol-1-phosphate dehydrogenase (M1PDH) is a key enzyme in Staphylococcus aureus mannitol metabolism, but its roles in pathophysiological settings have not been established. We performed comprehensive structure-function analysis of M1PDH from S. aureus USA300, a strain of community-associated methicillin-resistant S. aureus, to evaluate its roles in cell viability and virulence under pathophysiological conditions. On the basis of our results, we propose M1PDH as a potential antibacterial target. In vitro cell viability assessment of ΔmtlD knockout and complemented strains confirmed that M1PDH is essential to endure pH, high-salt, and oxidative stress and thus that M1PDH is required for preventing osmotic burst by regulating pressure potential imposed by mannitol. The mouse infection model also verified that M1PDH is essential for bacterial survival during infection. To further support the use of M1PDH as an antibacterial target, we identified dihydrocelastrol (DHCL) as a competitive inhibitor of S. aureus M1PDH (SaM1PDH) and confirmed that DHCL effectively reduces bacterial cell viability during host infection. To explain physiological functions of SaM1PDH at the atomic level, the crystal structure of SaM1PDH was determined at 1.7-Å resolution. Structure-based mutation analyses and DHCL molecular docking to the SaM1PDH active site followed by functional assay identified key residues in the active site and provided the action mechanism of DHCL. Collectively, we propose SaM1PDH as a target for antibiotic development based on its physiological roles with the goals of expanding the repertory of antibiotic targets to fight antimicrobial resistance and providing essential knowledge for developing potent inhibitors of SaM1PDH based on structure-function studies.IMPORTANCE Due to the shortage of effective antibiotics against drug-resistant Staphylococcus aureus, new targets are urgently required to develop next-generation antibiotics. We investigated mannitol-1-phosphate dehydrogenase of S. aureus USA300 (SaM1PDH), a key enzyme regulating intracellular mannitol levels, and explored the possibility of using SaM1PDH as a target for developing antibiotic. Since mannitol is necessary for maintaining the cellular redox and osmotic potential, the homeostatic imbalance caused by treatment with a SaM1PDH inhibitor or knockout of the gene encoding SaM1PDH results in bacterial cell death through oxidative and/or mannitol-dependent cytolysis. We elucidated the molecular mechanism of SaM1PDH and the structural basis of substrate and inhibitor recognition by enzymatic and structural analyses of SaM1PDH. Our results strongly support the concept that targeting of SaM1PDH represents an alternative strategy for developing a new class of antibiotics that cause bacterial cell death not by blocking key cellular machinery but by inducing cytolysis and reducing stress tolerance through inhibition of the mannitol pathway.

A thermostable mannitol-1-phosphate dehydrogenase is required in mannitol metabolism of the thermophilic acetogenic bacterium Thermoanaerobacter kivui.

Acetogenic bacteria recently attracted attention because they reduce carbon dioxide (CO2 ) with hydrogen (H2 ) to acetate or to other products such as ethanol. Besides gases, acetogens use a broad range of substrates, but conversion of the sugar alcohol mannitol has rarely been reported. We found that the thermophilic acetogenic bacterium Thermoanaerobacter kivui grew on mannitol with a specific growth rate of 0.33 h-1 to a final optical density (OD600 ) of 2.2. Acetate was the major product formed. A lag phase was observed only in cultures pre-grown on glucose, not in those pre-grown on mannitol, indicating that mannitol metabolism is regulated. Mannitol-1-phosphate dehydrogenase (MtlD) activity was observed in cell-free extracts of cells grown on mannitol only. A gene cluster (TKV_c02830-TKV_c02860) for mannitol uptake and conversion was identified in the T. kivui genome, and its involvement was confirmed by deleting the mtlD gene (TKV_c02860) encoding the key enzyme MtlD. Finally, we overexpressed mtlD, and the recombinant MtlD carried out the reduction of fructose-6-phosphate with NADH, at a high VMAX of 1235 U mg-1 at 65°C. The enzyme was thermostable for 40 min at 75°C, thereby representing the first characterized MtlD from a thermophile.

Identification of dicarbonyl and L-xylulose reductase as a therapeutic target in human chronic kidney disease.

An imbalance of nephroprotective factors and renal damaging molecules contributes to development and progression of chronic kidney disease (CKD). We investigated associations of renoprotective factor gene expression patterns with CKD severity and outcome. Gene expression profiles of 197 previously reported renoprotective factors were analyzed in a discovery cohort in renal biopsies of 63 CKD patients. Downregulation of dicarbonyl and L-xylulose reductase (DCXR) showed the strongest association with disease progression. This significant association was validated in an independent set of 225 patients with nephrotic syndrome from the multicenter NEPTUNE cohort. Reduced expression of DCXR was significantly associated with degree of histological damage as well as with lower estimated glomerular filtration rate and increased urinary protein levels. DCXR downregulation in CKD was confirmed in 3 publicly available transcriptomics data sets in the context of CKD. Expression of DCXR showed positive correlations to enzymes that are involved in dicarbonyl stress detoxification based on transcriptomics profiles. The sodium glucose cotransporter-2 (SGLT2) inhibitors canagliflozin and empagliflozin showed a beneficial effect on renal proximal tubular cells under diabetic stimuli-enhanced DCXR gene expression. In summary, lower expression of the renoprotective factor DCXR in renal tissue is associated with more severe disease and worse outcome in human CKD.

Homologous overexpression of hydrogenase and glycerol dehydrogenase in Clostridium pasteurianum to enhance hydrogen production from crude glycerol.

This study reports engineering of a hypertransformable variant of C. pasteurianum for bioconversion of glycerol into hydrogen (H2). A functional glycerol-triggered hydrogen pathway was engineered based on two approaches: (1) increasing product yield by overexpression of immediate enzyme catalyzing H2 production, (2) increasing substrate uptake by overexpression of enzymes involved in glycerol utilization. The first strategy aimed at overexpression of hydA gene encoding hydrogenase, and the second one, through combination of overexpression of dhaD1 and dhaK genes encoding glycerol dehydrogenase and dihydroxyacetone kinase. These genetic manipulations resulted in two recombinant strains (hydA++/dhaD1K++) capable of producing 97% H2 (v/v), with yields of 1.1 mol H2/mol glycerol in hydA overexpressed strain, and 0.93 mol H2/mol glycerol in dhaD1K overexpressed strain, which was 1.5 fold higher than wild type. Among two strains, dhaD1K++ consumed more glycerol than hydA++ which proves that overexpression of glycerol enzymes has enhanced glycerol intake rate.

Molecular chaperone prefoldin-assisted biosynthesis of gold nanoparticles with improved size distribution and dispersion.

Here we report a novel aspect of molecular chaperone prefoldin (PFD) as a biomaterial in the biocatalytic synthesis of gold nanoparticles (AuNPs) using glycerol dehydrogenase (GLD). We found that PFD could inhibit the aggregation of AuNPs during the biosynthesis, leading to the formation of AuNPs with controlled size distribution.