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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 β-hairpin, and that during the ordered progression of the kinetic mechanism, the flexibility of the β-hairpin is reduced after NAD + binding. It was also observed that this alterable flexibility of the β-hairpin contributes to the cofactor binding and possibly to the catalytic efficiency of GDH. These findings suggest the importance of the flexible β-hairpin to GDH enzymatic activity and shed new light on the kinetic mechanism of GDH., (© 2023 Federation of European Biochemical Societies.)

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 helix–loop–helix (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.

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 k cat /K m value for glycerol, which is three times higher than for racemic 2,3-butanediol and 32 times higher than for ethylene glycol. The k cat 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 T m 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.

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., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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 Δ mtlD 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 ( Sa M1PDH) and confirmed that DHCL effectively reduces bacterial cell viability during host infection. To explain physiological functions of Sa M1PDH at the atomic level, the crystal structure of Sa M1PDH was determined at 1.7-Å resolution. Structure-based mutation analyses and DHCL molecular docking to the Sa M1PDH active site followed by functional assay identified key residues in the active site and provided the action mechanism of DHCL. Collectively, we propose Sa M1PDH 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 Sa M1PDH 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 ( Sa M1PDH), a key enzyme regulating intracellular mannitol levels, and explored the possibility of using Sa M1PDH 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 Sa M1PDH inhibitor or knockout of the gene encoding Sa M1PDH results in bacterial cell death through oxidative and/or mannitol-dependent cytolysis. We elucidated the molecular mechanism of Sa M1PDH and the structural basis of substrate and inhibitor recognition by enzymatic and structural analyses of Sa M1PDH. Our results strongly support the concept that targeting of Sa M1PDH 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., (Copyright © 2019 Nguyen et al.)

Recently, increased attention has been drawn to application of graphene and its derivatives for construction of biosensors, since they can be used to rapidly detect the presence of bio-analytes. Present paper establishes the preparation of a unique transducer which relies on toluidine blue (TB), absorbed by electrochemically reduced graphene oxide (ERGO) transparent thin film onto the surface of the indium tin-oxide (ITO) glass electrode. The proposed TB/ERGO/ITO electrode shows excellent reversible electro-chemical properties. The novel platform has been explored to fabricate a triglyceride (TG) biosensor via co-immobilizing of lipase (LIP) and glycerol dehydrogenase (GDH) onto TB/ERGO/ITO electrode surface. The fabricated bioelectrode (LIP-GDH/TB/ERGO/ITO) directly oxidizes glycerol (produced by catalytic hydrolysis of tributyrin acting as a model TG) in the presence of GDH. The developed bioelectrode replaces unstable biological irreversible redox mediators NAD+/NADH, involved in the triglyceride breakdown reaction. NADH causes fouling on the bioelectrode surface in bi-enzymatic estimation of TG and reduces the shelf-life of biosensor. Electrochemical response studies carried out using cyclic voltammetry reveal that the fabricated electrode can detect tributyrin in the range of 50-400 mg dL-1 with high sensitivity of 29 pA mg-1 dL, low response time of 12 s, long-term stability and a low apparent Michaelis-Menten constant (Kappm) of 0.18 mM, indicating high enzyme affinity of LIP-GDH/TB/ERGO/ITO bioelectrode towards tributyrin. Furthermore, this modified bioelectrode has been explored for estimation of TG with negligible interference in human serum samples. The proposed bi-enzymatic bioelectrode for TG analysis offers an efficient and novel interface for application of graphene and its derivatives in the field of bioelectronic devices.

Escherichia coli (strain K-12, substrain MG1655) glycerol dehydrogenase (GldA) is required to catalyze the first step in fermentative glycerol metabolism. The protein was expressed and purified to homogeneity using a simple combination of heat-shock and chromatographic methods. The high yield of the protein (∼250 mg per litre of culture) allows large-scale production for potential industrial applications. Purified GldA exhibited a homogeneous tetrameric state (∼161 kDa) in solution and relatively high thermostability (T m = 65.6°C). Sitting-drop sparse-matrix screens were used for protein crystallization. An optimized condition with ammonium sulfate (2 M) provided crystals suitable for diffraction, and a binary structure containing glycerol in the active site was solved at 2.8 Å resolution. Each GldA monomer consists of nine β-strands, thirteen α-helices, two 3 10 -helices and several loops organized into two domains, the N- and C-terminal domains; the active site is located in a deep cleft between the two domains. The N-terminal domain contains a classic Rossmann fold for NAD + binding. The O 1 and O 2 atoms of glycerol serve as ligands for the tetrahedrally coordinated Zn 2+ ion. The orientation of the glycerol within the active site is mainly stabilized by van der Waals and electrostatic interactions with the benzyl ring of Phe245. Computer modeling suggests that the glycerol molecule is sandwiched by the Zn 2+ and NAD + ions. Based on this, the mechanism for the relaxed substrate specificity of this enzyme is also discussed.

Levoglucosan is the 1,6-anhydrosugar of d-glucose formed by pyrolysis of glucans and is found in the environment and industrial waste. Two types of microbial levoglucosan metabolic pathways are known. Although the eukaryotic pathway involving levoglucosan kinase has been well-studied, the bacterial pathway involving levoglucosan dehydrogenase (LGDH) has not been well-investigated. Here, we identified and cloned the lgdh gene from the bacterium Pseudarthrobacter phenanthrenivorans and characterized the recombinant protein. The enzyme exhibited high substrate specificity toward levoglucosan and NAD + for the oxidative reaction and was confirmed to be LGDH. LGDH also showed weak activities (∼4%) toward l-sorbose and 1,5-anhydro-d-glucitol. The reverse (reductive) reaction using 3-keto-levoglucosan and NADH exhibited significantly lower K m and higher k cat values than those of the forward reaction. The crystal structures of LGDH in the apo and complex forms with NADH, NADH + levoglucosan, and NADH + l-sorbose revealed that LGDH has a typical fold of Gfo/Idh/MocA family proteins, similar to those of scyllo -inositol dehydrogenase, aldose-aldose oxidoreductase, 1,5-anhydro-d-fructose reductase, and glucose-fructose oxidoreductase. The crystal structures also disclosed that the active site of LGDH is distinct from those of these enzymes. The LGDH active site extensively recognized the levoglucosan molecule with six hydrogen bonds, and the C3 atom of levoglucosan was closely located to the C4 atom of NADH nicotinamide. Our study is the first molecular characterization of LGDH, providing evidence for C3-specific oxidation and representing a starting point for future biotechnological use of LGDH and levoglucosan-metabolizing bacteria., (© 2018 Sugiura et al.)

Archaeal/fungal Rib7 and eubacterial RibG possess a reductase domain for ribosyl reduction in the second and third steps, respectively, of riboflavin biosynthesis. These enzymes are specific for an amino and a carbonyl group of the pyrimidine ring, respectively. Here, several crystal structures of Methanosarcina mazei Rib7 are reported at 2.27-1.95 Å resolution, which are the first archaeal dimeric Rib7 structures. Mutational analysis displayed that no detectable activity was observed for the Bacillus subtilis RibG K151A, K151D, and K151E mutants, and the M. mazei Rib7 D33N, D33K, and E156Q variants, while 0.1-0.6% of the activity was detected for the M. mazei Rib7 N9A, S29A, D33A, and D57N variants. Our results suggest that Lys151 in B. subtilis RibG, while Asp33 together with Arg36 in M. mazei Rib7, ensure the specific substrate recognition. Unexpectedly, an endogenous NADPH cofactor is observed in M. mazei Rib7, in which the 2'-phosphate group interacts with Ser88, and Arg91. Replacement of Ser88 with glutamate eliminates the endogenous NADPH binding and switches preference to NADH. The lower melting temperature of ∼10 °C for the S88E and R91A mutants suggests that nature had evolved a tightly bound NADPH to greatly enhance the structural stability of archaeal Rib7., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Sorbitol-6-phosphate 2-dehydrogenases (S6PDH) catalyze the interconversion of d-sorbitol 6-phosphate to d-fructose 6-phosphate. In the plant pathogen Erwinia amylovora the S6PDH SrlD is used by the bacterium to utilize sorbitol, which is used for carbohydrate transport in the host plants belonging to the Amygdaloideae subfamily (e.g., apple, pear, and quince). We have determined the crystal structure of S6PDH SrlD at 1.84 Å resolution, which is the first structure of an EC 1.1.1.140 enzyme. Kinetic data show that SrlD is much faster at oxidizing d-sorbitol 6-phosphate than in reducing d-fructose 6-phosphate, however, equilibrium analysis revealed that only part of the d-sorbitol 6-phosphate present in the in vitro environment is converted into d-fructose 6-phosphate. The comparison of the structures of SrlD and Rhodobacter sphaeroides sorbitol dehydrogenase showed that the tetrameric quaternary structure, the catalytic residues and a conserved aspartate residue that confers specificity for NAD + over NADP + are preserved. Analysis of the SrlD cofactor and substrate binding sites identified residues important for the formation of the complex with cofactor and substrate and in particular the role of Lys42 in selectivity towards the phospho-substrate. The comparison of SrlD backbone with the backbone of 302 short-chain dehydrogenases/reductases showed the conservation of the protein core and identified the variable parts. The SrlD sequence was compared with 500 S6PDH sequences selected by homology revealing that the C-terminal part is more conserved than the N-terminal, the consensus of the catalytic tetrad (Y[SN]AGXA) and a not previously described consensus for the NAD(H) binding., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Immobilization of glycerol dehydrogenase (GDH) from Serratia marcescens H30 onto epoxy functional magnetic nanoparticles by covalent attachment was carried out. The optimal immobilization conditions were obtained as follows: enzyme/support 6.08 mg/g, temperature 25 °C, pH 7.0 and time 8 h. Under these conditions, a high immobilization yield above 90% was obtained. The characterization of the immobilized GDH indicated that enhanced pH and thermal stability were achieved. Kinetic parameters Km of free and immobilized GDH were determined as 10.35 mM and 15.76 mM, respectively. The immobilized GDH retained about 85% initial activity after ten cycles. These results suggested that GDH immobilized onto magnetic nanoparticles is a simple and efficient way for preparation of stable enzyme. And the immobilized GDH has potential applications in the production of DHA.

For about 70 years, L-glucose had been considered non-metabolizable by either mammalian or bacterial cells. Recently, however, an L-glucose catabolic pathway has been discovered in Paracoccus laeviglucosivorans, and the genes responsible cloned. Scyllo-inositol dehydrogenase is involved in the first step in the pathway that oxidizes L-glucose to produce L-glucono-1,5-lactone with concomitant reduction of NAD+ dependent manner. Here, we report the crystal structure of the ternary complex of scyllo-inositol dehydrogenase with NAD+ and L-glucono-1,5-lactone at 1.8 Å resolution. The enzyme adopts a homo-tetrameric structure, similar to those of the inositol dehydrogenase family, and the electron densities of the bound sugar was clearly observed, allowing identification of the residues responsible for interaction with the substrate in the catalytic site. In addition to the conserved catalytic residues (Lys106, Asp191, and His195), another residue, His318, located in the loop region of the adjacent subunit, is involved in substrate recognition. Site-directed mutagenesis confirmed the role of these residues in catalytic activity. We also report the complex structures of the enzyme with myo-inositol and scyllo-inosose. The Arg178 residue located in the flexible loop at the entrance of the catalytic site is also involved in substrate recognition, and plays an important role in accepting both L-glucose and inositols as substrates. On the basis of these structural features, which have not been identified in the known inositol dehydrogenases, and a phylogenetic analysis of IDH family enzymes, we suggest a novel subfamily of the GFO/IDH/MocA family. Since many enzymes in this family have not biochemically characterized, our results could promote to find their activities with various substrates., Competing Interests: The authors have declared that no competing interests exist.

Glycyl radical enzymes (GREs) are important biological catalysts in both strict and facultative anaerobes, playing key roles both in the human microbiota and in the environment. GREs contain a backbone glycyl radical that is post-translationally installed, enabling radical-based mechanisms. GREs function in several metabolic pathways including mixed acid fermentation, ribonucleotide reduction and the anaerobic breakdown of the nutrient choline and the pollutant toluene. By generating a substrate-based radical species within the active site, GREs enable C-C, C-O and C-N bond breaking and formation steps that are otherwise challenging for nonradical enzymes. Identification of previously unknown family members from genomic data and the determination of structures of well-characterized GREs have expanded the scope of GRE-catalyzed reactions as well as defined key features that enable radical catalysis. Here, we review the structures and mechanisms of characterized GREs, classifying members into five categories. We consider the open questions about each of the five GRE classes and evaluate the tools available to interrogate uncharacterized GREs.

The determination of the crystal structure of a mutant protein using phases based on a previously determined crystal structure of the wild-type protein is often a straightforward molecular-replacement protocol. Such a structure determination may be difficult if there are large-scale structural differences between the wild-type and mutant proteins. In this manuscript, an interesting case is presented of the unintentional crystallization of a contaminant protein which shared some structural features with the presumed target protein, leading to difficulties in obtaining a completely satisfactory molecular-replacement structure solution. It was not immediately evident that the initial structure solution was incorrect owing to the poor quality of the X-ray diffraction data and low resolution. The structure was subsequently determined by improving the quality of the data and following a sequence-independent MarathonMR protocol. The structure corresponded to that of glycerol dehydrogenase, which crystallized as a contaminant, instead of the presumed mutant of a survival protein encoded by Salmonella typhimurium. The reasons why a solution that appeared to be reasonable was obtained with an incorrect protein model are discussed. The results presented here show that a degree of caution is warranted when handling large-scale structure-determination projects.

An improved glycerol biosensor was developed via direct attachment of NAD + -glycerol dehydrogenase coenzyme-apoenzyme complex onto supporting gold electrodes, using novel inorganic iron (II) sulfide (FeS)-based single molecular wires. Sensing performance factors, i.e., sensitivity, a detection limit and response time of the FeS and conventional pyrroloquinoline quinone (PQQ)-based biosensor were evaluated by dynamic constant potential amperometry at 1.3V under non-buffered conditions. For glycerol concentrations ranging from 1 to 25mM, a 77% increase in sensitivity and a 53% decrease in detection limit were observed for the FeS-based biosensor when compared to the conventional PQQ-based counterpart. The electrochemical behavior of the FeS-based glycerol biosensor was analyzed at different concentrations of glycerol, accompanied by an investigation into the effects of applied potential and scan rate on the current response. Effects of enzyme stimulants ((NH 4 ) 2 SO 4 and MnCl 2 ·4H 2 O) concentrations and buffers/pH (potassium phosphate buffer pH 6-8, Tris buffer pH 8-10) on the current responses generated by the FeS-based glycerol biosensor were also studied. The optimal detection conditions were 0.03M (NH 4 ) 2 SO 4 and 0.3µm MnCl 2 ·4H 2 O in non-buffered aqueous electrolyte under stirring whereas under non-stirring, Tris buffer at pH 10 with 0.03M (NH 4 ) 2 SO 4 and 30µm MnCl 2 ·4H 2 O were found to be optimal detection conditions. Interference by glucose, fructose, ethanol, and acetic acid in glycerol detection was studied. The observations indicated a promising enhancement in glycerol detection using the novel FeS-based glycerol sensing electrode compared to the conventional PQQ-based one. These findings support the premise that FeS-based bioanodes are capable of biosensing glycerol successfully and may be applicable for other enzymatic biosensors., (Copyright © 2016 Elsevier B.V. All rights reserved.)

A novel oxidation of D-pentonates to 4-keto-D-pentonates was analyzed with Gluconobacter thailandicus NBRC 3258. D-Pentonate 4-dehydrogenase activity in the membrane fraction was readily inactivated by EDTA and it was reactivated by the addition of PQQ and Ca 2+ . D-Pentonate 4-dehydrogenase was purified to two different subunits, 80 and 14 kDa. The absorption spectrum of the purified enzyme showed no typical absorbance over the visible regions. The enzyme oxidized D-pentonates to 4-keto-D-pentonates at the optimum pH of 4.0. In addition, the enzyme oxidized D-fructose to 5-keto-D-fructose, D-psicose to 5-keto-D-psicose, including the other polyols such as, glycerol, D-ribitol, D-arabitol, and D-sorbitol. Thus, D-pentonate 4-dehydrogenase was found to be identical with glycerol dehydrogenase (GLDH), a major polyol dehydrogenase in Gluconobacter species. The reaction versatility of quinoprotein GLDH was notified in this study.

As methods for the use of anhydrosugars in chemical and biofuel production continue to develop, our collective knowledge of anhydrosugar processing enzymes continues to improve, including their mechanistic details, structural dynamics and modes of substrate binding. Of particular interest, anhydrosugar kinases, such as levoglucosan kinase (LGK) and 1,6-anhydro-N-acetylmuramic acid kinase (AnmK), utilize an unusual mechanism whereby the sugar substrate is both cleaved and phosphorylated. The phosphorylated sugar can then be routed to other metabolic pathways, thereby allowing its further bioconversion. Advanced engineering efforts to improve the catalytic efficiency and stability of LGK have been steadily progressing. Other enzymes that cleave the glycosidic bond of disaccharide sugars containing an anhydrosugar component are also being identified and characterized. Accordingly, the potential future use of these enzymes in large-scale production strategies is becoming increasingly viable. Here, a mini-review of the observed characteristics of anhydrosugar processing enzymes is presented along with recent developments in the bioconversion of these sugars. © 2016 IUBMB Life 68(9):700-708, 2016., (© 2016 International Union of Biochemistry and Molecular Biology.)

Glycerol dehydrogenases (GlyDHs) are essential for glycerol metabolism in vivo, catalyzing its reversible reduction to 1,3-dihydroxypropranone (DHA). The gldA gene encoding a putative GlyDH was cloned from Thermoanaerobacterium thermosaccharolyticum DSM 571 (TtGlyDH) and expressed in Escherichia coli. The presence of Mn(2+) enhanced its enzymatic activity by 79.5%. Three highly conserved residues (Asp(171), His(254), and His(271)) in TtGlyDH were associated with metal ion binding. Based on an investigation of glycerol oxidation and DHA reduction, TtGlyDH showed maximum activity towards glycerol at 60°C and pH 8.0 and towards DHA at 60°C and pH 6.0. DHA reduction was the dominant reaction, with a lower Km(DHA) of 1.08 ± 0.13 mM and Vmax of 0.0053 ± 0.0001 mM/s, compared with glycerol oxidation, with a Km(glycerol) of 30.29 ± 3.42 mM and Vmax of 0.042 ± 0.002 mM/s. TtGlyDH had an apparent activation energy of 312.94 kJ/mol. The recombinant TtGlyDH was thermostable, maintaining 65% of its activity after a 2-h incubation at 60°C. Molecular modeling and site-directed mutagenesis analyses demonstrated that TtGlyDH had an atypical dinucleotide binding motif (GGG motif) and a basic residue Arg(43), both related to dinucleotide binding.

We introduce active surfaces generated by immobilizing protein-polymer nanoreactors on a solid support for sensitive sugar alcohols detection. First, such selective nanoreactors were engineered in solution by simultaneous encapsulation of specific enzymes in copolymer polymersomes, and insertion of membrane proteins for selective conduct of sugar alcohols. Despite the artificial surroundings, and the thickness of the copolymer membrane, functionality of reconstituted Escherichia coli glycerol facilitator (GlpF) was preserved, and allowed selective diffusion of sugar alcohols to the inner cavity of the polymersome, where encapsulated ribitol dehydrogenase (RDH) enzymes served as biosensing entities. Ribitol, selected as a model sugar alcohol, was detected quantitatively by the RDH-nanoreactors with GlpF-mediated permeability in a concentration range of 1.5-9 mM. To obtain "active surfaces" for detecting sugar alcohols, the nanoreactors optimized in solution were then immobilized on a solid support: aldehyde groups exposed at the compartment external surface reacted via an aldehyde-amino reaction with glass surfaces chemically modified with amino groups. The nanoreactors preserved their architecture and activity after immobilization on the glass surface, and represent active biosensing surfaces for selective detection of sugar alcohols, with high sensitivity., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

We have recently developed a simple, reusable and coupled whole-cell biocatalytic system with the capability of cofactor regeneration and biocatalyst immobilization for improved production yield and sustained synthesis. Described herewith is the experimental procedure for the development of such a system consisting of two E. coli strains that express functionally complementary enzymes. Together, these two enzymes can function co-operatively to mediate the regeneration of expensive cofactors for improving the product yield of the bioreaction. In addition, the method of synthesizing an immobilized form of the coupled biocatalytic system by encapsulation of whole cells in calcium alginate beads is reported. As an example, we present the improved biosynthesis of L-xylulose from L-arabinitol by coupling E. coli cells expressing the enzymes L-arabinitol dehydrogenase or NADH oxidase. Under optimal conditions and using an initial concentration of 150 mM L-arabinitol, the maximal L-xylulose yield reached 96%, which is higher than those reported in the literature. The immobilized form of the coupled whole-cell biocatalysts demonstrated good operational stability, maintaining 65% of the yield obtained in the first cycle after 7 cycles of successive re-use, while the free cell system almost completely lost the catalytic activity. Therefore, the methods reported here provides two strategies that could help improve the industrial production of L-xylulose, as well as other value-added compounds requiring the use of cofactors in general.

When redox enzymes are wired to electrodes outside a living cell (ex vivo), their ability to produce a sufficiently powerful electrical current diminishes significantly due to the thermodynamic and kinetic limitations associated with the wiring systems. Therefore, we are yet to harness the full potential of redox enzymes for the development of self-powering bioelectronics devices (such as sensors and fuel cells). Interestingly, nature uses iron-sulfur complexes ([Fe-S]), to circumvent these issues in vivo. Yet, we have not been able to utilize [Fe-S]-based chains ex vivo, primarily due to their instability in aqueous media. Here, a simple technique to attach iron (II) sulfide (FeS) to a gold surface in ethanol media and then complete the attachment of the enzyme in aqueous media is reported. Cyclic voltammetry and spectroscopy techniques confirmed the concatenation of FeS and glycerol-dehydrogenase/nicotinamide-adenine-dinucleotide (GlDH-NAD(+)) apoenzyme-coenzyme molecular wiring system on the base gold electrode. The resultant FeS-based enzyme electrode reached an open circuit voltage closer to its standard potential under a wide range of glycerol concentrations (0.001-1M). When probed under constant potential conditions, the FeS-based electrode was able to amplify current by over 10 fold as compared to electrodes fabricated with the conventional pyrroloquinoline quinone-based composite molecular wiring system. These improvements in current/voltage responses open up a wide range of possibilities for fabricating self-powering, bio-electronic devices., (Copyright © 2015 Elsevier B.V. All rights reserved.)

In this study, a novel, simple and generally applicable strategy for multimeric oxidoreductase immobilization with multi-levels interactions was developed and involved activity and stability enhancements. Linear polyethyleneimines (PEIs) are flexible cationic polymers with molecular weights that span a wide range and are suitable biomimic polypeptides for biocompatible frameworks for enzyme immobilization. Metal ion-chelated linear PEIs were applied as a heterofunctional framework for glycerol dehydrogenase (GDH) immobilization by hydrogen bonds, electrostatic forces and coordination bonds interactions. Nanoparticles with diameters from 250-650 nm were prepared that exhibited a 1.4-fold enhancement catalytic efficiency. Importantly, the half-life of the immobilized GDH was enhanced by 5.6-folds in aqueous phase at 85 °C. A mechanistic illustration of the formation of multi-level interactions in the PEI-metal-GDH complex was proposed based on morphological and functional studies of the immobilized enzyme. This generally applicable strategy offers a potential technique for multimeric enzyme immobilization with the advantages of low cost, easy operation, high activity reservation and high stability.

Background: Imbalance in cofactors causing the accumulation of intermediates in biosynthesis pathways is a frequently occurring problem in metabolic engineering when optimizing a production pathway in a microorganism. In our previous study, a single knock-out Citrobacter werkmanii ∆dhaD was constructed for improved 1,3-propanediol (PDO) production. Instead of an enhanced PDO concentration on this strain, the gene knock-out led to the accumulation of the toxic intermediate 3-hydroxypropionaldehyde (3-HPA). The hypothesis was emerged that the accumulation of this toxic intermediate, 3-HPA, is due to a cofactor imbalance, i.e. to the limited supply of reducing equivalents (NADH). Here, this bottleneck is alleviated by rationally engineering cell metabolism to balance the cofactor supply., Results: By eliminating non-essential NADH consuming enzymes (such as lactate dehydrogenase coded by ldhA, and ethanol dehydrogenase coded by adhE) or by increasing NADH producing enzymes, the accumulation of 3-HPA is minimized. Combining the above modifications in C. werkmanii ∆dhaD resulted in the strain C. werkmanii ∆dhaD∆ldhA∆adhE::ChlFRT which provided the maximum theoretical yield of 1.00 ± 0.03 mol PDO/mol glycerol when grown on glucose/glycerol (0.33 molar ratio) on flask scale under anaerobic conditions. On bioreactor scale, the yield decreased to 0.73 ± 0.01 mol PDO/mol glycerol although no 3-HPA could be measured, which indicates the existence of a sink of glycerol by a putative glycerol dehydrogenase, channeling glycerol to the central metabolism., Conclusions: In this study, a multiple knock-out was created in Citrobacter species for the first time. As a result, the concentration of the toxic intermediate 3-HPA was reduced to below the detection limit and the maximal theoretical PDO yield on glycerol was reached.

Archaea use glycolytic pathways distinct from those found in bacteria and eukaryotes, where unique enzymes catalyze each reaction step. In this study, we isolated three isozymes of glyceraldehyde oxidoreductase (GAOR1, GAOR2 and GAOR3) from the thermoacidophilic archaeon Sulfolobus tokodaii. GAOR1-3 belong to the xanthine oxidoreductase superfamily, and are composed of a molybdo-pyranopterin subunit (L), a flavin subunit (M), and an iron-sulfur subunit (S), forming an LMS hetero-trimer unit. We found that GAOR1 is a tetramer of the STK17810/STK17830/STK17820 hetero-trimer, GAOR2 is a dimer of the STK23390/STK05620/STK05610 hetero-trimer, and GAOR3 is the STK24840/STK05620/STK05610 hetero-trimer. GAOR1-3 exhibited diverse substrate specificities for their electron donors and acceptors, due to their different L-subunits, and probably participate in the non-phosphorylative Entner-Doudoroff glycolytic pathway. We determined the crystal structure of GAOR2, as the first three-dimensional structure of an archaeal molybdenum-containing hydroxylase, to obtain structural insights into their substrate specificities and subunit assemblies. The gene arrangement and the crystal structure suggested that the M/S-complex serves as a structural scaffold for the binding of the L-subunit, to construct the three enzymes with different specificities. Collectively, our findings illustrate a novel principle of a prokaryotic multicomponent isozyme system.

A novel strategy for the preparation of protein-decorated gold nanoparticles (Au NPs) was developed inside Escherichia coli cells, where an artificial oxidoreductase, composed of antibody-binding protein (pG), Bacillus stearothermophilus glycerol dehydrogenase (BsGLD) and a peptide tag with gold-binding affinity (H6C), was overexpressed in the cytoplasm. In situ formation of Au NPs was promoted by a natural electron-donating cofactor, nicotinamide adenine dinucleotide (NAD), which was regenerated to the reduced form of NADH by the catalytic activity of the fusion protein (pG-BsGLD-H6C) overexpressed in the cytoplasm of E. coli, with the concomitant addition of exogenous glycerol to the reaction system. The fusion protein was self-immobilized on Au NPs inside the E. coli cells, which was confirmed by SDS-PAGE and western blotting analyses of the resultant Au NPs. Finally, the IgG binding ability of the pG moiety displayed on Au NPs was evaluated by an enzyme-linked immunosorbent assay.

The glyceraldehyde dehydrogenase from Thermoplasma acidophilum (TaAlDH) is a microbial enzyme that catalyzes the oxidation of D-glyceraldehyde to D-glycerate in the artificial enzyme cascade designed for the conversion of glucose to the organic solvents isobutanol and ethanol. Various mutants of TaAlDH were constructed by a random approach followed by site-directed and saturation mutagenesis in order to improve the properties of the enzyme that are essential for its functioning within the cascade. Two enzyme variants, wild-type TaAlDH (TaAlDHwt) and an F34M+S405N variant (TaAlDH F34M+S405N), were successfully crystallized. Crystals of TaAlDHwt belonged to the monoclinic space group P1211 with eight molecules per asymmetric unit and diffracted to a resolution of 1.95 Å. TaAlDH F34M+S405N crystallized in two different space groups: triclinic P1 with 16 molecules per asymmetric unit and monoclinic C121 with four molecules per asymmetric unit. These crystals diffracted to resolutions of 2.14 and 2.10 Å for the P1 and C121 crystals, respectively.

The sugar alcohol mannitol is important in the food, pharmaceutical, medical and chemical industries. It is one of the most commonly occurring polyols in nature, with the exception of Archaea and animals. It has a range of physiological roles, including as carbon storage, compatible solute, and osmolyte. Mannitol is present in large amounts in brown algae, where its synthesis involved two steps: a mannitol-1-phosphate dehydrogenase (M1PDH) catalyzes a reversible reaction between fructose-6-phosphate (F6P) and mannitol-1-phosphate (M1P) (EC 1.1.1.17), and a mannitol-1-phosphatase hydrolyzes M1P to mannitol (EC 3.1.3.22). Analysis of the model brown alga Ectocarpus sp. genome provided three candidate genes for M1PDH activities. We report here the sequence analysis of Ectocarpus M1PDHs (EsM1PDHs), and the biochemical characterization of the recombinant catalytic domain of EsM1PDH1 (EsM1PDH1cat). Ectocarpus M1PDHs are representatives of a new type of modular M1PDHs among the polyol-specific long-chain dehydrogenases/reductases (PSLDRs). The N-terminal domain of EsM1PDH1 was not necessary for enzymatic activity. Determination of kinetic parameters indicated that EsM1PDH1cat displayed higher catalytic efficiency for F6P reduction compared to M1P oxidation. Both activities were influenced by NaCl concentration and inhibited by the thioreactive compound pHMB. These observations were completed by measurement of endogenous M1PDH activity and of EsM1PDH gene expression during one diurnal cycle. No significant changes in enzyme activity were monitored between day and night, although transcription of two out of three genes was altered, suggesting different levels of regulation for this key metabolic pathway in brown algal physiology., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

An NAD(+)-dependent ribitol dehydrogenase from Enterobacter aerogenes KCTC 2190 (EaRDH) was cloned and successfully expressed in Escherichia coli. The complete 729-bp gene was amplified, cloned, expressed, and subsequently purified in an active soluble form using nickel affinity chromatography. The enzyme had an optimal pH and temperature of 11.0 and 45°C, respectively. Among various polyols, EaRDH exhibited activity only toward ribitol, with Km, Vmax, and kcat/Km values of 10.3mM, 185Umg(-1), and 30.9s(-1)mM(-1), respectively. The enzyme showed strong preference for NAD(+) and displayed no detectable activity with NADP(+). Homology modeling and sequence analysis of EaRDH, along with its biochemical properties, confirmed that EaRDH belongs to the family of NAD(+)-dependent ribitol dehydrogenases, a member of short-chain dehydrogenase/reductase (SCOR) family. EaRDH showed the highest activity and unique substrate specificity among all known RDHs. Homology modeling and docking analysis shed light on the molecular basis of its unusually high activity and substrate specificity., (Copyright © 2015 Elsevier Inc. All rights reserved.)

An aldo-keto reductase AKR5C3 from Gluconobacter oxydans (designated as Gox0644) is a useful enzyme with various substrates, including aldehydes, diacetyl, keto esters, and α-ketocarbonyl compounds. The crystal structures of AKR5C3 in apoform in complex with NADPH and the D53A mutant (AKR5C3(-D53A) ) in complex with NADPH are presented herein. Structure comparison and site-directed mutagenesis combined with biochemical kinetics analysis reveal that the conserved Asp53 in the AKR5C3 catalytic tetrad has a crucial role in securing active pocket conformation. The gain-of-function Asp53 to Ala mutation triggers conformational changes on the Trp30 and Trp191 side chains, improving NADPH affinity to AKR5C3, which helps increase catalytic efficiency. The highly conserved Trp30 and Trp191 residues interact with the nicotinamide moiety of NADPH and help form the NADPH-binding pocket. The AKR5C3(-W30A) and AKR5C3(-W191Y) mutants show decreased activities, confirming that both residues facilitate catalysis. Residue Trp191 is in the loop structure, and the AKR5C3(-W191Y) mutant does not react with benzaldehyde, which might also determine substrate recognition. Arg192, which is involved in the substrate binding, is another important residue. The introduction of R192G increases substrate-binding affinity by improving hydrophobicity in the substrate-binding pocket. These results not only supplement the AKRs superfamily with crystal structures but also provide useful information for understanding the catalytic properties of AKR5C3 and guiding further engineering of this enzyme., (© 2014 The Protein Society.)

The meso-2,3-butanediol dehydrogenase (meso-BDH) from S. marcescens H30 is responsible for converting acetoin into 2,3-butanediol during sugar fermentation. Inactivation of the meso-BDH encoded by budC gene does not completely abolish 2,3-butanediol production, which suggests that another similar enzyme involved in 2,3-butanediol formation exists in S. marcescens H30. In the present study, a glycerol dehydrogenase (GDH) encoded by gldA gene from S. marcescens H30 was expressed in Escherichia coli BL21(DE3), purified and characterized for its properties. In vitro conversion indicated that the purified GDH could catalyze the interconversion of (3S)-acetoin/meso-2,3-butanediol and (3R)-acetoin/(2R,3R)-2,3-butanediol. (2S,3S)-2,3-Butanediol was not a substrate for the GDH at all. Kinetic parameters of the GDH enzyme showed lower K m value and higher catalytic efficiency for (3S/3R)-acetoin in comparison to those for (2R,3R)-2,3-butanediol and meso-2,3-butanediol, implying its physiological role in favor of 2,3-butanediol formation. Maximum activity for reduction of (3S/3R)-acetoin and oxidations of meso-2,3-butanediol and glycerol was observed at pH 8.0, while it was pH 7.0 for diacetyl reduction. The enzyme exhibited relative high thermotolerance with optimum temperature of 60 °C in the oxidation-reduction reactions. Over 60 % of maximum activity was retained at 70 °C. Additionally, the GDH activity was significantly enhanced for meso-2,3-BD oxidation in the presence of Fe(2+) and for (3S/3R)-acetoin reduction in the presence of Mn(2+), while several cations inhibited its activity, particularly Fe(2+) and Fe(3+) for (3S/3R)-acetoin reduction. The properties provided potential application for single configuration production of acetoin and 2,3-butanediol .

NAD-dependent Thermotoga maritima glycerol dehydrogenase (TmGlyDH) converts glycerol into dihydroxyacetone (DHA), a valuable synthetic precursor and sunless tanning agent. In this work, recombinant TmGlyDH was characterized to determine if it can be used to catalyze DHA production. The pH optima for glycerol oxidation and DHA reduction at 50 °C were 7.9 and 6.0, respectively. Under the conditions tested, TmGlyDH had a linear Arrhenius plot up to 80 °C. TmGlyDH was more thermostable than other glycerol dehydrogenases, remaining over 50 % active after 7 h at 50 °C. TmGlyDH was active on racemic 1,2-propanediol and produced (R)-1,2-propanediol from hydroxyacetone with an enantiomeric excess above 99 %, suggesting that TmGlyDH can also be used for chiral synthesis. (R)-1,2-propanediol production from hydroxyacetone was demonstrated for the first time in a one-enzyme cycling reaction using glycerol as the second substrate. Negative cooperativity was observed with glycerol and DHA, but not with the cofactor. Apparent kinetic parameters for glycerol, DHA, and NAD(H) were determined over a broad pH range. TmGlyDH showed little activity with N(6)-carboxymethyl-NAD(+) (N(6)-CM-NAD), an NAD(+) analog modified for easy immobilization to amino groups, but the double mutation V44A/K157G increased catalytic efficiency with N(6)-CM-NAD(+) ten-fold. Finally, we showed for the first time that a GlyDH is active with immobilized N(6)-CM-NAD(+), suggesting that N(6)-CM-NAD(+) can be immobilized on an electrode to allow TmGlyDH activity in a system that reoxidizes the cofactor electrocatalytically.

The sorbitol-6-phosphate dehydrogenase (S6PDH) is a key enzyme for sorbitol synthesis and plays an important role in the alleviation of salinity stress in plants. Despite the huge significance, the structure and the mode of action of this enzyme are still not known. In the present study, sequence analysis, cloning, expression, activity assays and enzyme kinetics using various substrates (glucose-6-phosphate, sorbitol-6-phosphate and mannose-6-phosphate) were performed to establish the functional role of S6PDH protein from rice (Oryza sativa). For the structural analysis of the protein, a comparative homology model was prepared on the basis of percentage sequence identity and substrate similarity using the crystal structure of human aldose reductase in complex with glucose-6-phosphate and NADP(+) (PDB ID: 2ACQ) as a template. Molecular docking was performed for studying the structural details of substrate binding and possible enzyme mechanism. The cloned sequence resulted into an active recombinant protein when expressed into a bacterial expression system. The purified recombinant protein was found to be active with glucose-6-phosphate and sorbitol-6-phosphate; however, activity against mannose-6-phosphate was not found. The K m values for glucose-6-phosphate and sorbitol-6-phosphate were found to be 15.9 ± 0.2 and 7.21 ± 0.5 mM, respectively. A molecular-level analysis of the active site of OsS6PDH provides valuable information about the enzyme mechanism and requisite enantioselectivity for its physiological substrates. Thus, the fundamental studies of structure and function of OsS6PDH could serve as the basis for the future studies of bio-catalytic applications of this enzyme.

An intracellular alcohol oxidase (AOX) was isolated from the white-rot basidiomycete Phanerochaete chrysosporium (Pch), grown on l-lactate induction medium, and purified to electrophoretic homogeneity. The dimeric protein consisted of two identical 75kDa subunits. The open reading frame of 1,956bp resulted in a monomer consisting of 651 amino acids. The enzyme showed a pI at 5.4, a pH optimum of 9, a temperature optimum at 50°C, possessed putative conserved domains of the GMC superfamily, a FAD binding domain, and showed up to 86% homology to alcohol oxidase sequences of Gloeophyllum trabeum and Coprinopsis cinerea. As was shown for the first time for an AOX from a basidiomycete, not only methanol, but also lower primary alcohols and glycerol were accepted as substrates. An assay based on aldehyde dehydrogenase confirmed d-glyceraldehyde as the product of the reaction. A bioprocess based on this enzyme could alleviate the problems associated with the huge side-stream of glycerol occurring during the manufacture of biodiesel, yielding the green oxidant hydrogen peroxide., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Lactobacillus casei BL23 contains two genes, iolG1 and iolG2, homologous with inositol dehydrogenase encoding genes from many bacteria. Inositol dehydrogenase catalyzes the oxidation of inositol with concomitant reduction of NAD+. The protein encoded by iolG2, LcIDH2, has been purified to homogeneity, crystallized and cryoprotected for diffraction at 77 K. The crystals had a high mosaicity and poor processing statistics. Subsequent diffraction measurements were performed without cryoprotectant at room temperature. These crystals were radiation-resistant and a full diffraction data set was collected at room temperature to 1.6 Å resolution.

Mechanistic insights regarding the activity enhancement of dehydrogenase by metal ion substitution were investigated by a simple method using a kinetic and thermodynamic analysis. By profiling the binding energy of both the substrate and product, the metal ion's role in catalysis enhancement was revealed. Glycerol dehydrogenase (GDH) from Klebsiella pneumoniae sp., which demonstrated an improvement in activity by the substitution of a zinc ion with a manganese ion, was used as a model for the mechanistic study of metal ion substitution. A kinetic model based on an ordered Bi-Bi mechanism was proposed considering the noncompetitive product inhibition of dihydroxyacetone (DHA) and the competitive product inhibition of NADH. By obtaining preliminary kinetic parameters of substrate and product inhibition, the number of estimated parameters was reduced from 10 to 4 for a nonlinear regression-based kinetic parameter estimation. The simulated values of time-concentration curves fit the experimental values well, with an average relative error of 11.5% and 12.7% for Mn-GDH and GDH, respectively. A comparison of the binding energy of enzyme ternary complex for Mn-GDH and GDH derived from kinetic parameters indicated that metal ion substitution accelerated the release of dioxyacetone. The metal ion's role in catalysis enhancement was explicated.

Galactitol 2-dehydrogenase (GDH) belongs to the protein subfamily of short-chain dehydrogenases/reductases and can be used to produce optically pure building blocks and for the bioconversion of bioactive compounds. An NAD(+)-dependent GDH from Rhizobium leguminosarum bv. viciae 3841 (RlGDH) was cloned and overexpressed in Escherichia coli. The RlGDH protein was purified as an active soluble form using His-tag affinity chromatography. The molecular mass of the purified enzyme was estimated to be 28kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 114kDa by gel filtration chromatography, suggesting that the enzyme is a homotetramer. The enzyme has an optimal pH and temperature of 9.5 and 35°C, respectively. The purified recombinant RlGDH catalyzed the oxidation of a wide range of substrates, including polyvalent aliphatic alcohols and polyols, to the corresponding ketones and ketoses. Among various polyols, galactitol was the preferred substrate of RlGDH with a Km of 8.8mM, kcat of 835min(-1) and a kcat/Km of 94.9min(-1)mM(-1). Although GDHs have been characterized from a few other sources, RlGDH is distinguished from other GDHs by its higher specific activity for galactitol and broad substrate spectrum, making RlGDH a good choice for practical applications., (Copyright © 2014 Elsevier Inc. All rights reserved.)

A gene in Bradyrhizobium japonicum USDA 110, annotated as a ribitol dehydrogenase (RDH), had 87 % sequence identity (97 % positives) to the N-terminal 31 amino acids of an L-glucitol dehydrogenase from Stenotrophomonas maltophilia DSMZ 14322. The 729-bp long RDH gene coded for a protein consisting of 242 amino acids with a molecular mass of 26.1 kDa. The heterologously expressed protein not only exhibited the main enantio selective activity with D-glucitol oxidation to D-fructose but also converted L-glucitol to D-sorbose with enzymatic cofactor regeneration and a yield of 90 %. The temperature stability and the apparent K m value for L-glucitol oxidation let the enzyme appear as a promising subject for further improvement by enzyme evolution. We propose to rename the enzyme from the annotated RDH gene (locus tag bll6662) from B. japonicum USDA as a D-sorbitol dehydrogenase (EC 1.1.1.14).

Glycerol dehydrogenase (GDH) is an important polyol dehydrogenase for glycerol metabolism in diverse microorganisms and for value-added utilization of glycerol in the industry. Two GDHs from Klebsiella pneumoniae, DhaD and GldA, were expressed in Escherichia coli, purified and characterized for substrate specificity and kinetic parameters. Both DhaD and GldA could catalyze the interconversion of (3R)-acetoin/(2R,3R)-2,3-butanediol or (3S)-acetoin/meso-2,3-butanediol, in addition to glycerol oxidation. Although purified GldA appeared more active than DhaD, in vivo inactivation and quantitation of their respective mRNAs indicate that dhaD is highly induced by glycerol and plays a dual role in glycerol metabolism and 2,3-butanediol formation. Complementation in K. pneumoniae further confirmed the dual role of DhaD. Promiscuity of DhaD may have vital physiological consequences for K. pneumoniae growing on glycerol, which include balancing the intracellular NADH/NAD(+) ratio, preventing acidification, and storing carbon and energy. According to the kinetic response of DhaD to modified NADH concentrations, DhaD appears to show positive homotropic interaction with NADH, suggesting that the physiological role could be regulated by intracellular NADH levels. The co-existence of two functional GDH enzymes might be due to a gene duplication event. We propose that whereas DhaD is specialized for glycerol utilization, GldA plays a role in backup compensation and can turn into a more proficient catalyst to promote a survival advantage to the organism. Revelation of the dual role of DhaD could further the understanding of mechanisms responsible for enzyme evolution through promiscuity, and guide metabolic engineering methods of glycerol metabolism.

The 1.90 Å resolution X-ray crystal structure of glycerol dehydrogenase derived from contaminating bacteria present during routine Escherichia coli protein expression is presented. This off-target enzyme showed intrinsic affinity for Ni(2+)-Sepharose, migrated at the expected molecular mass for the target protein during gel filtration and was crystallized before it was realised that contamination had occurred. In this study, it is shown that liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) can efficiently identify the protein composition of crystals in a crystallization experiment as part of a structure-determination pipeline for an unknown protein. The high-resolution X-ray data enabled sequencing directly from the electron-density maps, allowing the source of contamination to be placed within the Serratia genus. Incorporating additional protein-identity checks, such as tandem LC-MS/MS, earlier in the protein expression, purification and crystallization workflow may have prevented the unintentional structure determination of this metabolic enzyme, which represents the first enterobacterial glycerol dehydrogenase reported to date.

L-Xylulose is a potential starting material for therapeutics. However, its translation into clinical practice has been hampered by its inherently low bioavailability. In addition, the high cost associated with the production of L-xylulose is a major factor hindering its rapid deployment beyond the laboratory. In the current study, L-arabinitol 4-dehydrogenase from Hypocrea jecorina (HjLAD), which catalyzes the conversion of L-arabinitol into L-xylulose, was immobilized onto various carriers, and the immobilized enzymes were characterized. HjLAD covalently immobilized onto silicon oxide nanoparticles showed the highest immobilization efficiency (94.7 %). This report presents a comparative characterization of free and immobilized HjLAD, including its thermostability and kinetic parameters. The thermostability of HjLAD immobilized on silicon oxide nanoparticles was more than 14.2-fold higher than free HjLAD; the t1/2 of HjLAD at 25 °C was enhanced from 190 min (free) to 45 h (immobilized). In addition, the immobilized HjLAD retained 94 % of its initial activity after 10 cycles. When the immobilized HjLAD was used to catalyze the biotransformation of L-arabinitol to L-xylulose, 66 % conversion and a productivity of 7.9 g · h(-1) · L(-1) were achieved. The enhanced thermostability and reusability of HjLAD suggest that immobilization of HjLAD onto silicon oxide nanoparticles has the potential for use in the industrial production of rare sugars.

Dicarbonyl/L-xylulose reductase (DCXR) is a highly conserved and phylogenetically widespread enzyme converting L-xylulose into xylitol. It also reduces highly reactive α-dicarbonyl compounds, thus performing a dual role in carbohydrate metabolism and detoxification. Enzymatic properties of DCXR from yeast, fungi and mammalian tissue extracts are extensively studied. Deficiency of the DCXR gene causes a human clinical condition called pentosuria and low DCXR activity is implicated in age-related diseases including cancers, diabetes, and human male infertility. While mice provide a model to study clinical condition of these diseases, it is necessary to adopt a physiologically tractable model in which genetic manipulations can be readily achieved to allow the fast genetic analysis of an enzyme with multiple biological roles. Caenorhabditis elegans has been successfully utilized as a model to study DCXR. Here, we discuss the biochemical properties and significance of DCXR activity in various human diseases, and the utility of C. elegans as a research platform to investigate the molecular and cellular mechanism of the DCXR biology., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The primary role of yeast Ara1, previously mis-annotated as a D-arabinose dehydrogenase, is to catalyze the reduction of a variety of toxic α,β-dicarbonyl compounds using NADPH as a cofactor at physiological pH levels. Here, crystal structures of Ara1 in apo and NADPH-complexed forms are presented at 2.10 and 2.00 Å resolution, respectively. Ara1 exists as a homodimer, each subunit of which adopts an (α/β)8-barrel structure and has a highly conserved cofactor-binding pocket. Structural comparison revealed that induced fit upon NADPH binding yielded an intact active-site pocket that recognizes the substrate. Moreover, the crystal structures combined with computational simulation defined an open substrate-binding site to accommodate various substrates that possess a dicarbonyl group.

Two different methods, enzyme-mediated reactions and biotrasformations with microorganisms, were applied to obtain optically pure cis- and trans-isomers of whisky lactone 4a and 4b. In the first method, eight alcohol dehydrogenases were investigated as biocatalysts to enantioselective oxidation of racemic erythro- and threo-3-methyloctane-1,4-diols (1a and 1b). Oxidation processes with three of them, alcohol dehydrogenases isolated from horse liver (HLADH) as well as recombinant from Escherichia coli and primary alcohol dehydrogenase (PADH I), were characterized by the highest degree of conversion with moderate enantioselectivity (ee=27-82%) of the reaction. In all enzymatic reactions enantiomerically enriched not naturally occurring isomers of trans-(-)-(4R,5S)-4b or cis-(+)-(4R,5R)-4a were formed preferentially. In the second strategy, based on microbial lactonization of γ-oxoacids, naturally occurring opposite isomers of whisky lactones were obtained. Trans-(+)-(4S,5R)-isomer (ee=99%) of whisky lactone 4b was stereoselectively formed as the only product of biotransformations of 3-methyl-4-oxooctanoic acid (5) catalyzed by Didimospheria igniaria KCH6651, Laetiporus sulphurens AM525, Chaetomium sp.1 KCH6670 and Saccharomyces cerevisiae AM464. Biotransformation of γ-oxoacid 5, in the culture of Beauveria bassiana AM278 and Pycnidiella resinae KCH50 afforded a mixtures of trans-(+)-(4S,5R)-4b with enantiomeric excess ee=99% and cis-(-)-(4S,5S)-4a with enantiomeric excesses ee=77% and ee=45% respectively., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Dicarbonyl/l-xylulose reductase (DCXR), mainly catalysing the reduction of α-dicarbonyl compounds and l-xylulose, belongs to the short-chain dehydrogenase/reductase superfamily. Its enzyme activity can be inhibited by short-chain fatty acids. In this study, a novel DCXR inhibitor named (-)-epigallocatechin-3-gallate (EGCG) was reported. First, we overexpressed recombinant human DCXR in Escherichia coli, purified the enzyme by affinity chromatography and measured its activity. The inhibition effects of EGCG and its analogues on DCXR were determined subsequently, and EGCG showed the strongest inhibition with 50% inhibition concentration value of 78.8 μM. The surface plasmon resonance analysis also indicated that the equilibrium dissociation constant (KD) reached to 7.11 × 10(-8) M, which implied a high affinity between EGCG and DCXR. From enzyme kinetic analysis, EGCG acted as a mixed inhibitor against its forward and reverse substrates and the coenzyme, reduced nicotinamide adenine dinucleotide phosphate (NADPH). However, the inhibition is pH dependent. The molecular docking finally showed that EGCG formed several hydrogen bonds with the Thr190 residue of DCXR, and the model was further verified by site-directed mutagenesis. Therefore, EGCG is a potential inhibitor to human DCXR.

YisP is an enzyme involved in the pathway of biofilm formation in bacteria and is predicted to possess squalene synthase activity. A BlastP search using the YisP protein sequence from Bacillus subtilis subsp. subtilis strain 168 shows that it shares 23% identity with the dehydrosqualene synthase from Staphylococcus aureus. The YisP from B. subtilis 168 was expressed in Escherichia coli and the recombinant protein was purified and crystallized. The crystals, which belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 43.966, b = 77.576, c = 91.378 Å, were obtained by the sitting-drop vapour-diffusion method and diffracted to 1.92 Å resolution. Structure determination using MAD and MIR methods is in progress.

Endogenous galactitol-1-phosphate 5-dehydrogenase (GPDH) (EC 1.1.1.251) from Escherichia coli spontaneously interacts with Ni(2+)-NTA matrices becoming a potential contaminant for recombinant, target His-tagged proteins. Purified recombinant, untagged GPDH (rGPDH) converted galactitol into tagatose, and d-tagatose-6-phosphate into galactitol-1-phosphate, in a Zn(2+)- and NAD(H)-dependent manner and readily crystallized what has permitted to solve its crystal structure. In contrast, N-terminally His-tagged GPDH was marginally stable and readily aggregated. The structure of rGPDH revealed metal-binding sites characteristic from the medium-chain dehydrogenase/reductase protein superfamily which may explain its ability to interact with immobilized metals. The structure also provides clues on the harmful effects of the N-terminal His-tag., (Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

In addition to the well established Leloir pathway for the catabolism of d-galactose in fungi, the oxidoreductive pathway has been recently identified. In this oxidoreductive pathway, D-galactose is converted via a series of NADPH-dependent reductions and NAD(+)-dependent oxidations into D-fructose. The pathway intermediates include galactitol, L-xylo-3-hexulose, and d-sorbitol. This study identified the missing link in the pathway, the L-xylo-3-hexulose reductase that catalyzes the conversion of L-xylo-3-hexulose to D-sorbitol. In Trichoderma reesei (Hypocrea jecorina) and Aspergillus niger, we identified the genes lxr4 and xhrA, respectively, that encode the l-xylo-3-hexulose reductases. The deletion of these genes resulted in no growth on galactitol and in reduced growth on D-galactose. The LXR4 was heterologously expressed, and the purified protein showed high specificity for L-xylo-3-hexulose with a K(m) = 2.0 ± 0.5 mm and a V(max) = 5.5 ± 1.0 units/mg. We also confirmed that the product of the LXR4 reaction is D-sorbitol.

Microfloccules of TiO(2) nanoparticles, on which glycerol-dehydrogenase (GDH), 1,3-propanediol-oxidoreductase (PDOR), and glycerol-dehydratase (GDHt) were coimmobilized, were prepared by adsorption-flocculation with polyacrylamide (PAM). The catalytic activity of immobilized enzyme in the glycerol redox reaction system, the enzyme leakage, stabilities of pH and temperature, as well as catalytic kinetics of immobilized enzymes relative to the free enzymes were evaluated. Enzyme loading on the microfloccules as much as 104.1 mg/g TiO(2) (>90% loading efficiency) was obtained under the optimal conditions. PAM played a key role for the formation of microfloccules with relatively homogeneous distribution of size and reducing the enzyme leakage from the microfloccules during the catalysis reaction. The stabilities of GDH against pH and temperature was significantly higher than that those of free GDH. Kinetic study demonstrated that simultaneous NAD(H) regeneration was feasible in glycerol redox system catalysted by these multienzyme microfloccules and the yield of 1, 3-popanediol (1, 3-PD) was up to 11.62 g/L. These results indicated that the porous and easy-separation microfloccules of TiO(2) nanoparticles with immobilized multienzymes were efficient in term of catalytic activity as much as the free enzymes. Moreover, compared with free enzyme, the immobilized multienzymes system exhibited the broader pH, higher temperature stability.

Vibrio cholerae utilizes mannitol through an operon of the phosphoenolpyruvate-dependent phosphotransferase (PTS) type. A gene, mtlD, encoding mannitol-1-phosphate dehydrogenase was identified within the 3.9 kb mannitol operon of V. cholerae. The mtlD gene was cloned from V. cholerae O395, and the recombinant enzyme was functionally expressed in E. coli as a 6×His-tagged protein and purified to homogeneity. The recombinant protein is a monomer with a molecular mass of 42.35 kDa. The purified recombinant MtlD reduced fructose 6-phosphate (F6P) using NADH as a cofactor with a K(m) of 1.54 +/- 0.1 mM and V(max) of 320.8 +/- 7.81 micronmol/min/mg protein. The pH and temperature optima for F6P reduction were determined to be 7.5 and 37°C, respectively. Using quantitative real-time PCR analysis, mtlD was found to be constitutively expressed in V. cholerae, but the expression was up-regulated when grown in the presence of mannitol. The MtlD expression levels were not significantly different between V. cholerae O1 and non-O1 strains.