Function Of Enzyme Concentration Research Articles

Cardoon seed oil cake proteins as substrate for microbial transglutaminase: Their application as matrix for bio-based packaging to extend the shelf-life of peanuts

In this study, microbial transglutaminase (mTGase), an enzyme that can alter the biological properties of various proteins by creating isopeptide bonds between glutamine and lysine residues, was tested for the first time on proteins from cardoon oil cakes (CPs). mTGase crosslinked CPs were used as matrix for preparing bio-based materials further evaluated for their technological characteristics, such as mechanical and hydrophilicity behavior, gas barrier and thermal properties, color and opacity. All the performed analyses demonstrated that the microbial enzyme affected the film features by making them more resistant (Young's modulus and tensile strength were 52 MPa and 1.7 MPa for the film manufactured in the absence of enzyme and 98 MPa and 3.3 MPa for the crosslinked film, respectively), less hydrophilic and permeable to gases likely due to the formation of a more compact structure due to isopeptide bonds formed by mTGase as demonstrated by scanning electron microscope analyses. For example, the water vapor permeability of neat films was 8.37 g mm m−2 d−1 kPa−1, whereas that performed by mTGase-crosslinked material was equal to 5.16 g mm m−2 d−1 kPa-1. Bio-disintegration test proved that the materials were able to be decomposed under soil-like conditions. Thermal analyses also showed that the crosslinked materials had a matrix more stable over different temperatures, as a function of enzyme concentrations. Finally, the prepared bioplastics were used as sachets to protect peanuts chosen as an example of a high-oil content snack. The findings revealed that during a 30-day period, peroxidase levels and water content of peanuts packed in sachets manufactured from mTGase-crosslinked CP were lower than those packed in neat film and unpacked, and similar to those packed in LDPE, indicating that the novel bioplastics may be employed as biodegradable materials to prolong the shelf-life of such food goods.

Active and Intelligent Packaging for Enhancing Modified Atmospheres and Monitoring Quality and Shelf Life of Packed Gilthead Seabream Fillets at Isothermal and Variable Temperature Conditions.

The study investigated the effect of active modified atmosphere packaging (20% CO2–60% N2–20% O2) with CO2 emitters (MAP-PAD) and conventional MAP (MAP) on the quality and shelf-life of gilthead seabream fillets during chill storage, while the most appropriate enzymatic Time Temperature Integrators (TTI) were selected for monitoring their shelf-life at isothermal and variable temperature storage conditions (Teff = 4.8 °C). The concentration of CO2 and O2 in the headspace of the package, volatile compounds and of the microbial population were monitored during storage. The kinetic parameters for bacterial growth were estimated at 0–10 °C using the Baranyi growth model. The MAP-PAD samples presented significantly lower microbial growth rates and longer lag phases compared to the MAP samples, leading to significant shelf-life extension: 2 days of extension at 2.5 °C and 5 °C, while 50% extension at variable conditions (Teff = 4.8 °C). CO2 emitters in the package improved the chemical freshness (K-values) and volatile compounds (characterizing freshness). The responses of different enzymatic TTI were modeled as the function of enzyme concentration, temperature and storage time. The activation energy (Ea) ranged from 97 to 148 kJ mol−1, allowing the selection of appropriate TTIs for the shelf-life monitoring of each fish product: LP-150U for the MAP and M-25U for the MAP-PAD samples. The validation experiment at Teff = 4.8 °C confirmed the applicability of Arrhenius-type models, as well as the use of TTIs as effective chill chain management tools during distribution and storage.

Human Salivary α-Amylase and Starch Digestion: A Simple and Inexpensive At-Home Laboratory Experience in Times of the COVID-19 Pandemic.

The first case of coronavirus disease 2019 in Colombia was detected on March 6, 2020. Subsequently, schools, colleges, and universities were closed on March 26, which forced a massive migration to virtual education and impacted laboratory-based teaching courses. The teaching of biochemistry requires an experimental component that virtual laboratories cannot emulate. To address this concern, the article describes an at-home biochemistry laboratory experience that explores the hydrolysis of starch by α-amylase as a function of enzyme concentration, reaction time, and pH. The general success of the experience was assessed through the quality of information submitted through laboratory reports and feedback from students. A total of 19 laboratory reports were reviewed, and 50 students were surveyed. The analysis indicated that approximately 90% of students expressed favorable opinions about the experience. They understood the objective of the practice, identified the function of each material, and explained the relationship between the obtained results and concepts of enzyme activity presented in theoretical classes. Finally, the study concluded that the at-home laboratory experience is inexpensive and easy to perform outside the traditional laboratory. Furthermore, it enables a genuine practical experience with observations, data collection, analysis, and discussion of results, which meets the expectations for pharmaceutical chemistry students at the Universidad El Bosque in Bogotá, Colombia.

Fluorescence Detection of MMP-9. II. Ratiometric FRET-Based Sensing With Dually Labeled Specific Peptide

In our previous paper we showed that the MMP-9 enzyme recognizes a specific peptide sequence, Lys-Gly- Pro-Arg-Ser-Leu-Ser-Gly-Lys, and cleaves the peptide into two parts [1]. In this study, the peptide is labeled with two dyes, carboxyfluorescein (5-FAM) and Cy5. A highly efficient energy transfer of over 80% results in a dominant emission of Cy5 at ~670 nm with an excitation of 470 nm. Severance of the peptide by the MMP-9 enzyme eliminates Förster Resonance Energy Transfer (FRET) and strongly increases the fluorescence of the 5-FAM dye. In this manuscript we describe the strategy for a FRET-based method for MMP-9 enzyme detection. The basic aim is to apply a ratio-metric sensing technique in which a ratio of green/red fluorescence intensity is measured as a function of enzyme concentration. The ratio-metric method eliminates many experimental variables and enables accurate MMP-9 detection.

Regulation of intestinal calcium absorption by luminal calcium content: role of intestinal alkaline phosphatase.

Intestinal alkaline phosphatase is a brush border enzyme that is stimulated by calcium. Inhibition of intestinal alkaline phosphatase increases intestinal calcium absorption. We hypothesized that intestinal alkaline phosphatase acts as a minute-to-minute regulatory mechanism of calcium entry. The aim of this study was to evaluate the mechanism by which intestinal luminal calcium controls intestinal calcium absorption. We performed kinetic studies with purified intestinal alkaline phosphatase and everted duodenal sacs and showed that intestinal alkaline phosphatase modifies the luminal pH as a function of enzyme concentration and calcium luminal content. A decrease in pH occurred simultaneously with a decrease in calcium absorption. The inhibition of intestinal alkaline phosphatase by l-phenylalanine caused an increase in calcium absorption. This effect was also confirmed in calcium uptake experiments with isolated duodenal cells. Changes in luminal pH arising from intestinal alkaline phosphatase activity induced by luminal calcium concentration modulate intestinal calcium absorption.

Protein Gels Degradation by Proteases

Tissues and organs are formed by specialized cells and by extracellular matrix (ECM) organized in 3-dimensional (3D) structure. ECM is a mix of various components, mainly of proteins. ECM remodeling is a physiological and pathological phenomenon (Raeber et al 2005). During tumor dissemination, invasive cells must liquefy the matrix to invade other tissues and establish distant metastases. Many groups study cancer cell behavior; however, up to now, few experimental data are focused on understanding the physical aspect of ECM degradation by enzymes.The aim of this work is to find general behavior able to describe the physical mechanism of ECM enzymatic degradation.Previous experimental studies have shown that the gelatin-gel degradation kinetics by thermolysin, a metalloproteinase, is diffusion limited (Lairez et al, 2007). A power law dependence on degradation time as a function of enzyme concentration was found and associated to a self-attracting enzyme random walk, leading to a continuum percolation model for proteolysis. Recently, we have demonstrated the same behavior with 2 serine proteases, trypsin and proteinase K (Breton et al, in preparation).Here we study protein gel degradation by papain (a cystein protease) and chymotrypsin (a serine protease) by varying solvent viscosity, gelatin and enzyme concentrations. We obtain either a linear dependency or a power law of the degradation time (tc) as a function of enzyme concentrations: tc α 1/[papain]1.1+/-0.1 and tc α 1/[chymotrypsin]1.9+/-0.15. In the presence of glycerol, gel degradation kinetics with papain is reaction limited and enzyme diffusion is Brownian. With chymotrypsin, tc increase and can be superimposed to those obtained without glycerol by taking into account both, the enzyme activity decrease and the solvent viscosity increase. Gel proteolysis kinetics is therefore diffusion limited and the diffusion is anomalous. We discuss possible explanation for these different mechanisms.

The kinetics of p-nitrophenyl-β- d-cellobioside hydrolysis and transglycosylation by Thermobifida fusca Cel5Acd

The hydrolysis of p-nitrophenyl-β-1,4-cellobioside (pNP-G2) by the catalytic domain of the retaining-family 5-2 endocellulase Cel5A from Thermobifida fusca (Cel5Acd) was studied. The dominant reaction pathway involves hydrolysis of the aglyconic bond, producing cellobiose (G2) and a ‘reporter’ species p-nitrophenol (pNP), which was monitored spectrophotometrically to track the reaction. We also detected the production of cellotriose (G3) and p-nitrophenyl-glucoside (pNP-G1), confirming the presence of a competing transglycosylation pathway. We use a mechanistic model of hydrolysis and transglycosylation to derive an expression for the rate of pNP-formation as a function of enzyme concentration, substrate concentration, and several lumped kinetics parameters. The derivation assumes that the quasi-steady-state assumption (QSSA) applies for three intermediate species in the mechanism; we determine conditions under which this assumption is rigorously justified. We integrate the rate expression and compare its integral form to pNP-versus-time data collected for a range of enzyme and substrate concentrations. The integral comparison gives a stringent test of the mechanistic model, and it serves to quantify the lumped kinetics parameters with good statistical precision, particularly a previously unidentified parameter that determines the selectivity of hydrolysis versus transglycosylation. The integrated rate expression accounts well for pNP-versus-time data under all circumstances we have investigated.

A Substrate Serves as a Hydrogen Atom Donor in the Enzyme-Initiated Catalytic Mechanism of Dual Positional Specific Maize Lipoxygenase-1

The maize lipoxgyenase-1 is a non-traditional dual positional specific enzyme and the reaction proceeds via enzyme-initiated catalysis. Bioinformatic analysis indicated that the maize lipoxygenase-1 is structurally more similar to soybean LOX1 than pea LOXN2 in that it has an additional external loop (residues 318-351) in the carboxy-terminal catalytic domain. We analyzed the dependence of product distribution on concentration of linoleic acid and monitored the formation of hydroperoxyoctadecadienoic acid as a function of enzyme concentration. Product distribution was strongly influenced by substrate concentration, such that kinetically-controlled regioisomers were enriched and thermodynamically-controlled regioisomers were depleted at high substrate concentration. Kinetic studies indicated that the formation of hydroperoxyoctadecadienoic acid saturated rapidly in an enzyme concentration-dependent manner, which implied that reactivation by reoxidation of inactive Fe(II) failed to occur. Our results support the previously proposed enzyme-initiated catalytic mechanism of the maize lipoxgyenase-1 and reveals that a substrate molecule serves as a hydrogen atom donor in its enzyme-initiated catalysis.

Detection of Transglucosidase-Catalyzed Polysaccharide Synthesis on a Surface in Real Time Using Surface Plasmon Resonance Spectroscopy

Biological systems that involve enzyme catalysis at surfaces, particularly strategically important ones that involve insoluble substrates/products such as the cell wall and the starch granule, require analyses beyond classical solution state enzymology. Using a model system, we have demonstrated the real-time measurement of transglucosidase activity on a surface using surface plasmon resonance (SPR) spectroscopy. We monitored the extension of a (partially carboxymethylated) dextran surface with alternansucrase and sucrose as a glycosyl donor. Conditions were used where surface polymer synthesis rates were a function of enzyme concentration and proportional to the extent of enzyme binding to the surface. A method to determine the turnover number of the enzyme on the surface was also developed. The presence of a new amorphous polysaccharide was observed optically, detected by lectin binding and imaged by atomic force microscopy. This surface method will have utility in a wide range of carbohydrate enzyme systems including screens.

A modelling framework describing the enzyme regulation of membrane lipids underlying gradient perception in Dictyostelium cells II: Input–output analysis

Spatial sensing in Dictyostelium involves localization of the phosphoinositide lipids PI(3,4,5)P 3 and PI(3,4)P 2 at the leading edge of the cell in response to an external gradient. We have previously proposed a modelling framework describing the regulation of these lipids by the enzymes PI3K and PTEN. In this paper we analyse this regulation from an input–output perspective. When the inputs are homogeneous, we obtain explicit analytical expressions for the lipid concentrations as a function of enzyme concentrations and model parameters. We also show that the system can be cast as an open-loop bilinear control system, and employ control engineering tools to show that a local three-dimensional region in the four-dimensional phase space can be accessed by temporally varying either or both enzyme concentrations. For spatially graded enzyme profiles, we show that diffusion limits the extent to which lipid profiles can be manipulated by enzymes. However, we also demonstrate that for certain ranges of network parameters, increasing lipid diffusion can lead to an increase in steady-state leading-edge concentrations of PI(3,4,5)P 3 or PI(3,4)P 2, even though all lipid diffusion coefficients are equal. Finally, in order to determine the extent to which lipid profiles can be regulated by the enzymes, we formulate and solve inverse problems, where we determine the enzyme profiles required to realize particular lipid profiles at steady state.

Human RECQ5beta, a protein with DNA helicase and strand-annealing activities in a single polypeptide.

Proteins belonging to the highly conserved RecQ helicase family are essential for the maintenance of genomic stability. Here, we describe the biochemical properties of the human RECQ5beta protein. Like BLM and WRN, RECQ5beta is an ATP-dependent 3'-5' DNA helicase that can promote migration of Holliday junctions. However, RECQ5beta required the single-stranded DNA-binding protein RPA in order to mediate the efficient unwinding of oligonucleotide-based substrates. Surprisingly, we found that RECQ5beta possesses an intrinsic DNA strand-annealing activity that is inhibited by RPA. Analysis of deletion variants of RECQ5beta revealed that the DNA helicase activity resides in the conserved N-terminal portion of the protein, whereas strand annealing is mediated by the unique C-terminal domain. Moreover, the strand-annealing activity of RECQ5beta was strongly inhibited by ATPgammaS, a poorly hydrolyzable analog of ATP. This effect was alleviated by mutations in the ATP-binding motif of RECQ5beta, indicating that the ATP-bound form of the protein cannot promote strand annealing. This is the first demonstration of a DNA helicase with an intrinsic DNA strand-annealing function residing in a separate domain.

In Vitro and in Vivo Dimerization of Human Endonuclease III Stimulates Its Activity

Human endonuclease III (hNTH1), a DNA glycosylase with associated abasic lyase activity, repairs various mutagenic and toxic-oxidized DNA lesions, including thymine glycol. We demonstrate for the first time that the full-length hNTH1 positively cooperates in product formation as a function of enzyme concentration. The protein concentrations that caused cooperativity in turnover also exhibited dimerization, independent of DNA binding. Earlier we had found that the hNTH1 consists of two domains: a well conserved catalytic domain, and an inhibitory N-terminal tail. The N-terminal truncated proteins neither undergo dimerization, nor do they show cooperativity in turnover, indicating that the homodimerization of hNTH1 is specific and requires the N-terminal tail. Further kinetic analysis at transition states reveals that this homodimerization stimulates an 11-fold increase in the rate of release of the final product, an AP-site with a 3'-nick, and that it does not affect other intermediate reaction rates, including those of DNA N-glycosylase or AP lyase activities that are modulated by previously reported interacting proteins, YB-1, APE1, and XPG. Thus, the site of modulating action of the dimer on the hNTH1 reaction steps is unique. Moreover, the high intranuclear (2.3 microM) and cytosolic (0.65 microM) concentrations of hNTH1 determined here support the possibility of in vivo dimerization; indeed, in vivo protein cross-linking showed the presence of the dimer in the nucleus of HeLa cells. Therefore, it is likely that the dimerization of hNTH1 involving the N-terminal tail masks the inhibitory effect of this tail and plays a critical role in its catalytic turnover in the cell.

Localization of the Deoxyribose Phosphate Lyase Active Site in Human DNA Polymerase ι by Controlled Proteolysis

Human DNA polymerase iota (pol iota) is a member of the Y-family of low fidelity lesion bypass DNA polymerases. In addition to a probable role in DNA lesion bypass, this enzyme has recently been shown to be required for somatic hypermutation in human B-cells. We found earlier that human pol iota has deoxyribose phosphate (dRP) lyase activity and unusual specificity for activity during DNA synthesis, suggesting involvement in specialized forms of base excision repair (BER). Here, mapping of the domain structure of human pol iota by controlled proteolysis revealed that the enzyme has a 48-kDa NH2-terminal domain and a protease resistant 40-kDa "core domain" spanning residues Met79 to approximately Met445. A covalently cross-linked pol iota-DNA complex, representing a trapped intermediate in the dRP lyase reaction, was subjected to controlled proteolysis. Cross-linking was mapped to the 40-kDa core domain, indicating that the dRP lyase active site is in this region. To further evaluate the BER capacity of the enzyme, the dRP lyase and DNA polymerase activities were characterized on DNA substrates representing BER intermediates, and we found that pol iota was able to complement the in vitro single-nucleotide BER deficiency of a DNA polymerase beta null cell extract.

Development of photo-polymerisable polyvinyl alcohol for biotechnological applications

Polyvinyl alcohol (PVA) containing styryl pyridinium groups has been used as photosensitive polymer. After dilution into deionised water, thin PVA films (with a thickness less than 1 μm) have been deposited by spin coating and patterned by ultraviolet (UV) photolithography. The impact of the different technological parameters (dilution rate, rotation speed, illumination power and duration, etc.) has been studied in so as to optimise the whole process. Results evidence the realisation of high quality PVA patterns whose area ranges from 0.1×0.8 to 10×10 m 2. The deposition of several thin PVA films in a row has also been investigated, allowing the fabrication of thick multilayers. Finally, the optimised technological process has been used for the deposition of enzymatic PVA films. Urease activities into a thin PVA films have been studied by enzyme-linked immunoabsorbent assay (ELISA) characterisations. Results are analysed as a function of enzyme concentration into the films, dilution medium and storage conditions. This technological process will be advantageously used for the development of low cost biochemical layers.

Concentration-dependent dissociation/association of human prostatic acid phosphatase.

The apparent molecular mass of human prostatic acid phosphatase (PAP) was estimated over a wide range of enzyme concentrations using equilibrium centrifugation in the "Airfuge" tabletop ultracentrifuge. We show that the average mass of all active PAP species steeply increases at enzyme concentrations around 100 nM. The data indicate that at lower concentrations, active monomer prevail, whereas at concentrations above 100 nM, PAP active dimers are formed. These findings were confirmed by measurements of fluorescence emission intensity as a function of enzyme concentration. A shift of the normalized PAP fluorescence intensity around 100 nM independently indicates that a major structural change of the PAP protein occurs in that range of concentrations. From these findings, we conclude that in dilute solutions, several active PAP species exist, which are involved in concentration-dependent dissociation/association equilibria.

High stability binding of poly(ADPribose) polymerase-like thermozyme from S. solfataricus with circular DNA.

The poly(ADPribose) polymerase-like thermozyme from the hyperthermophilic archaeon S. solfataricus was found to bind DNA with high affinity and non-specifically. Binding was independent of base composition and length of the nucleic acid, and the protein showed a slight preference for the circular structure. By using pCMV-Neo-Bam plasmid as experimental model, the behaviour of the thermozyme upon binding with either circular or linear plasmid was analyzed. pCMV-Neo-Bam has a single HindIII site that allows to obtain the linear structure after digestion with the restriction enzyme. Intrinsic tryptophan-dependent fluorescence of poly(ADPribose) polymerase-like thermozyme noticeably changed upon addition of either circular or linear plasmid, showing the same binding affinity (K=2 x 10(9) M-1). However, experiments of protection against temperature and DNase I gave evidence that the thermozyme formed more stable complexes with the circular structure than with the linear pCMV-Neo-Bam. Increasing temperature at various DNA/protein ratios had a double effect to reduce the amount of circular DNA undergoing denaturation and to split the melting point towards higher temperatures. Nil or irrelevant effect was observed with the linear form. Similarly, DNase acted preferentially on the linear plasmid/protein complexes, producing an extensive digestion even at high protein/DNA ratios, whereas the circular plasmid was protected by the thermozyme in a dose-dependent manner. The complexes formed by archaeal poly(ADPribose) polymerase (PARPss) with the circular plasmid were visualized by bandshift experiments both with ethidium bromide staining and by labelling the circular plasmid with 32P. The stability of complexes was tested as a function of enzyme concentration and in the presence of a cold competitor and of 0.1% SDS. From the performed experiments, a number of 3-10 base pairs bound per molecule of enzyme was calculated, indicating a high frequency of binding. The presence of circular DNA was also able to increase by 80% the poly(ADPribose)polymerase-like activity, as compared to 25% activation induced by the linear pCMV-Neo-Bam.

Calculation of Degradation Rate of Pectic Substances in Ramie and Linen Fabrics.

Ramie and linen fabrics were treated with a commercial pectinase under various conditions. The effects of temperature, enzyme concentration, and treating time on degradation rate of pectic substances in the fabrics were evaluated. Equation to calculate their degradation rate was established as a function of enzyme concentration and treating time based on the obtained results.

Nucleotide release and associated conformational changes regulate function in the COOH-terminal Src kinase, Csk.

The COOH-terminal Src kinase (Csk) regulates a broad array of cellular processes via the specific phosphorylation and downregulation of Src family protein kinases. While Csk has been a topic for steady-state kinetic studies, the individual steps associated with substrate phosphorylation have not been investigated. To understand active-site phenomena, pre-steady-state and transient-state kinetic methods were applied to develop a catalytic pathway for substrate processing. Rapid quench flow techniques show that the phosphorylation of a substrate peptide, generated from a random library, occurs in two kinetic phases: a rapid, exponential "burst" phase followed by a slow, linear phase. The amplitude of the burst phase increases as a function of enzyme concentration, indicating that the biphasic kinetics are not the result of product inhibition. Analysis of the burst rate as a function of substrate concentration indicates that the phosphoryl transfer step is fast (k3 > or = 140 s(-1) and highly favorable (k3/k-3 > or = 6). The apparent dissociation rate constant for ADP (0.6 s(-1), measured using stopped-flow kinetic methods and a fluorescent trapping agent, mant-ATP, is close to kcat. Since the substrate dissociation constant is high, the release of phosphopeptide is not likely to limit turnover. These findings indicate that Csk rapidly delivers the gamma-phosphate of ATP to the substrate and rapidly releases the phosphoproduct. Overall rate limitation in the steady state is then attributed to the slow, net dissociation of ADP. Viscosometric studies suggest that this final event in the catalytic cycle is coupled with slow conformational changes.

Novel substrates of Escherichia coli nth protein and its kinetics for excision of modified bases from DNA damaged by free radicals.

Escherichia coli Nth protein (endonuclease III) is a DNA glycosylase with a broad substrate specificity for pyrimidine derivatives. We discovered novel substrates of E. coli Nth protein using gas chromatography/isotope-dilution mass spectrometry and DNA samples, which were damaged by gamma-irradiation or by H(2)O(2)/Fe(III)-EDTA/ascorbic acid. These were 4, 6-diamino-5-formamidopyrimidine, 5,6-dihydroxyuracil, and 5, 6-dihydroxycytosine. The first compound was recognized for the first time as a purine-derived substrate of the enzyme. We also investigated kinetics of excision of a multitude of modified bases from three damaged DNA substrates. Excision of modified bases was determined as a function of enzyme concentration, incubation time, and substrate concentration. Excision followed Michaelis-Menten kinetics. Kinetic parameters were determined for the following modified bases: 4,6-diamino-5-formamidopyrimidine, cis- and trans-thymine glycols, 5-hydroxycytosine, cis- and trans-uracil glycols, 5-hydroxyuracil, 5-hydroxy-5-methylhydantoin, alloxan, 5, 6-dihydroxycytosine, 5,6-dihydroxyuracil, 5-hydroxy-6-hydrothymine, and 5-hydroxy-6-hydrouracil. The results show that three newly discovered substrates were excised by the enzyme with a preference similar to excision of its known major substrates such as thymine glycol and 5-hydroxycytosine. Excision kinetics significantly depended on the nature of the damaged DNA substrates in agreement with previous results on other DNA glycosylases. Specificity constants (k(cat)/K(M)) of E. coli Nth protein were compared to those of its previously investigated functional homologues such as human and Schizosaccharomyces pombe Nth proteins and Saccharomyces cerevisiae Ntg1 and Ntg2 proteins. This comparison shows that significant differences exist with respect to substrate specificity and kinetic parameters despite extensive structural conservation among the Nth homologues.

Substrate specificity of Deinococcus radiodurans Fpg protein.

A DNA repair enzyme has recently been isolated from the ionizing radiation-resistant bacterium Deinococcus radiodurans [Bauche, C., and Laval, J. (1999) J. Bacteriol. 181, 262-269]. This enzyme is a homologue of the Fpg protein of Escherichia coli. We investigated the substrate specificity of this enzyme for products of oxidative DNA base damage using gas chromatography/isotope-dilution mass spectrometry and DNA substrates, which were either gamma-irradiated or treated with H(2)O(2)/Fe(III)-EDTA/ascorbic acid. Excision of purine lesions 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua), 4,6-diamino-5-formamidopyrimidine (FapyAde), and 8-hydroxyguanine (8-OH-Gua) was observed among 17 lesions detected in damaged DNA substrates. The extent of excision was determined as a function of enzyme concentration, time, and substrate concentration. FapyGua and FapyAde were excised with similar specificities from three DNA substrates, whereas 8-OH-Gua was the least preferred lesion. The results show that D. radiodurans Fpg protein and its homologue E. coli Fpg protein excise the same modified DNA bases, but the excision rates of these enzymes are significantly different. Formamidopyrimidines are preferred substrates of D. radiodurans Fpg protein over 8-OH-Gua, whereas E. coli Fpg protein excises these three lesions with similar efficiencies from various DNA substrates. Substrate specificities of these enzymes were also compared with that of Saccharomyces cerevisiae Ogg1 protein, which excises FapyGua and 8-OH-Gua, but not FapyAde.