Inactive Apoenzyme Research Articles

Functional contacts for activation of urease from Helicobacter pylori: an integrated approach using evolutionary couplings, in-cell enzymatic assays, and computational docking

Introduction: Urease is an enzyme exploited by many virulent bacteria and fungi to infect the host and exert their virulence. The Gram-negative bacterium Helicobacter pylori relies on the activity of urease to infect the highly acidic human stomach. The activity of urease depends on the presence of a catalytic site containing two Ni(II) ions. In vivo, urease is initially synthesized as an inactive apo-enzyme and requires a post-translational activation process that involves the incorporation of the metal ions into its buried active site. In H. pylori, as well as in other bacteria, this activation process is mediated by four accessory proteins, named UreD, UreF, UreG, and UreE. Targeting the interactions between urease chaperones could potentially inhibit the activation of urease through blocking the Ni(II) ions incorporation, providing a route for the development of antimicrobial strategies against ureolytic pathogens.Methods: In this paper, an evolutionary couplings (EC) approach was adopted to determine the interaction surface between urease and UreD, the first protein that binds the enzyme, preparing it for the subsequent activation steps. Site-directed mutagenesis and an in-cell assay were used to detect urease activity in recombinant bacteria expressing the mutated operon. The obtained data were used to drive a protein-protein docking computational approach.Results and Discussion: The EC prediction retrieved ten pairs of residues lying at the interface between UreD and the urease subunit UreB, likely involved in contacts essential to build the protein complex. These contacts were largely confirmed experimentally, leading to the obtainment of a model for the urease-UreD complex that agrees well with the recently reported experimental cryo-EM structure. This work represents a proof of concept for the calculation of reliable models of protein interaction surfaces in the absence of experimental structures of critical assemblies.

Enzyme Activation with a Synthetic Catalytic Co-enzyme Intermediate: Nucleotide Methylation by Flavoenzymes.

To facilitate production of functional enzymes and to study their mechanisms, especially in the complex cases of coenzyme-dependent systems, activation of an inactive apoenzyme preparation with a catalytically competent coenzyme intermediate is an attractive strategy. This is illustrated with the simple chemical synthesis of a flavin-methylene iminium compound previously proposed as a key intermediate in the catalytic cycle of several important flavoenzymes involved in nucleic acid metabolism. Reconstitution of both flavin-dependent RNA methyltransferase and thymidylate synthase apoproteins with this synthetic compound led to active enzymes for the C5-uracil methylation within their respective transfer RNA and dUMP substrate. This strategy is expected to be of general application in enzymology.

Enzyme Activation with a Synthetic Catalytic Co‐enzyme Intermediate: Nucleotide Methylation by Flavoenzymes

AbstractTo facilitate production of functional enzymes and to study their mechanisms, especially in the complex cases of coenzyme‐dependent systems, activation of an inactive apoenzyme preparation with a catalytically competent coenzyme intermediate is an attractive strategy. This is illustrated with the simple chemical synthesis of a flavin‐methylene iminium compound previously proposed as a key intermediate in the catalytic cycle of several important flavoenzymes involved in nucleic acid metabolism. Reconstitution of both flavin‐dependent RNA methyltransferase and thymidylate synthase apoproteins with this synthetic compound led to active enzymes for the C5‐uracil methylation within their respective transfer RNA and dUMP substrate. This strategy is expected to be of general application in enzymology.

Enhancement of potency and stability of human extracellular superoxide dismutase

Cells express several antioxidant enzymes to scavenge reactive oxygen species (ROS) responsible for oxidative damages and various human diseases. Therefore, antioxidant enzymes are considered biomedicine candidates. Among them, extracellular superoxide dismutase (SOD3) had showed prominent efficacy against asthma and inflammation. Despite its advantages as a biomedicine, the difficulty in obtaining large quantity of active recombinant human SOD3 (rhSOD3) has limited its clinical applications. We found that a significant fraction of overexpressed rhSOD3 was composed of the inactive apo-enzyme and its potency against inflammation depended on the rate of metal incorporation. Also, purified rhSOD3 was unstable and lost its activity very quickly. Here, we suggest an ideal preparative method to express, purify, and store highly active rhSOD3. The enzymatic activity of rhSOD3 was maximized by incorporating metal ions into rhSOD3 after purification. Also, albumin or polyethylene glycol prevented rapid inactivation or degradation of rhSOD3 during preparative procedures and long-term storage. [BMB Reports 2015; 48(2): 91-96]

Arginine aminopeptidase from white shrimp (Litopenaeus vannamei) muscle: purification and characterization

Aminopeptidases act on N-terminal of proteins and peptides produce free amino acids making an impact on the final flavor of foods. An arginine aminopeptidase (RAP) which preferred to hydrolyze basic amino acids from N-termini of peptides and proteins was purified to homogeneity from white shrimp (Litopenaeus vannamei) muscle. The molecular mass of RAP was estimated as 100 kDa on SDS-PAGE. Peptide mass fingerprinting analysis obtained 95 amino acid residues which was 100 and 77.9 % identical to puromycin-sensitive aminopeptidases from insect and zebrafish, respectively. Optimum pH and temperature of the RAP were 7.0 and 30 °C. RAP rapidly hydrolyzed fluorogenic substrates l-arginine 4-methylcoumaryl-7-amide (Arg-MCA) and Lys-MCA with Km values of 2.7 and 4.9 μM, respectively. The enzyme can be strongly inhibited by puromycin, bestatin, and 1,10-phenanthroline and partially inhibited by ethylenediaminetetraacetic acid (EDTA) and ethylene glycol-bis (2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA). Moreover, the competitive inhibition of puromycin for RAP was confirmed, and Ki value was calculated as 0.07 nM. Metal ions of Zn2+ and Mn2+ significantly reactivated the inactive apoenzyme activity dialyzed by EDTA. All these results indicated that the purified enzyme is a metalloaminopeptidase which would possibly contribute to flavor development in shrimp muscle.

The M17 Leucine Aminopeptidase of the Malaria Parasite Plasmodium falciparum: Importance of Active Site Metal Ions in the Binding of Substrates and Inhibitors

The M17 leucine aminopeptidase of the intraerythrocytic stages of the malaria parasite Plasmodium falciparum (PfLAP) plays a role in releasing amino acids from host hemoglobin that are used for parasite protein synthesis, growth, and development. This enzyme represents a target at which new antimalarials could be designed since metalloaminopeptidase inhibitors prevent the growth of the parasites in vitro and in vivo. A study on the metal ion binding characteristics of recombinant P. falciparum M17 leucine aminopeptidase (rPfLAP) shows that the active site of this exopeptidase contains two metal-binding sites, a readily exchangeable site (site 1) and a tight binding site (site 2). The enzyme retains activity when the metal ion is removed from site 1, while removal of metal ions from both sites results in an inactive apoenzyme that cannot be reactivated by the addition of divalent metal cations. The metal ion at site 1 is readily exchangeable with several divalent metal ions and displays a preference in the order of preference Zn(2+) > Mn(2+) > Co(2+) > Mg(2+). While it is likely that native PfLAP contains a Zn(2+) in site 2, the metal ion located in site 1 may be dependent on the type and concentration of metal ions in the cytosolic compartment of the parasite. Importantly, the type of metal ion present at site 1 influences not only the catalytic efficiency of the enzyme for peptide substrates but also the mode of binding by bestatin, a metal-chelating inhibitor of M17 aminopeptidases with antimalarial activity.

Glucose Modulation of Glucokinase Activation by Small Molecules

Small molecule activators of glucokinase (GK) were used in kinetic and equilibrium binding studies to probe the biochemical basis for their allosteric effects. These small molecules decreased the glucose K 0.5 ( approximately 1 mM vs approximately 8 mM) and the glucose cooperativity (Hill coefficient of 1.2 vs 1.7) and lowered the k cat to various degrees (62-95% of the control activity). These activators relieved GK's inhibition from glucokinase regulatory protein (GKRP) in a glucose-dependent manner and activated GK to the same extent as control reactions in the absence of GKRP. In equilibrium binding studies, the intrinsic glucose affinity to the activator-bound enzyme was determined and demonstrated a 700-fold increase relative to the apoenzyme. This is consistent with a reduction in apparent glucose K D and the steady-state parameter K 0.5 as a result of enzyme equilibrium shifting to the activator-bound form. The binding of small molecules to GK was dependent on glucose, consistent with the structural evidence for an allosteric binding site which is present in the glucose-induced, active enzyme form of GK and absent in the inactive apoenzyme [Kamata et al. (2004) Structure 12, 429-438]. A mechanistic model that brings together the kinetic and structural data is proposed which allows qualitative and quantitative analysis of the glucose-dependent GK regulation by small molecules. The regulation of GK activation by glucose may have an important implication for the discovery and design of GK activators as potential antidiabetic agents.

D-Ribulose 5-Phosphate 3-Epimerase: Functional and Structural Relationships to Members of the Ribulose-Phosphate Binding (β/α)8-Barrel Superfamily,

The "ribulose phosphate binding" superfamily defined by the Structural Classification of Proteins (SCOP) database is considered the result of divergent evolution from a common (beta/alpha)(8)-barrel ancestor. The superfamily includes d-ribulose 5-phosphate 3-epimerase (RPE), orotidine 5'-monophosphate decarboxylase (OMPDC), and 3-keto-l-gulonate 6-phosphate decarboxylase (KGPDC), members of the OMPDC suprafamily, as well as enzymes involved in histidine and tryptophan biosynthesis that utilize phosphorylated metabolites as substrates. We now report studies of the functional and structural relationships of RPE to the members of the superfamily. As suggested by the results of crystallographic studies of the RPEs from rice [Jelakovic, S., Kopriva, S., Suss, K. H., and Schulz, G. E. (2003) J. Mol. Biol. 326, 127-35] and Plasmodium falciparum [Caruthers, J., Bosch, J., Bucker, F., Van Voorhis, W., Myler, P., Worthey, E., Mehlin, C., Boni, E., De Titta, G., Luft, J., Kalyuzhniy, O., Anderson, L., Zucker, F., Soltis, M., and Hol, W. G. J. (2006) Proteins 62, 338-42], the RPE from Streptococcus pyogenes is activated by Zn(2+) which binds with a stoichiometry of one ion per polypeptide. Although wild type RPE has a high affinity for Zn(2+) and inactive apoenzyme cannot be prepared, the affinity for Zn(2+) is decreased by alanine substitutions for the two histidine residues that coordinate the Zn(2+) ion (H34A and H67A); these mutant proteins can be prepared in an inactive, metal-free form and activated by exogenous Zn(2+). The crystal structure of the RPE was solved at 1.8 A resolution in the presence of d-xylitol 5-phosphate, an inert analogue of the d-xylulose 5-phosphate substrate. This structure suggests that the 2,3-enediolate intermediate in the 1,1-proton transfer reaction is stabilized by bidentate coordination to the Zn(2+) that also is liganded to His 34, Asp 36, His 67, and Asp 176; the carboxylate groups of the Asp residues are positioned also to function as the acid/base catalysts. Although the conformation of the bound analogue resembles those of ligands bound in the active sites of OMPDC and KGPDC, the identities of the active site residues that coordinate the essential Zn(2+) and participate as acid/base catalysts are not conserved. We conclude that only the phosphate binding motif located at the ends of the seventh and eighth beta-strands of the (beta/alpha)(8)-barrel is functionally conserved among RPE, OMPDC, and KGPDC, consistent with the hypothesis that the members of the "ribulose phosphate binding" (beta/alpha)(8)-barrel "superfamily" as defined by SCOP have not evolved by evolutionary processes involving the intact (beta/alpha)(8)-barrel. Instead, this "superfamily" may result from assembly from smaller modules, including the conserved phosphate binding motif associated with the C-terminal (beta/alpha)(2)-quarter barrel.

Bacillus subtilis 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase revisited: resolution of two long-standing enigmas

The mono/bifunctional and metallo/non-metallo properties of Bacillus subtilis DAHPS (3-deoxy-D-arabino-heptulosonate 7-phosphate synthase) have been controversial for several decades. The present study investigated the DAHPSs from both the B. subtilis parent Marburg strain and the derivative strain 168 in detail and clarified the above two long-standing questions. The DAHPSs from the parent and the derivative 168 strains have identical sequence and are both bifunctional enzymes with a CM (chorismate mutase) activity and a DAHPS activity. The parent strain expresses a second independent monofunctional CM, encoded by aroH, that is highly active, while the 168 strain expresses an aroH containing a single residue mutation (A112V) that is significantly less active thus leading to previous confusion regarding the mono/bifunctionality of DAHPS. Metal analysis showed that B. subtilis DAHPS as isolated contained iron and zinc and is inactivated by dipicolinic acid; the inactive apoenzyme can be reactivated by bivalent metal ions, indicating that the enzyme is a metalloenzyme. The enzyme-bound metal is insensitive to EDTA treatment, leading to the previous conclusion that this DAHPS does not require a metal. The enzyme displays a homotetrameric structure in solution and appears to follow Michaelis-Menten kinetics with K(m)(PEP)=139+/-11.4 microM for phosphoenolpyruvate, K(m)(E4P)=1760+/-110 microM for D-erythrose 4-phosphate, kcat=4.6+/-0.1 s(-1) for DAHPS activity and K(m)(chorismate)=850+/-97 microM, kcat=0.41+/-0.01 s(-1) for CM activity. B. subtilis DAHPS is inhibited by the Shikimate pathway intermediates prephenate and chorismate.

Modulation of cooperativity in Mycobacterium tuberculosis NADPH‐ferredoxin reductase: Cation‐and pH‐induced alterations in native conformation and destabilization of the NADP+‐binding domain

FprA, a Mycobacterium tuberculosis NADPH-ferredoxin reductase, consists of two structural domains, a FAD-binding and a NADP-binding domain, respectively. For the first time, we demonstrated that native FprA, on thermal treatment underwent partial denaturation with unfolding of only the FAD-binding domain and release of the protein-bound flavin. The NADP-binding domain of this protein is highly resistant to denaturation under these conditions. However, the presence of either 150 mM NaCl or KCl or 10 muM MgCl(2) or CaCl(2) or slightly acidic pH of 6.0 resulted in a highly cooperative and complete thermal unfolding of the protein. Physicochemical investigations showed that the monovalent cations or low concentrations of divalent cations induced compaction of the protein conformation. However, divalent cations at higher concentrations resulted in FAD release leading to stabilization of an enzymatically inactive apoenzyme. Detailed thermal denaturation studies on the native protein and the isolated NADP-binding domain showed that cations and pH 6.0 destabilized only the heat-stable NADP-binding domain. The experimental studies demonstrate that modulation of intramolecular ionic interactions induce significant conformational changes in the NADP-binding domain of FprA, resulting in a substantial increase in the structural cooperativity of the whole molecule. The results presented in this paper are of importance as they demonstrate alterations in the native three-dimensional structure of FprA and cooperativity in protein molecule on slight alteration of pH or modification of ionic interactions in protein.

Removal of the tightly bound zinc from Escherichia coli trypsin-modified methionyl-tRNA synthetase.

The study of the behaviour of Escherichia coli methionyl-tRNA synthetase with chelating agents has shown that only 1,10-phenanthroline has an inhibitory effect on the tRNAMet aminoacylation activity. Under identical buffer conditions the isotopic [32P]PPi-ATP exchange activity is insensitive. Dialysis of the enzyme against 1,10-phenanthroline causes a slow loss of zinc from the enzyme which is paralleled by an irreversible loss of both the aminoacylation and isotopic exchange activities. The loss of zinc becomes faster upon the addition of small amounts of guanidine hydrochloride to the dialysis buffer containing phenanthroline, presumably by partially unfolding the protein. Studies of the reversible denaturation of the enzyme by 5 M guanidine hydrochloride shows that the inclusion of EDTA produces an enzyme species that has lost both zinc and activity. The inactive apoenzyme prepared in guanidine and EDTA can regain activity by dilution in a zinc-containing buffer.

Protein-Based Biosensors for Diabetic Patients

In this article we show the recent progress in the field of glucose sensing based on the utilization of enzymes and proteins as probes for stable and non-consuming fluorescence biosensors. We developed a new methodology for glucose sensing using inactive forms of enzymes such as the glucose oxidase from Aspergillus niger, the glucose dehydrogenase from the thermophilic microorganism Thermoplasma acidophilum, and the glucokinase from the thermophilic eubacterium Bacillus stearothermophilus. Glucose oxidase was rendered inactive by removal of the FAD cofactor. The resulting apo-glucose oxidase still binds glucose as observed from a decrease in its intrinsic tryptophan fluorescence. 8-Anilino-1-naphthalene sulfonic acid was found to bind spontaneously to apo-glucose oxidase as seen from an enhancement of the ANS fluorescence. The steady state intensity of the bound ANS decreased 25% upon binding of glucose, and the mean lifetime of the bound ANS decreased about 40%. These spectral changes occurred with a midpoint from 10 to 20 mM glucose, which is comparable to the KD of holo-glucose oxidase. The ANS-labeled apo-glucose dehydrogenase from Thermoplasma acidophilum also displayed an approximate 25% decrease in emission intensity upon binding glucose. This decrease can be also used to measure the glucose concentration. The thermophilic apo-glucose dehydrogenase was also stable in the presence of organic solvents, allowing determination of glucose in the presence of acetone. The third enzyme used for glucose sensing was the glucokinase from Bacillus stearothermophilus. A fluorescence competitive assay for the determination of glucose was developed based on the utilization of this thermostable enzyme. Taken together, our results show that enzymes which use glucose as their substrate can be used as reversible and non-consuming glucose biosensors in the absence of required co-factors. Moreover, the possibility of using inactive apo-enzymes for a reversible sensor greatly expands the range of proteins which can be used as sensors, not only for glucose, but for a wide variety of biochemically relevant analytes.

Unusual Structural, Functional, and Stability Properties of Serine Hydroxymethyltransferase from Mycobacterium tuberculosis

From the genome analysis of the Mycobacterium tuberculosis two putative genes namely GlyA and GlyA2 have been proposed to encode for the enzyme serine hydroxymethyltransferase. We have cloned, overexpressed, and purified to homogeneity their respective protein products, serine hydroxymethyltransferase, SHM1 and SHM2. The recombinant SHM1 and SHM2 exist as homodimers of molecular mass about 90 kDa under physiological conditions, however, SHM2 has more compact conformation and higher thermal stability than SHM1. The most interesting structural observation was that the SHM1 contains 1 mol of pyridoxal 5'-phosphate (PLP)/mol of enzyme dimer. This is the first report of such a unique stoichiometry of PLP and enzyme dimer for SHMT. The SHM2 contains 2 mol of PLP/mol of enzyme dimer, which is the usual stoichiometry reported for SHMT. Functionally both the recombinant enzymes showed catalysis of reversible interconversion of serine and glycine and aldol cleavage of a 3-hydroxyamino acid. However, unlike SHMT from other sources both SHM1 and SHM2 do not undergo half-transamination reaction with d-alanine resulting in formation of apoenzyme but l-cysteine removed the prosthetic group, PLP, from both the recombinant enzymes leaving the respective inactive apoenzymes. Comparative structural studies on the two enzymes showed that the SHM1 is resistant to alkaline denaturation up to pH 10.5, whereas the native SHM2 dimer dissociates into monomer at pH 9. Urea- and guanidinium chloride-induced two-step unfolding of SHM1 and SHM2 with the first step being dissociation of dimer into apomonomer at low denaturant concentrations followed by unfolding of the stabilized monomer at higher denaturant concentrations.

Improved recovery of active recombinant laccase from maize seed.

Lignolytic enzymes such as laccase have been difficult to over-express in an active form. This paper describes the expression, characterization, and application of a fungal laccase in maize seed. The transgenic seed contains immobilized and extractable laccase. Fifty ppm dry weight of aqueously extractable laccase was obtained, and the remaining solids contained a significant amount of immobilized laccase that was active. Although a portion of the extractable laccase was produced as inactive apoenzyme, laccase activity was recovered by treatment with copper and chloride. In addition to allowing the apoenzyme to regain activity, treatment with copper also provided a partial purification step by precipitating other endogenous corn proteins while leaving >90% of the laccase in solution. The data also demonstrate the application of maize-produced laccase as a polymerization agent. The apparent concentration of laccase in ground, defatted corn germ is approximately 0.20% of dry weight.

Recognition of Hybrid Peptidyl Carrier Proteins/Acyl Carrier Proteins in Nonribosomal Peptide Synthetase Modules by the 4′-Phophopantetheinyl Transferases AcpS and Sfp

The acyl carrier proteins (ACPs) of fatty acid synthase and polyketide synthase as well as peptidyl carrier proteins (PCPs) of nonribosomal peptide synthetases are modified by 4'-phosphopantetheinyl transferases from inactive apo-enzymes to their active holo forms by transferring the 4'-phosphopantetheinyl moiety of coenzyme A to a conserved serine residue of the carrier protein. 4'-Phosphopantetheinyl transferases have been classified into two types; the AcpS type accepts ACPs of fatty acid synthase and some ACPs of type II polyketide synthase as substrates, whereas the Sfp type exhibits an extraordinarily broad substrate specificity. Based on the previously published co-crystal structure of Bacillus subtilis AcpS and ACP that provided detailed information about the interacting residues of the two proteins, we designed a novel hybrid PCP by replacing the Bacillus brevis TycC3-PCP helix 2 with the corresponding helix of B. subtilis ACP that contains the interacting residues. This was performed for the PCP domain as a single protein as well as for the TycA-PCP domain within the nonribosomal peptide synthetase module TycA from B. brevis. Both resulting proteins, designated hybrid PCP (hPCP) and hybrid TycA (hTycA), were modified in vivo during heterologous expression in Escherichia coli (hPCP, 51%; hTycA, 75%) and in vitro with AcpS as well as Sfp to 100%. The designated hTycA module contains two other domains: an adenylation domain (activating phenylalanine to Phe-AMP and afterward transferring the Phe to the PCP domain) and an epimerization domain (converting the PCP-bound l-Phe to d-Phe). We show here that the modified PCP domain of hTycA communicates with the adenylation domain and that the co-factor of holo-hPCP is loaded with Phe. However, communication between the hybrid PCP and the epimerization domain seems to be disabled. Nevertheless, hTycA is recognized by the next proline-activating elongation module TycB1 in vitro, and the dipeptide is formed and released as diketopiperazine.

Metabolic engineering of a methylmalonyl-CoA mutase-epimerase pathway for complex polyketide biosynthesis in Escherichia coli.

A barrier to heterologous production of complex polyketides in Escherichia coli is the lack of (2S)-methylmalonyl-CoA, a common extender substrate for the biosynthesis of complex polyketides by modular polyketide synthases. One biosynthetic route to (2S)-methylmalonyl-CoA involves the sequential actions of two enzymes, methylmalonyl-CoA mutase and methylmalonyl-CoA epimerase, which convert succinyl-CoA to (2R)- and then to (2S)-methylmalonyl-CoA. As reported [McKie, N., et al. (1990) Biochem. J. 269, 293-298; Haller, T., et al. (2000) Biochemistry 39, 4622-4629], when genes encoding coenzyme B(12)-dependent methylmalonyl-CoA mutases were expressed in E. coli, the inactive apo-enzyme was produced. However, when cells harboring the mutase genes from Propionibacterium shermanii or E. coli were treated with the B12 precursor hydroxocobalamin, active holo-enzyme was isolated, and (2R)-methylmalonyl-CoA represented approximately 10% of the intracellular CoA pool. When the E. coli BAP1 cell line [Pfeifer, B. A., et al. (2001) Science 291, 1790-1792] harboring plasmids that expressed P. shermanii methylmalonyl-CoA mutase, Streptomyces coelicolor methylmalonyl-CoA epimerase, and the polyketide synthase DEBS (6-deoxyerythronolide B synthase) was fed propionate and hydroxocobalamin, the polyketide 6-deoxyerythronolide B (6-dEB) was produced. Isotopic labeling studies using [(13)C]propionate showed that the starter unit for polyketide synthesis was derived exclusively from exogenous propionate, while the extender units stemmed from methylmalonyl-CoA via the mutase-epimerase pathway. Thus, the introduction of an engineered mutase-epimerase pathway in E. coli enabled the uncoupling of carbon sources used to produce starter and extender units of polyketides.

Molecular “Wiring” Enzymes in Organized Nanostructures

We report on the "molecular wiring" efficiency of glucose oxidase in organized self-assembled nanostructures comprised of enzyme layers alternating with layers of an osmium-derivatized poly(allylamine) cationic polyelectrolyte, acting as redox relays. Varying the relative position of the active enzyme layer in nanostructures alternating active enzyme and inactive apoenzyme we have demonstrated that the specific rate of bimolecular FADH(2) oxidation ("wiring efficiency") is limited by the diffusion-like electron hopping mechanism in the multilayers.

Iron Extraction from Soybean Lipoxygenase 3 and Reconstitution of Catalytic Activity from the Apoenzyme

Lipoxygenases contain a unique nonheme iron cofactor with a redox role in the catalyzed reaction. The conditions for the extraction of the metal atom were investigated for one of the soybean lipoxygenase isoenzymes. Removal of the iron by o-phenanthroline was attained in the presence of substrate under anaerobic conditions, but the apoenzyme could not be isolated and reconstituted. The freshly regenerated sodium form of Chelex-100 also removes the iron atom from native soybean lipoxygenase 3, but only in sodium bicarbonate buffer at pH 8.0. The soluble but inactive apoenzyme was reconstituted with ferric ammonium sulfate in Tris–HCl buffer at pH 7.0. Stoichiometric iron in the reconstituted enzyme was established using inductively coupled plasma-atomic emission spectroscopy. The reconstituted enzyme contained 90 ± 10% of the specific activity of the native enzyme. The native configuration of the reconstituted iron site was confirmed by electron paramagnetic resonance spectroscopy.

A Thermophilic Apoglucose Dehydrogenase as Nonconsuming Glucose Sensor

Blood glucose is a clinically important analytes for diabetic health care. In this preliminary report we describe a protein biosensor for d-glucose based on a thermostable glucose dehydrogenase. The glucose dehydrogenase was noncovalently labeled with 8-anilino-1-naphthalene sulfonic acid (ANS). The ANS-labeled enzyme displayed an approximate 25% decrease in emission intensity upon binding glucose. This decrease can be used to measure the glucose concentration. Our results suggest that enzymes which use glucose as their substrate can be used as reversible and nonconsuming glucose sensors in the absence of required cofactors. Moreover, the possibility of using inactive apoenzymes for a reversible sensor greatly expands the range of proteins which can be used as sensors, not only for glucose, but for a wide variety of biochemically relevant analytes.

Tight Binding of Pyridoxal 5′-Phosphate to Recombinant Escherichia coli Pyridoxine 5′-Phosphate Oxidase

Escherichia coli pyridoxine (pyridoxamine) 5′-phosphate oxidase (PNPOx) catalyzes the oxidation of pyridoxine 5′-phosphate and pyridoxamine 5′-phosphate to pyridoxal 5′-phosphate (PLP) using flavin mononucleotide (FMN) as the immediate electron acceptor and oxygen as the ultimate electron acceptor. This reaction serves as the terminal step in the de novo biosynthesis of PLP in E. coli. Removal of FMN from the holoenzyme results in a catalytically inactive apoenzyme. PLP molecules bind tightly to both apo- and holoPNPOx with a stoichiometry of one PLP per monomer. The unique spectral property of apoPNPOx-bound PLP suggests a non-Schiff base linkage. HoloPNPOx with tightly bound PLP shows normal catalytic activity, suggesting that the tightly bound PLP is at a noncatalytic site. The tightly bound PLP is readily transferred to aposerine hydroxymethyltransferase in dilute phosphate buffer. However, when the PNPOx · PLP complex was added to aposerine hydroxymethyltransferase suspended in an E. coli extract the rate of reactivation of the apoenzyme was several-fold faster than when free PLP was added. This suggests that PNPOx somehow targets PLP to aposerine hydroxymethyltransferase in vivo.