Monomeric Enzyme Research Articles

Biochemical studies on dipeptidyl peptidase I (cathepsin C) from germinated Vigna radiata seeds

Dipeptidyl peptidases (DPPs) are widely distributed exopeptidases that hydrolyse the dipeptide moieties from the N-termini of oligopeptide chains. In the present study, DPP-I was purified from germinated moong bean seeds via acid and ammonium sulphate precipitation followed by successive chromatographies, that is, gel filtration (pH 7.4), cation exchange (pH 5.9) and anion exchange (pH 7.5). The purity of the enzyme was confirmed by native polyacrylamide gel electrophoresis (PAGE) and in situ gel assay. Purified plant DPP-I is a monomeric enzyme with a molecular weight of 38kDa. It works optimally at pH 7.0 and 40°C, and it exhibits stability at pH ranging from acidic to slightly alkaline. Plant DPP-I preferentially hydrolyses glycine–arginine–4-methoxy-β-naphthylamide and various other synthetic dipeptidyl substrates, but none of the studied endopeptidase and monopeptidase substrates. Inhibitory studies revealed the role of Cys and His amino acids in the catalytic mechanism. Functional studies of DPP-I revealed the significant role of this glycoproteinous enzyme in protein mobilization during germination.

Apparent cooperativity and apparent hyperbolic behavior of enzyme mixtures acting on the same substrate

It is well known that a negative cooperative behavior displayed by a monomeric enzyme may be associated with the simultaneous presence of two enzymes acting on the same substrate. In this paper, emphasis is given to the effect exerted by a rapid equilibrium between the enzyme forms in leading to a hyperbolic behavior, thus masking the presence of multiple enzyme forms.

DISSOCIATION CONSTANTS FOR REVERSIBLE HOMODIMERS OF RECOMBINANT MONOMERIC HUMAN ACETYLCHOLINESTERASE

Size exclusion chromatography profiles of highly purified monomeric recombinant human acetylcholinesterase (hAChE; EC 3.1.1.7) at micro molar concentration range reveal elution volumes smaller than expected for a monomeric globular protein comparable in size to the bovine serum albumin. Dilution of hAChE to the nano molar concentration range results in the elution peak shifts towards larger volumes indicating formation of hAChE monomers. This observation is consistent with small angle X‐ray scattering (SAXS) experiments on hAChE that indicate formation of reversible homodimers of the micro molar monomeric recombinant enzyme in solution (Blumenthal et al., EB2016 abstract).FPLC chromatography of a series of concentrations of recombinant monomeric hAChE in the range of 0.00001 – 1 mg/ml (150 pM‐15uM) was performed to assess concentration dependent elution shifts and determine dimer dissociation constants in 50 mM Tris/HCl, 100 mM NaCl, pH 7.4 at 4 ºC. Peaks of enzyme activity in FPLC elution fractions were detected using Ellman spectrophotometric activity assay. Wild type hAChE constructs with and without N‐terminal FLAG tag sequence as well as Tyr337Ala/Phe338Ala double mutant hAChE not containing FLAG tag were assayed. Size of the hAChE active center gorge in the choline binding domain was increased in this double mutant. Corresponding dimer dissociation constants were determined to be in the range of 1 – 2 uM for both wild type and mutant hAChE, consistent with hAChE oligomerisation interface residing outside of the active center gorge opening as indicated by crystallographic dimers of hAChE.Support or Funding InformationThis work was supported by the Counter ACT Program, National Institutes of Health Office of the Director, Grant 1U01NS083451 from NINDS.

Single-Molecule Mechanistic Dissection of a Chromatin Remodeling Motor

Chromatin remodeling complexes are molecular motors that catalyze diverse structural rearrangements of the genetic material in eukaryotic cells. One such motor, human ACF, is involved in many fundamental gene regulatory processes, including transcriptional activation and repression. ACF is composed of a motor subunit called SNF2h, which is shared with several other remodeling complexes, and a non-catalytic subunit called Acf1. Both SNF2h alone and the ACF complex slide nucleosomes, the basic unit of chromatin, along DNA. Despite the prevalence of ACF and related complexes in genomic regulation, much remains unclear about the mechanism of their sliding activity. SNF2h and Acf1 contain a number of “moving parts” that contribute to significant conformational rearrangements of the enzyme during the remodeling cycle, making them challenging to study by ensemble methods. SNF2h and ACF also dimerize on nucleosomes, further complicating their study in asynchronous populations. Single-molecule techniques are therefore particularly well-suited to investigating such a system. We use single-molecule FRET to observe SNF2h and ACF remodeling individual nucleosomes, to address the following questions: (1) How does the Acf1 accessory subunit modify the basic motor properties of SNF2h, especially in terms of processivity, directional commitment, and remodeling efficiency? (2) How do the “moving” parts of SNF2h combine to regulate the basic activity of the motor? (3) How is protomer coordination achieved across the nucleosome to avoid a tug of war, since enzyme monomers do not directly contact each other?

Purification and Characterization of a New Thermostable, Haloalkaline, Solvent Stable, and Detergent Compatible Serine Protease from Geobacillus toebii Strain LBT 77.

A new thermostable, haloalkaline, solvent stable SDS-induced serine protease was purified and characterized from a thermophilic Geobacillus toebii LBT 77 newly isolated from a Tunisian hot spring. This study reveals the potential of the protease from Geobacillus toebii LBT 77 as an additive to detergent with spectacular proprieties described for the first time. The protease was purified to homogeneity by ammonium sulfate precipitation followed by Sephadex G-75 and DEAE-Cellulose chromatography. It was a monomeric enzyme with molecular weight of 30 kDa. The optimum pH, temperature, and NaCl for maximum protease activity were 13.0, 95°C, and 30%, respectively. Activity was stimulated by Ca2+, Mg2+, DTNB, β-mercaptoethanol, and SDS. The protease was extremely stable even at pH 13.25, 90°C, and 30% NaCl and in the presence of hydrophilic, hydrophobic solvents at high concentrations. The high compatibility with ionic, nonionic, and commercial detergents confirms the utility as an additive to cleaning products. Kinetic and thermodynamic characterization of protease revealed K m = 1 mg mL−1, V max = 217.5 U mL−1, K cat/K m = 99 mg mL−1 S−1, E a = 51.5 kJ mol−1, and ΔG ⁎ = 56.5 kJ mol−1.

Purification and characterization of an extracellular haloalkaline serine protease from the moderately halophilic bacterium, Bacillus iranensis (X5B)

This study reports the purification and characterization of an extracellular haloalkaline serine protease from the moderately halophilic bacterium, Bacillus iranensis, strain X5B. The enzyme was purified to homogeneity by acetone precipitation, ultrafiltration and carboxymethyl (CM) cation exchange chromatography, respectively. The purified protease was a monomeric enzyme with a relative molecular mass of 48-50 kDa and it was inhibited by PMSF indicating that it is a serine-protease. The optimum pH, temperature and NaCl concentration were 9.5, 35 °C and 0.98 M, respectively. The enzyme showed a significant tolerance to salt and alkaline pH. It retained approximately 50% of activity at 2.5 M NaCl and about 70% of activity at highly alkaline pH of 11.0; therefore, it was a moderately halophilic and also can be activated by metals, especially by Ca(2+). The specific activity of the purified protease was measured to be 425.23 μmol of tyrosine/min per mg of protein using casein as a substrate. The apparent K m and V max values were 0.126 mM and 0.523 mM/min, respectively and the accurate value of k cat was obtained as 3.284 × 10(-2) s(-1). These special and important characteristics make this serine protease as valuable tool for industrial applications.

GacA catalyzes the final step in dTDP-L-rhamnose biosynthesis which is critical for the biosynthesis of the Group A carbohydrate, a virulence determinant of the human pathogen Group A Streptococcus (GAS). GacA is a monomeric enzyme (in front of mirror), i

Molecular MicrobiologyVolume 98, Issue 5 p. i-i Front CoverFree Access GacA catalyzes the final step in dTDP-L-rhamnose biosynthesis which is critical for the biosynthesis of the Group A carbohydrate, a virulence determinant of the human pathogen Group A Streptococcus (GAS). GacA is a monomeric enzyme (in front of mirror), in contrast to the previously characterized RmlD dimer (in the mirror), and is essential for GAS survival (in background, left: wild-type cells, right: conditional knockout cells). For details, see the article by van der Beek et al. on pp. 946–962 of this issue. First published: 24 November 2015 https://doi.org/10.1111/mmi.13274AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume98, Issue5December 2015Pages i-i RelatedInformation

Physicochemical properties of thermotolerant extracellular β-glucosidase from Talaromyces thermophilus and enzymatic synthesis of cello-oligosaccharides

A thermophilic fungus, Talaromyces thermophilus that produces a novel thermotolerant extra-cellular β-glucosidase (Bgl.tls), was isolated from Tunisian soil samples. The enzyme was purified from the culture filtrates of T. thermophilus grown on lactose using gel filtration, ion exchange chromatography and FPLC. The monomeric enzyme had a molecular mass of 116.0 kDa and a high specific activity of 1429 UI/mg. Bgl.tls exhibited optimal activity at pH 5.0 and 65 °C. It was also stable over a wide range of pH (4.0–10.0) and stable at 50 °C for 34 h. Bgl.tls retained about 80% of its initial activity after 1.0 hours of preincubation at 60 °C. The Km and Vmax values recorded for pNPG were 0.25 mM and 228.7 µmol min−1, respectively. Bgl.tls was activated by Mn2+, Mg2+, Ca2+ and Co2+ but obviously inhibited by Fe2+ and Cu2+. It was able to hydrolyze a variety of aryl / alkyl -β-glucosides and disaccharides as well as (1→6) and (1→4)-β-glucosidic linkages and α-glycosidic substrates, thus providing evidence for its broad substrate specificity. The enzyme also displayed high hydrolytic and transglycosylation activities. Overall, this study is the first report on the purification and physicochemical properties of a β-glucosidase secreted by T. thermophilus. The cello-oligosaccharides synthesized by this enzyme within 2 h were mainly cellotriose, cellotetraose and cellopentaose identified by HPLC and ESI-MS techniques.

Deleting the Ig-Like Domain of Alicyclobacillus acidocaldarius Endoglucanase Cel9A Causes a Simultaneous Increase in the Activity and Stability.

Endoglucanase Cel9A from Alicyclobacillus acidocaldarius (AaCel9A) is a monomeric enzyme with 537 residues. This enzyme has an Ig-like domain in the N-terminus of the catalytic domain. In this study, the role of the Ig-like domain on the activity, stability, and structural rigidity of AaCel9A and the effect of calcium on enzyme activity and stability were examined by comparing a truncated enzyme with deletion of the Ig-like domain (AaCel9AΔN) to the wild-type enzyme. Our results showed that the deletion of the Ig-like domain increased the catalytic efficiency of the truncated enzyme up to threefold without any significant changes in the K m of the enzyme. Furthermore, pH and temperature optimum for activity were shifted from 6.5 to 7.5 and from 65 to 60°C, respectively, by deletion of the Ig-like domain. The thermal stability and fluorescence quenching results indicated that the stability and rigidity of the truncated enzyme have been more than that of the wild-type enzyme. Calcium similarly increased the catalytic efficiency of the enzymes (up to 40%) and remarkably raised the stability of the AaCel9A compared to the AaCel9AΔN. This shows that Ig-like domain has a role in the increase of the enzyme stability by calcium in the wild-type enzyme.

Functional characterization of a yellow laccase from Leucoagaricus gongylophorus.

In this work we have identified, using mass spectrometry, two laccases produced by Leucoagaricus gongylophorus. One of them, Lac1Lg, was isolated, purified and characterized. Lac1Lg, a monomeric enzyme, was studied using ABTS and syringaldazine substrates. Lac1Lg presented kcat/Km almost threefold higher for syringaldazine than for ABTS, showing a higher catalytic efficiency of Lac1Lg for syringaldazine. The interference of several metal ions and substances in the laccase activity were evaluated. Lac1Lg did not absorb at 600 nm, which is a characteristic of so-called yellow laccases. Lac1Lg also was able to oxidize non-phenolic substrate (anthracene) in the absence of an exogenous mediator, showing that the enzyme has potential to explore in biotechnological processes. Our Lac1Lg three-dimensional molecular model, constructed using homology modeling, showed that the Lac1Lg catalytic site is very closed to blue laccases.

GacA is essential for Group A Streptococcus and defines a new class of monomeric dTDP-4-dehydrorhamnose reductases (RmlD).

SummaryThe sugar nucleotide dTDP‐L‐rhamnose is critical for the biosynthesis of the Group A Carbohydrate, the molecular signature and virulence determinant of the human pathogen Group A S treptococcus (GAS). The final step of the four‐step dTDP‐L‐rhamnose biosynthesis pathway is catalyzed by dTDP‐4‐dehydrorhamnose reductases (RmlD). RmlD from the Gram‐negative bacterium S almonella is the only structurally characterized family member and requires metal‐dependent homo‐dimerization for enzymatic activity. Using a biochemical and structural biology approach, we demonstrate that the only RmlD homologue from GAS, previously renamed GacA, functions in a novel monomeric manner. Sequence analysis of 213 Gram‐negative and Gram‐positive RmlD homologues predicts that enzymes from all Gram‐positive species lack a dimerization motif and function as monomers. The enzymatic function of GacA was confirmed through heterologous expression of gac A in a S. mutans rml D knockout, which restored attenuated growth and aberrant cell division. Finally, analysis of a saturated mutant GAS library using Tn‐sequencing and generation of a conditional‐expression mutant identified gac A as an essential gene for GAS. In conclusion, GacA is an essential monomeric enzyme in GAS and representative of monomeric RmlD enzymes in Gram‐positive bacteria and a subset of Gram‐negative bacteria. These results will help future screens for novel inhibitors of dTDP‐L‐rhamnose biosynthesis.

Detoxification of hexavalent chromium by Leucobacter sp. uses a reductase with specificity for dihydrolipoamide.

Leucobacter sp. belongs to the metal stressed community and possesses higher tolerance to metals including chromium and can detoxify toxic hexavalent chromium by reduction to less toxic trivalent chromium. But, the mechanism of reduction of hexavalent chromium by Leucobacter sp. has not been studied. Understanding the enzyme catalyzing reduction of chromium is important to improve the species for application in bioremediation. Hence, a soluble reductase catalyzing the reduction of hexavalent chromium was purified from a Leucobacter sp. and characterized. The pure chromate reductase was obtained from the cell-free extract through hydrophobic interaction and gel filtration column chromatographic methods. It was a monomeric enzyme and showed similar molecular weights in both gel filtration (∼68 KDa) and SDS-PAGE (64 KDa). It reduced Cr(VI) using both NADH and NADPH as the electron donor, but exhibited higher activity with NADH. The optimal activity was found at pH 5.5 and 30 °C. The K(m) and V(max) for Cr(VI) reduction with NADH were 46.57 μM and 0.37 μmol min(-1) (mg protein) (-1), respectively. The activity was inhibited by p-hydroxy mercury benzoate, Ag(2+) and Hg(2+) indicating the role of thiol groups in the catalysis. The spectrophotometric analysis of the purified enzyme showed the absence of bound flavin in the enzyme. The N-terminal amino acid sequence and LC/MS analysis of trypsin digested purified enzyme showed similarity to dihydrolipoyl dehydrogenase. The purified enzyme had dihydrolipoyl dehydrogenase activity with dihydrolipoamide as the substrate, which suggested that Leucobacter sp. uses reductase with multiple substrate specificity for reduction of Cr(VI) detoxification.

A molecular dynamics study of Beta-Glucosidase B upon small substrate binding

Paenibacillus polymyxa β-glucosidase B (BglB), belongs to a GH family 1, is a monomeric enzyme that acts as an exo-β-glucosidase hydrolysing cellobiose and cellodextrins of higher degree of polymerization using retaining mechanism. A molecular dynamics (MD) simulation was performed at 300 K under periodic boundary condition for 5 ns using the complexes structure obtained from previous docking study, namely BglB-Beta-d-glucose and BglB-Cellobiose. From the root-mean-square deviation analysis, both enzyme complexes were reported to deviate from the initial structure in the early part of the simulation but it was stable afterwards. The root-mean-square fluctuation analysis revealed that the most flexible regions comprised of the residues from 26 to 29, 43 to 53, 272 to 276, 306 to 325 and 364 to 367. The radius of gyration analysis had shown the structure of BglB without substrate became more compact towards the end of the simulation compare to other two complexes. The residues His122 and Trp410 were observed to form stable hydrogen bond with occupancy higher than 10%. In conclusion, the behaviour of BglB enzyme towards the substrate binding was successfully explored via MD simulation approaches.

Tandem Benzophenone Amino Pyridines, Potent and Selective Inhibitors of Human Leukotriene C4 Synthase.

Cysteinyl leukotrienes (cys-LTs) are lipid mediators of inflammation. The enzyme catalyzing synthesis of cys-LTs, leukotriene C4 synthase (LTC4S), is considered an important drug target. Here we report the synthesis and characterization of three tandem benzophenone amino pyridines as inhibitors of LTC4S in vitro and in vivo. The inhibitors were characterized in vitro using recombinant human LTC4S, MonoMac 6 cells, and a panel of peripheral human immune cells. In vivo, the compounds were tested in the Zymosan A-induced peritonitis mouse model. The molecules, denoted TK04, TK04a, and TK05, were potent and selective inhibitors of LTC4S with IC50 values of 116, 124, and 95 nM, respectively. Molecular docking revealed binding in a hydrophobic crevice between two enzyme monomers and interaction with two catalytic residues, Arg104 and Arg31. The TK compounds potently inhibited cys-LT biosynthesis in immune cells. In coincubations of platelets and polymorphonuclear leukocytes, inhibition of LTC4S led to shunting of LTA4 toward anti-inflammatory lipoxin A4, which was significantly enhanced by simultaneous inhibition of LTA4H. Finally, we found that TK05 (6 mg⋅kg(-1)⋅body weight) reduces LTE4 levels in peritoneal lavage fluid by 88% and significantly decreases vascular permeability in vivo. Our findings indicate that the TK compounds are valuable experimental tools in eicosanoid research in vitro and in vivo. Their chemical structures may serve as leads for further inhibitor design. Novel drugs depleting cys-LT production could be beneficial for treatment of inflammatory diseases associated with overexpression of LTC4S.

Structure of α-carbonic anhydrase from the human pathogen Helicobacter pylori.

The crystal structure of α-carbonic anhydrase, an enzyme present in the periplasm of Helicobacter pylori, a bacterium that affects humans and that is responsible for several gastric pathologies, is described. Two enzyme monomers are present in the asymmetric unit of the monoclinic space group P21, forming a dimer in the crystal. Despite the similarity of the enzyme structure to those of orthologues from other species, the H. pylori protein has adopted peculiar features in order to allow the bacterium to survive in the difficult environment of the human stomach. In particular, the crystal structure shows how the bacterium has corrected for the mutation of an essential amino acid important for catalysis using a negative ion from the medium and how it localizes close to the inner membrane in the periplasm. Since carbonic anhydrase is essential for the bacterial colonization of the host, it is a potential target for antibiotic drugs. The definition of the shape of the active-site entrance and cavity constitutes a basis for the design of specific inhibitors.

Evidence of Kinetic Cooperativity in Dimeric Ketopantoate Reductase from Staphylococcus aureus.

Ketopantoate reductase (KPR) catalyzes the NADPH-dependent production of pantoate, an essential precursor in the biosynthesis of coenzyme A. Previous structural studies have been limited to Escherichia coli KPR, a monomeric enzyme that follows a sequential ordered mechanism. Here we report the crystal structure of the Staphylococcus aureus enzyme at 1.8 Å resolution, the first description of a dimeric KPR. Using sedimentation velocity analysis, we show that the S. aureus KPR dimer is stable in solution. In fact, our structural analysis shows that the dimeric assembly we identify is present in the majority of KPR crystal structures. Steady state analysis of S. aureus KPR reveals strong positive cooperativity with respect to NADPH (Hill coefficient of 2.5). In contrast, high concentrations of the substrate ketopantoate (KP) inhibit the activity of the enzyme. These observations are consistent with a random addition mechanism in which the initial binding of NADPH is the kinetically preferred path. In fact, Förster resonance energy transfer studies of the equilibrium binding of NADPH show only a small degree of cooperativity between subunits (Hill coefficient of 1.3). Thus, the apparently strong cooperativity observed in substrate saturation curves is due to a kinetic process that favors NADPH binding first. This interpretation is consistent with our analysis of the A181L substitution, which increases the Km of ketopantoate 844-fold, without affecting kcat. The crystal structure of KPRA181L shows that the substitution displaces Ser239, which is known to be important for the binding affinity of KP. The decrease in KP affinity would enhance the already kinetically preferred NADPH binding path, making the random mechanism appear to be sequentially ordered and reducing the kinetic cooperativity. Consistent with this interpretation, the NADPH saturation curve for KPRA181L is hyperbolic.

Nano-rheology measurements reveal that the hydration layer of enzymes partially controls conformational dynamics

For a typical (20 kD, 4 nm size) monomeric enzyme, more than 50% of the residues are at the surface. The mechanics of these soft, heterogeneous nanoparticles was recently shown to be viscoelastic. Here, we explore the contribution of the enzyme's surface to the mechanics of the molecule. Nano-rheology provides sub-Å resolution measurements of the reversible deformation of the enzyme subject to an oscillatory mechanical stress. We perturb the surface of the enzyme by adding small amounts of DMSO (dimethyl sulfoxide), believed to affect ordering of the enzyme–water interface. We observe a dramatic though reversible change in the mechanics of the enzyme, which becomes more viscous. On the other hand, the catalytic speed is unaffected, while at higher DMSO concentrations (>1 %) it even increases.

Fluorescent Silica Nanoparticles with Multivalent Inhibitory Effects towards Carbonic Anhydrases.

Multifunctional silica nanoparticles decorated with fluorescent and sulfonamide carbonic anhydrase (CA) inhibitors were prepared and investigated as multivalent enzyme inhibitors against the cytosolic isoforms hCA I and II and the transmembrane tumor-associated ones hCA IX and XII. Excellent inhibitory effects were observed with these nanoparticles, with KI values in the low nanomolar range (6.2-0.67 nM) against all tested isozymes. A significant multivalency effect was seen for the inhibition of the monomeric enzymes hCA I and II compared to the dimeric hCA IX and hCA XII isoforms, where no multivalent effect was observed, suggesting that the multivalent binding is occurring through enzyme clustering.

Too packed to change: side-chain packing and site-specific substitution rates in protein evolution.

In protein evolution, due to functional and biophysical constraints, the rates of amino acid substitution differ from site to site. Among the best predictors of site-specific rates are solvent accessibility and packing density. The packing density measure that best correlates with rates is the weighted contact number (WCN), the sum of inverse square distances between a site’s Cα and the Cα of the other sites. According to a mechanistic stress model proposed recently, rates are determined by packing because mutating packed sites stresses and destabilizes the protein’s active conformation. While WCN is a measure of Cα packing, mutations replace side chains. Here, we consider whether a site’s evolutionary divergence is constrained by main-chain packing or side-chain packing. To address this issue, we extended the stress theory to model side chains explicitly. The theory predicts that rates should depend solely on side-chain contact density. We tested this prediction on a data set of structurally and functionally diverse monomeric enzymes. We compared side-chain contact density with main-chain contact density measures and with relative solvent accessibility (RSA). We found that side-chain contact density is the best predictor of rate variation among sites (it explains 39.2% of the variation). Moreover, the independent contribution of main-chain contact density measures and RSA are negligible. Thus, as predicted by the stress theory, site-specific evolutionary rates are determined by side-chain packing.

Methemoglobin reduction by NADH-cytochrome b(5) reductase in Propsilocerus akamusi larvae.

For oxygen respiration, a methemoglobin (metHb) reduction system is needed in the cell because metHb cannot bind oxygen. We examined the insect Propsilocerus akamusi larvae to elucidate the metHb reduction system in an organism that inhabits an oxygen-deficient environment. NADH-dependent reduction of metHb in coelomic fluid suggested the coexistence of cytochrome b5 reductase (b5R) and cytochrome b5 with hemoglobin in the fluid and that these proteins were involved in physiological metHb reduction in the larvae. The presence of b5R was revealed by purifying b5R to homogeneity from the midge larvae. Using purified components, we showed that larval metHb was reduced via the NADH-b5R (FAD)-cytochrome b5-metHb pathway, a finding consistent with that in aerobic vertebrates, specifically humans and rabbits, and b5R function between mammal and insect was conserved. b5R was identified as a monomeric FAD-containing enzyme; it had a molecular mass of 33.2 kDa in gel-filtration chromatography and approximately 37 kDa in SDS-PAGE analysis. The enzyme's optimal pH and temperature were 6.4 and 25 °C, respectively. The apparent Km and Vmax values were 345 μM and 160 μmol min(-1) mg(-1), respectively, for ferricyanide and 328 μM and 500 μmol min(-1) mg(-1), respectively, for 2,6-dichlorophenolindophenol. The enzyme reaction was inhibited by benzoate, p-hydroxymercuribenzoate, iodoacetamide, and iodoacetate, and was not inhibited by metal ions or EDTA.