ß-Hydroxybutyrate dehydrogenase functionalized two-dimensional photonic crystals for quantitative and visual sensing of ketone bodies.

ß-Hydroxybutyrate (BHB) is a substantial physiological ketone body. Its elevated concentration causes ketoacidosis, which is a disorder with a high mortality rate. Therefore, there is an urgent need to develop a simple method for the in-situ monitoring of BHB in urine. In this study, a photonic crystal hydrogel (PCH) sensing material for the detection of urinary ketones was prepared by embedding a two-dimensional polystyrene photonic crystal array (PCA) in a hydrogel functionalized with ß-hydroxybutyrate dehydrogenase (BHBDH). BHBDH catalyzes the interconversion between ß-hydroxybutyrate and acetoacetic acid and relies on the cofactor nicotinamide adenine dinucleotide (NAD+) to participate in the reaction process. The catalytic cycle of converting ß-hydroxybutyrate to acetoacetate generates H+, which reduces the electrostatic repulsion between the carboxyl groups in the hydrogel network, ultimately leading to the shrinkage of the hydrogel volume. The hydrogel volume change was detected by measuring the diameter of the Debye diffraction ring, thus reflecting the concentration of BHB. When the concentration of BHB was increased from 0 to 10 mM, the reflection spectrum of PCH shifted for 117 nm within 60 min, consequently, the structural color of PCH changed from red to green and finally to blue. The material was used for quantitative detection of BHB with a detection limit of 48.94 µM. Then it was used for detection in artificial urine samples. While, this smart and reusable sensing material could provide a more convenient and efficient strategy for the ketone body detection in clinical diagnosis and point-of-care monitoring.

Recharacterization of the mammalian cytosolic type 2 (R)-ß-hydroxybutyrate dehydrogenase as 4-oxo-l-proline reductase (EC 1.1.1.104).

Early studies revealed that chicken embryos incubated with a rare analog of l-proline, 4-oxo-l-proline, showed increased levels of the metabolite 4-hydroxy-l-proline. In 1962, 4-oxo-l-proline reductase, an enzyme responsible for the reduction of 4-oxo-l-proline, was partially purified from rabbit kidneys and characterized biochemically. However, only recently was the molecular identity of this enzyme solved. Here, we report the purification from rat kidneys, identification, and biochemical characterization of 4-oxo-l-proline reductase. Following mass spectrometry analysis of the purified protein preparation, the previously annotated mammalian cytosolic type 2 (R)-ß-hydroxybutyrate dehydrogenase (BDH2) emerged as the only candidate for the reductase. We subsequently expressed rat and human BDH2 in Escherichia coli, then purified it, and showed that it catalyzed the reversible reduction of 4-oxo-l-proline to cis-4-hydroxy-l-proline via chromatographic and tandem mass spectrometry analysis. Specificity studies with an array of compounds carried out on both enzymes showed that 4-oxo-l-proline was the best substrate, and the human enzyme acted with 12,500-fold higher catalytic efficiency on 4-oxo-l-proline than on (R)-ß-hydroxybutyrate. In addition, human embryonic kidney 293T (HEK293T) cells efficiently metabolized 4-oxo-l-proline to cis-4-hydroxy-l-proline, whereas HEK293T BDH2 KO cells were incapable of producing cis-4-hydroxy-l-proline. Both WT and KO HEK293T cells also produced trans-4-hydroxy-l-proline in the presence of 4-oxo-l-proline, suggesting that the latter compound might interfere with the trans-4-hydroxy-l-proline breakdown in human cells. We conclude that BDH2 is a mammalian 4-oxo-l-proline reductase that converts 4-oxo-l-proline to cis-4-hydroxy-l-proline and not to trans-4-hydroxy-l-proline, as originally thought. We also hypothesize that this enzyme may be a potential source of cis-4-hydroxy-l-proline in mammalian tissues.

Transition States for Psychrophilic and Mesophilic (R)-3-Hydroxybutyrate Dehydrogenase-Catalyzed Hydride Transfer at Sub-zero Temperatures.

(R)-3-Hydroxybutyrate dehydrogenase (HBDH) catalyzes the NADH-dependent reduction of 3-oxocarboxylates to (R)-3-hydroxycarboxylates. The active sites of a pair of cold- and warm-adapted HBDHs are identical except for a single residue, yet kinetics evaluated at -5, 0, and 5 °C show a much higher steady-state rate constant (kcat) for the cold-adapted than for the warm-adapted HBDH. Intriguingly, single-turnover rate constants (kSTO) are strikingly similar between the two orthologues. Psychrophilic HBDH primary deuterium kinetic isotope effects on kcat (Dkcat) and kSTO (DkSTO) decrease at lower temperatures, suggesting more efficient hydride transfer relative to other steps as the temperature decreases. However, mesophilic HBDH Dkcat and DkSTO are generally temperature-independent. The DkSTO data allowed calculation of intrinsic primary deuterium kinetic isotope effects. Intrinsic isotope effects of 4.2 and 3.9 for cold- and warm-adapted HBDH, respectively, at 5 °C, supported by quantum mechanics/molecular mechanics calculations, point to a late transition state for both orthologues. Conversely, intrinsic isotope effects of 5.7 and 3.1 for cold- and warm-adapted HBDH, respectively, at -5 °C indicate the transition state becomes nearly symmetric for the psychrophilic enzyme, but more asymmetric for the mesophilic enzyme. His-to-Asn and Asn-to-His mutations in the psychrophilic and mesophilic HBDH active sites, respectively, swap the single active-site position where these orthologues diverge. At 5 °C, the His-to-Asn mutation in psychrophilic HBDH decreases Dkcat to 3.1, suggesting a decrease in transition-state symmetry, while the His-to-Asn mutation in mesophilic HBDH increases Dkcat to 4.4, indicating an increase in transition-state symmetry. Hence, temperature adaptation and a single divergent active-site residue may influence transition-state geometry in HBDHs.

ß-Hydroxybutyrate dehydrogenase decorated MXene nanosheets for the amperometric determination of ß-hydroxybutyrate.

MXene nanosheets of type Ti3C2Tx were modified with ß-hydroxybutyrate dehydrogenase and then used as a biosensor for amperometric sensing of ß-hydroxybutyrate. The MXene and the nanocomposite were characterized by X-ray photoelectron spectroscopy, field-emission scanning electron microscopy, X-ray diffraction, and Fourier transform infrared spectroscopy. The MXene has a layered structure and proved to be an excellent immobilization matrix providing good compatibility with the enzyme ß-hydroxybutyrate dehydrogenase. The MXene-based biosensor, best operated at a potential of - 0.35 V (vs. Ag/AgCl), displays a wide linear range (0.36 to 17.9 mM), a sensitivity of 0.480 µA mM-1 cm-2, and a low detection limit (45 µM). The biosensor was successfully applied to the determination of ß-hydroxybutyrate in (spiked) real serum samples. Graphical abstract Schematic representation of the synthesis and decoration of Mxene 2D sheets with ß-hydroxybutyrate dehydrogenase for the amperometric determination of ß-hydroxybutyric acid.

Kinetics aspects of Gamma-hydroxybutyrate dehydrogenase.

Two groups of metabolically related enzymes, the Group III family of Fe2+-dependent alcohol dehydrogenases (ADHs) and the separate subfamily of nucleoside diphosphates linked to x (nudix) hydrolases that activate Group III ADHs are under-characterized. Here we report the steady-state initial-velocity forward direction (alcohol → aldehyde) reaction of a Group III ADH, namely gamma-hydroxybutyrate dehydrogenase (GHBDH, UniProt: Q59104), cloned from Cupriavidus necator as a fusion protein. We also report the effects of nudix hydrolases on the GHBDH reaction. At optimal pH 9.0, the GHBDH reaction is activated ~2-fold by two different saturating purified nudix hydrolases, namely Bacillus methanolicus activator (ACT, UniProt: I3EA59) and Escherichia coli NudF (UniProt Q93K97) proteins. At physiological pH values of ~7.0, ACT activates by >3.5-fold. Initial-rate characterization at pH 9.0 of the forward direction un-activated and ACT-activated reactions show for both cases competitive inhibition by the product succinic semialdehyde versus GHB, and noncompetitive inhibitions by the three other substrate-product combinations. This pattern is consistent with NAD+ binding first in Mono-Iso Theorell-Chance kinetics. Mutants of some possibly important residues in GHBDH also were characterized. H265, conserved among all Group III ADHs and previously proposed to be a critical general base, is only ~4-fold helpful for GHBDH activity relevant to H265A. The four previously proposed conserved Fe2+ chelators (D193, H197, H261 and H280) each are essential for GHBDH activity. A 2-step explanation for cross-species stimulation by sub-stoichiometric ACT in the forward direction and confirmed lack of ACT stimulation in the reverse direction reaction is proposed.

Linear Eyring Plots Conceal a Change in the Rate-Limiting Step in an Enzyme Reaction.

The temperature dependence of psychrophilic and mesophilic ( R)-3-hydroxybutyrate dehydrogenase steady-state rates yields nonlinear and linear Eyring plots, respectively. Solvent viscosity effects and multiple- and single-turnover pre-steady-state kinetics demonstrate that while product release is rate-limiting at high temperatures for the psychrophilic enzyme, either interconversion between enzyme-substrate and enzyme-product complexes or a step prior to it limits the rate at low temperatures. Unexpectedly, a similar change in the rate-limiting step is observed with the mesophilic enzyme, where a step prior to chemistry becomes rate-limiting at low temperatures. This observation may have implications for past and future interpretations of temperature-rate profiles.

Structural insights into the catalytic reaction trigger and inhibition of D-3-hydroxybutyrate dehydrogenase.

D-3-Hydroxybutyrate dehydrogenase catalyzes the reversible conversion of acetoacetate and D-3-hydroxybutyrate. These ketone bodies are both energy-storage forms of acetyl-CoA. In order to clarify the structural mechanisms of the catalytic reaction with the cognate substrate D-3-hydroxybutyrate and of the inhibition of the reaction by inhibitors, the enzyme from Alcaligenes faecalis has been analyzed by X-ray crystallography in liganded states with the substrate and with two types of inhibitor: malonate and methylmalonate. In each subunit of the tetrameric enzyme, the substrate is trapped on the nicotinamide plane of the bound NAD(+). An OMIT map definitively shows that the bound ligand is D-3-hydroxybutyrate and not acetoacetate. The two carboxylate O atoms form four hydrogen bonds to four conserved amino-acid residues. The methyl group is accommodated in the nearby hydrophobic pocket so that the formation of a hydrogen bond from the OH group of the substrate to the hydroxy group of Tyr155 at the active centre is facilitated. In this geometry, the H atom attached to the C(3) atom of the substrate in the sp(3) configuration is positioned at a distance of 3.1 Šfrom the nicotinamide C(4) atom in the direction normal to the plane. In addition, the donor-acceptor relationship of the hydrogen bonds suggests that the Tyr155 OH group is allowed to ionize by the two donations from the Ser142 OH group and the ribose OH group. A comparison of the protein structures with and without ligands indicates that the Gln196 residue of the small movable domain participates in the formation of additional hydrogen bonds. It is likely that this situation can facilitate H-atom movements as the trigger of the catalytic reaction. In the complexes with inhibitors, however, their principal carboxylate groups interact with the enzyme in a similar way, while the interactions of other groups are changed. The crucial determinant for inhibition is that the inhibitors have no active H atom at C(3). A second determinant is the Tyr155 OH group, which is perturbed by the inhibitors to donate its H atom for hydrogen-bond formation, losing its nucleophilicity.

Succinic semialdehyde reductase Gox1801 from Gluconobacter oxydans in comparison to other succinic semialdehyde-reducing enzymes.

Gluconobacter oxydans is an industrially important bacterium that possesses many uncharacterized oxidoreductases, which might be exploited for novel biotechnological applications. In this study, gene gox1801 was homologously overexpressed in G. oxydans and it was found that the relative expression of gox1801 was 13-fold higher than that in the control strain. Gox1801 was predicted to belong to the 3-hydroxyisobutyrate dehydrogenase-type proteins. The purified enzyme had a native molecular mass of 134 kDa and forms a homotetramer. Analysis of the enzymatic activity revealed that Gox1801 is a succinic semialdehyde reductase that used NADH and NADPH as electron donors. Lower activities were observed with glyoxal, methylglyoxal, and phenylglyoxal. The enzyme was compared to the succinic semialdehyde reductase GsSSAR from Geobacter sulfurreducens and the γ-hydroxybutyrate dehydrogenase YihU from Escherichia coli K-12. The comparison revealed that Gox1801 is the first enzyme from an aerobic bacterium reducing succinic semialdehyde with high catalytic efficiency. As a novel succinic semialdehyde reductase, Gox1801 has the potential to be used in the biotechnological production of γ-hydroxybutyrate.

3-Hydroxybutyrate oligomer hydrolase and 3-hydroxybutyrate dehydrogenase participate in intracellular polyhydroxybutyrate and polyhydroxyvalerate degradation in Paracoccus denitrificans.

Genes encoding 3-hydroxybutyrate oligomer hydrolase (PhaZc) and 3-hydroxybutyrate dehydrogenase (Hbd) were isolated from Paracoccus denitrificans. PhaZc and Hbd were overproduced as His-tagged proteins in Escherichia coli and purified by affinity and gel filtration chromatography. Purified His-tagged proteins had molecular masses of 31 kDa and 120 kDa (a tetramer of 29-kDa subunits). The His-tagged PhaZc hydrolyzed not only 3-hydroxybutyrate oligomers but also 3-hydroxyvalerate oligomers. The His-tagged Hbd catalyzed the dehydrogenation of 3-hydroxyvalerate as well as 3-hydroxybutyrate. When both enzymes were included in the same enzymatic reaction system with 3-hydroxyvalerate dimer, sequential reactions occurred, suggesting that PhaZc and Hbd play an important role in the intracellular degradation of poly(3-hydroxyvalerate). When the phaZc gene was disrupted in P. denitrificans by insertional inactivation, the mutant strain lost PhaZc activity. When the phaZc-disrupted P. denitrificans was complemented with phaZc, PhaZc activity was restored. These results suggest that P. denitrificans carries a single phaZc gene. Disruption of the phaZc gene in P. denitrificans affected the degradation rate of PHA.

Development of 3-hydroxybutyrate dehydrogenase enzyme biosensor based on carbon nanotube-modified screen-printed electrode.

Precise detection of 3-hydroxybutyrate (HB) in biological samples is of great importance for management of diabetic patients. In this study, an HB biosensor based on single-walled carbon nanotubes (SWCNTs)-modified screen-printed electrode (SPE) was developed to determine the concentration of HB in serum. The specific detecting enzyme, HB dehydrogenase, was physically immobilised on SWCNTs deposited on the surface of SPEs. The electrochemical measurement of HB that involved cyclic voltammetry was based on the sAgnal produced by j3-nicotinamide adenine dinucleotide (NADH), one of the products of the enzymatic reaction. The application of SWCNT reduced the oxidation potential of NADH to about -0.05 V. Electrochemical measurements showed that the response of this biosensor had relevant good linearity in the range of 0.1-2 mM with a low detection limit of 0.009 mM. Investigation of biosensor response in the presence of interfering molecules verified its specificity. Furthermore, the study of long-term stability demonstrated the acceptable efficiency of this biosensor for about 100 days.

A novel, disposable, screen-printed amperometric biosensor for ketone 3-ß-hydroxybutyrate fabricated using a 3-ß-hydroxybutyrate dehydrogenase-mesoporous silica conjugate.

A disposable amperometric biosensor for ketone 3-ß-hydroxybutyrate (3HB) has been developed successfully. The sensor is based on a screen-printed carbon electrode containing Meldola's Blue (MB) and sensing components containing nicotinamide adenine dinucleotide (NAD(+)) and 3-ß-hydroxybutyrate dehydrogenase (3HBDH) immobilized in mesoporous silica (FSM8.0) using an aqueous photo-cross-linkable polymer matrix of polyvinyl alcohol (O-391), and it requires only a small sample volume of 10 µL for the measurement. The behavior of a resulting biosensor, i.e., 3HBDH-FSM8.0/NAD(+)/MB-SPCE, was examined in terms of NAD(+) concentration for construction, pH, applied potential, operational range, selectivity, and storage stability. The sensor showed an optimum response at a pH of 7.6 and at an applied potential of -50 mV. The determination range and the response time for 3HB were from 30 µM to 8 mM and approximately 30 s, respectively. In addition, the sensor was quite stable and maintained >90% of its initial response after being stored for over 6 months. This result implies that our method provides a novel biosensor for ketone 3-ß-hydroxybutyrate which is easy-to-use, cost-effective, and has good reproducibility, which are vital for commercial purposes.

Identification of succinic semialdehyde reductases from Geobacter: expression, purification, crystallization, preliminary functional, and crystallographic analysis.

Succinic semialdehyde reductase (SSAR) is an important enzyme involved in γ-aminobutyrate (GABA) metabolism. By converting succinic semialdehyde (SSA) to γ-hydroxybutyrate (GHB), the SSAR facilitates an alternative pathway for GABA degradation. In this study, we identified SSARs from Geobacter sulfurreducens and Geobacter metallireducens (GsSSAR and GmSSAR, respectively). The enzymes were over-expressed in Escherichia coli and purified to near homogeneity. Both GsSSAR and GmSSAR showed the activity of reducing SSA using nicotinamide adenine dinucleotide phosphate as a co-factor. The oligomeric sizes of GsSSAR and GmSSAR, as determined by analytical size exclusion chromatography, suggest that the enzymes presumably exist as tetramers in solution. The recombinant GsSSAR and GmSSAR crystallized in the presence of NADP(+), and the resulting crystals diffracted to 1.89 Å (GsSSAR) and 2.25 Å (GmSSAR) resolution. The GsSSAR and GmSSAR crystals belong to the space groups P2(1)22(1) (a= 99.61 Å, b= 147.49 Å, c= 182.47 Å) and P1 (a= 75.97 Å, b= 79.14 Å, c= 95.47 Å, α = 82.15°, ß = 88.80°, γ = 87.66°), respectively. Preliminary crystallographic data analysis suggests the presence of eight protein monomers in the asymmetric units for both GsSSAR and GmSSAR.

Development of a sensitive long-wavelength fluorogenic probe for nitroreductase: a new fluorimetric indictor for analyte determination by dehydrogenase-coupled biosensors.

Nitroreductase (NTR) is a flavin-containing enzyme that uses NADH as the electron source to reduce nitroaromatic compounds to the corresponding amines. Previous studies have shown that nitroreductase-targeted latent fluorophores exhibit low solubility in the aqueous media and fluoresce at lower wavelengths upon uncloaking, thus limiting their effective applications. Here, we have prepared a new switch-on long-wavelength latent fluorogenic substrate, NTRLF (4), for NTR. In the presence of NADH, NTR catalyzes the reduction of the nitroaromatic moiety in NTRLF (4), followed by the cascade reaction, 1,6-rearrangement-elimination reaction, cyclic urea formation, and concomitant ejects a long-wavelength fluorescence coumarin (8). However, this reaction was inhibited in the presence of nitroaromatic analogues. The fluorescence signal generated by the cascade reaction was specific and insensitive to various reductants. Accordingly, we propose that NTRLF and NTR in the presences of NADH constitute a useful switch-off high-throughput fluorescence sensor for screening nitroaromatic compounds. Furthermore, NTRLF in the NTR-coupled 3-hydroxybutyrate dehydrogenase and aldehyde dehydrogenase assay reactions was a sensitive fluorimetric indicator for the quantitatively measurement of 3-hydroxybutyrate and propionaldehyde, respectively within micromolar range. Our novel NTRLF and NTR-coupled dehydrogenase assay platform may thus be effectively applied for the quantitative estimation of a broad range of analytes.

cAMP receptor protein (CRP) positively regulates the yihU-yshA operon in Salmonella enterica serovar Typhi.

Salmonella enterica serovar Typhi (S. Typhi) is the aetiological agent of typhoid fever in humans. This bacterium is also able to persist in its host, causing a chronic disease by colonizing the spleen, liver and gallbladder, in the last of which the pathogen forms biofilms in order to survive the bile. Several genetic components, including the yihU-yshA genes, have been suggested to be involved in the survival of Salmonella in the gallbladder. In this work we describe how the yihU-yshA gene cluster forms a transcriptional unit regulated positively by the cAMP receptor global regulator CRP (cAMP receptor protein). The results obtained show that two CRP-binding sites on the regulatory region of the yihU-yshA operon are required to promote transcriptional activation. In this work we also demonstrate that the yihU-yshA transcriptional unit is carbon catabolite-repressed in Salmonella, indicating that it forms part of the CRP regulon in enteric bacteria.

A mammalian siderophore synthesized by an enzyme with a bacterial homolog involved in enterobactin production.

Intracellular iron homeostasis is critical for survival and proliferation. Lipocalin 24p3 is an iron-trafficking protein that binds iron through association with a bacterial siderophore, such as enterobactin, or a postulated mammalian siderophore. Here, we show that the iron-binding moiety of the 24p3-associated mammalian siderophore is 2,5-dihydroxybenzoic acid (2,5-DHBA), which is similar to 2,3-DHBA, the iron-binding component of enterobactin. We find that the murine enzyme responsible for 2,5-DHBA synthesis, BDH2, is the homolog of bacterial EntA, which catalyzes 2,3-DHBA production during enterobactin biosynthesis. RNA interference-mediated knockdown of BDH2 results in siderophore depletion. Mammalian cells lacking the siderophore accumulate abnormally high amounts of cytoplasmic iron, resulting in elevated levels of reactive oxygen species, whereas the mitochondria are iron deficient. Siderophore-depleted mammalian cells and zebrafish embryos fail to synthesize heme, an iron-dependent mitochondrial process. Our results reveal features of intracellular iron homeostasis that are conserved from bacteria through humans.

Structural and catalytic properties of the D-3-hydroxybutyrate dehydrogenase from Pseudomonas aeruginosa.

To put forward BDH from Pseudomonas aeruginosa's enzymatic properties, we report a two-step purification of BDH and its gene sequencing allowing the investigation of its structural properties. Purification of BDH was achieved, using ammonium sulfate fractionation and Blue Sepharose CL-6B affinity chromatography. SDS-PAGE analysis reveals a MM of 29 kDa, whereas the native enzyme showed a MM of 120 kDa suggesting a homotetrameric structure. BDH encoding gene sequence shows a nucleotide open reading frame sequence of 771 bp encoding a 265 amino acid residues polypeptide chain. The modeling analysis of the three dimensional structure fits with the importance of amino acids in the catalysis reaction especially a strictly conserved tetrad. Amino-acid residues in interaction with the coenzyme NAD(+) were also identified.

Development and characterization of an enzymatic method for the rapid determination of gamma hydroxybutyric acid.

Gamma hydroxybutyric acid (GHB) is a regulated therapeutic drug, which naturally occurs in mammalian brain tissues as an intermediate of the GABA (gamma aminobutyric acid) neurotransmitter metabolism. The increasing misuse of GHB as a narcotic or abusing drug in recent years calls for the development of a simple and rapid screening method as an alternative to the currently available, technically demanding diagnostic methods. We have developed a rapid enzymatic assay based on the GHB dehydrogenase of Ralstonia eutropha. The enzyme is expressed as a recombinant protein in Escherichia coli and characterized in terms of reaction mechanism and kinetic parameters for the catalysis of conversion of GHB into succinic semialdehyde (SSA). The concomitant NADH production enables spectrophotometric monitoring of the reaction and the quantification of GHB in physiological fluids depending on initial velocities. We have tested a panel of twelve serum and urine samples containing GHB concentrations from 0.0 to 2.1 mmol/L. GHB dehydrogenase activity obeys a non classical bi bi ping pong mechanism exhibiting substrate inhibition by NAD+. With an optimal NAD+ concentration of 3.7 mmol/L in the reaction, the enzyme yields a K(M) of 1.0 mmol/L for GHB and a Vmax of 3.37 mmol/min/mg. The assay shows a linear standard curve from 0.1 to at least 1 mmol/L of GHB. Spiking experiments result in mean recoveries of 92% for urine and 114% for serum, respectively. The comparison to an ion chromatographic reference method exhibits a mean difference of 10% divergence from the target values in urine and 9% in serum, respectively.

Flow injection analysis of angiotensin I-converting enzyme inhibitory activity with enzymatic reactors.

Assay of angiotensin I-converting enzyme (ACE) inhibitory activity always draws much attention because of diverse applications in the field of antihypertension and related pathogenesis. Recently, the use of a new synthetic substrate, 3-hydroxybutyrylglycyl-glycyl-glycine (3HB-GGG), for the assay of ACE inhibitory activity has been confirmed. To construct a rapid, economical, and automatic determination system of ACE inhibitory activity using 3HB-GGG, a flow injection analysis (FIA) system with enzymatic reactors was developed in this study. Enzyme reactors were composed of aminoacylase and 3-hydroxybutyrate dehydrogenase immobilized separately on CNBr-activated Sepharose 4B. The assay condition was optimized in terms of the conversion of 3HB-G into NADH by the enzymatic reactors when the reaction solution containing 3HB-G generated from 3HB-GGG (after the incubation with ACE) was repetitively injected into the FIA system. Under the optimized conditions, 3HB-G was converted to 3HB, and then 3HB was oxidized by NAD(+) to form NADH. The developed system successfully detected practical ACE inhibitors with a great sensitivity, high sampling frequency (10 samples h(-1)) and a durable stability of the enzymatic reactors.

Structure of D-3-hydroxybutyrate dehydrogenase prepared in the presence of the substrate D-3-hydroxybutyrate and NAD+.

D-3-hydroxybutyrate dehydrogenase from Alcaligenes faecalis catalyzes the reversible conversion between D-3-hydroxybutyrate and acetoacetate. The enzyme was crystallized in the presence of the substrate D-3-hydroxybutyrate and the cofactor NAD(+) at the optimum pH for the catalytic reaction. The structure, which was solved by X-ray crystallography, is isomorphous to that of the complex with the substrate analogue acetate. The product as well as the substrate molecule are accommodated well in the catalytic site. Their binding geometries suggest that the reversible reactions occur by shuttle movements of a hydrogen negative ion from the C3 atom of the substrate to the C4 atom of NAD(+) and from the C4 atom of NADH to the C3 atom of the product. The reaction might be further coupled to the withdrawal of a proton from the hydroxyl group of the substrate by the ionized Tyr155 residue. These structural features strongly support the previously proposed reaction mechanism of D-3-hydroxybutyrate dehydrogenase, which was based on the acetate-bound complex structure.

Closed complex of the D-3-hydroxybutyrate dehydrogenase induced by an enantiomeric competitive inhibitor.

D-3-Hydroxybutyrate dehydrogenase (HBDH) from Pseudomonas fragi showed a strict stereospecificity to the d-enantiomer of 3-hydroxybutyrate (d-3-HB) as a substrate. The l-enantiomer acts as a competitive inhibitor, with a K(i) value comparable to the K(m) value for d-3-HB. We have determined the crystal structures of the ternary complex of HBDH-NAD(+)-l-3-HB and the binary complex of HBDH-NAD(+). The former structure showed a so-called closed-form conformation, which is considered an active form for catalysis, while the latter stayed mostly in a open-form conformation. The determined structures along with the site-directed mutagenesis confirmed the substrate recognition mechanism that we proposed previously. The hydrogen bonding interaction between Gln196, located in the moving helix, and the carboxyl group of the substrate/inhibitor is important for the stable ternary complex formation. Finally, the crystal structures of the Thr190 mutants, T190S and T190A, indicate that the Thr190 is a key residue for the open-closed conformational change. T190S retained 37% of the activity. In T190A, however, the activity decreased to 0.1% that of the wild-type enzyme. Fixing the position of the hydroxyl group of Thr190 to form hydrogen bonds to the pyrophosphate moiety and the carboxamide of NAD(+) seems to be a significant factor for the open-closed conformational change.