High levels of cathepsins B, L and S in human seminal plasma and their association with prostasomes.

Semen is a heterogenous and complex fluid with different functions, some of them well known, others still obscure. The aim of this study was to investigate the presence of cathepsins B, S and L in human seminal plasma and their possible associations with other semen variables. Cathepsin B, L and S concentrations were measured in seminal plasma from 99 men utilising commercial ELISA kits. Seminal plasma cathepsin B was approximately 70 times higher, while the cathepsin L values were approximately 500 times higher and the cathepsin S values approximately 40 times higher in seminal plasma than in a group of serum samples. The study shows that seminal plasma contains high levels of cathepsins B, L and S. All three cathepsins were also bound to the surface of prostasomes.

Association of cystatin C with prostasomes in human seminal plasma.

It was recently elucidated that cystatin C, a protein targeted to the classical secretory pathway by its signal peptide sequence, can also be secreted in association with exosomes. Accordingly, we wanted to investigate whether there is a secretory link between cystatin C and prostasomes in human seminal plasma. Cystatin C concentrations in seminal plasma from 50 men including 6 vasectomized men were measured by turbidimetry on an Architect Ci8200. Some of the seminal plasma samples were also analysed utilizing an Epics Profile XL-MCL cytometer. We found high concentrations of cystatin C in seminal plasma. The 2.5-97.5 percentiles, performed by bootstrap estimation, were 25.8 [95% confidence interval (CI): 22.3-29.4] to 77.0 mg/L (95% CI: 71.9-82.1). Cystatin C is present in approximately 50 times higher concentration in seminal plasma compared with blood plasma. There was no clear difference as regards seminal plasma content of cystatin C between vasectomized men and the rest of the group. Immunoblot analysis with chicken anti-cystatin C antibody revealed a firm association of cystatin C with prostasomes. Flow cytometric analysis demonstrated that cystatin C was linked to prostasomes also meaning an at least partial prostasomal membrane surface localization.

The anaerobic ribonucleotide reductase from Lactococcus lactis. Interactions between the two proteins NrdD and NrdG.

Deoxyribonucleotide synthesis by anaerobic class III ribonucleotide reductases requires two proteins, NrdD and NrdG. NrdD contains catalytic and allosteric sites and, in its active form, a stable glycyl radical. This radical is generated by NrdG with its [4Fe-4S](+) cluster and S-adenosylmethionine. We now find that NrdD and NrdG from Lactobacillus lactis anaerobically form a tight alpha(2)beta(2) complex, suggesting that radical generation by NrdG and radical transfer to the specific glycine residue of NrdD occurs within the complex. Activated NrdD was separated from NrdG by anaerobic affinity chromatography on dATP-Sepharose without loss of its glycyl radical. NrdD alone then catalyzed the reduction of CTP with formate as the electron donor and ATP as the allosteric effector. The reaction required Mg(2+) and was stimulated by K(+) but not by dithiothreitol. Thus NrdD is the actual reductase, and NrdG is an activase, making class III reductases highly similar to pyruvate formate lyase and its activase and suggesting a common root for the two anaerobic enzymes during early evolution. Our results further support the contention that ribonucleotide reduction during transition from an RNA world to a DNA world started with a class III-like enzyme from which other reductases evolved when oxygen appeared on earth.

Cross-talk between the allosteric effector-binding sites in mouse ribonucleotide reductase.

We compared the allosteric regulation and effector binding properties of wild type R1 protein and R1 protein with a mutation in the "activity site" (D57N) of mouse ribonucleotide reductase. Wild type R1 had two effector-binding sites per polypeptide chain: one site (activity site) for dATP and ATP, with dATP-inhibiting and ATP-stimulating catalytic activity; and a second site (specificity site) for dATP, ATP, dTTP, and dGTP, directing substrate specificity. Binding of dATP to the specificity site had a 20-fold higher affinity than to the activity site. In all these respects, mouse R1 resembles Escherichia coli R1. Results with D57N were complicated by the instability of the protein, but two major changes were apparent. First, enzyme activity was stimulated by both dATP and ATP, suggesting that D57N no longer distinguished between the two nucleotides. Second, the two binding sites for dATP both had the same low affinity for the nucleotide, similar to that of the activity site of wild type R1. Thus the mutation in the activity site had decreased the affinity for dATP at the specificity site, demonstrating the interaction between the two sites.

The active form of the R2F protein of class Ib ribonucleotide reductase from Corynebacterium ammoniagenes is a diferric protein.

Corynebacterium ammoniagenes contains a ribonucleotide reductase (RNR) of the class Ib type. The small subunit (R2F) of the enzyme has been proposed to contain a manganese center instead of the dinuclear iron center, which in other class I RNRs is adjacent to the essential tyrosyl radical. The nrdF gene of C. ammoniagenes, coding for the R2F component, was cloned in an inducible Escherichia coli expression vector and overproduced under three different conditions: in manganese-supplemented medium, in iron-supplemented medium, and in medium without addition of metal ions. A prominent typical tyrosyl radical EPR signal was observed in cells grown in rich medium. Iron-supplemented medium enhanced the amount of tyrosyl radical, whereas cells grown in manganese-supplemented medium had no such radical. In highly purified R2F protein, enzyme activity was found to correlate with tyrosyl radical content, which in turn correlated with iron content. Similar results were obtained for the R2F protein of Salmonella typhimurium class Ib RNR. The UV-visible spectrum of the C. ammoniagenes R2F radical has a sharp 408-nm band. Its EPR signal at g = 2.005 is identical to the signal of S. typhimurium R2F and has a doublet with a splitting of 0.9 millitesla (mT), with additional hyperfine splittings of 0.7 mT. According to X-band EPR at 77-95 K, the inactive manganese form of the C. ammoniagenes R2F has a coupled dinuclear Mn(II) center. Different attempts to chemically oxidize Mn-R2F showed no relation between oxidized manganese and tyrosyl radical formation. Collectively, these results demonstrate that enzymatically active C. ammoniagenes RNR is a generic class Ib enzyme, with a tyrosyl radical and a diferric metal cofactor.

The anaerobic (class III) ribonucleotide reductase from Lactococcus lactis. Catalytic properties and allosteric regulation of the pure enzyme system.

Lactococcus lactis contains an operon with the genes (nrdD and nrdG) for a class III ribonucleotide reductase. Strict anaerobic growth depends on the activity of these genes. Both were sequenced, cloned, and overproduced in Escherichia coli. The corresponding proteins, NrdD and NrdG, were purified close to homogeneity. The amino acid sequences of NrdD (747 residues, 84.1 kDa) and NrdG (199 residues, 23.3 kDa) are 53 and 42% identical with the respective E. coli proteins. Together, they catalyze the reduction of ribonucleoside triphosphates to the corresponding deoxyribonucleotides in the presence of S-adenosylmethionine, reduced flavodoxin or reduced deazaflavin, potassium ions, dithiothreitol, and formate. EPR experiments demonstrated a [4Fe-4S](+) cluster in reduced NrdG and a glycyl radical in activated NrdD, similar to the E. coli NrdD and NrdG proteins. Different from E. coli, the two polypeptides of NrdD and the proteins in the NrdD-NrdG complex were only loosely associated. Also the FeS cluster was easily lost from NrdG. The substrate specificity and overall activity of the L. lactis enzyme was regulated according to the general rules for ribonucleotide reductases. Allosteric effectors bound to two separate sites on NrdD, one binding dATP, dGTP, and dTTP and the other binding dATP and ATP. The two sites showed an unusually high degree of cooperativity with complex interactions between effectors and a fine-tuning of their physiological effects. The results with the L. lactis class III reductase further support the concept of a common origin for all present day ribonucleotide reductases.

Allosteric control of three B12-dependent (class II) ribonucleotide reductases. Implications for the evolution of ribonucleotide reduction.

Three separate classes of ribonucleotide reductases are known, each with a distinct protein structure. One common feature of all enzymes is that a single protein generates each of the four deoxyribonucleotides. Class I and III enzymes contain an allosteric substrate specificity site capable of binding effectors (ATP or various deoxyribonucleoside triphosphates) that direct enzyme specificity. Some (but not all) enzymes contain a second allosteric site that binds only ATP or dATP. Binding of dATP to this site inhibits the activity of these enzymes. X-ray crystallography has localized the two sites within the structure of the Escherichia coli class I enzyme and identified effector-binding amino acids. Here, we have studied the regulation of three class II enzymes, one from the archaebacterium Thermoplasma acidophilum and two from eubacteria (Lactobacillus leichmannii and Thermotoga maritima). Each enzyme has an allosteric site that binds ATP or various deoxyribonucleoside triphosphates and that regulates its substrate specificity according to the same rules as for class I and III enzymes. dATP does not inhibit enzyme activity, suggesting the absence of a second active allosteric site. For the L. leichmannii and T. maritima enzymes, binding experiments also indicate the presence of only one allosteric site. Their primary sequences suggest that these enzymes lack the structural requirements for a second site. In contrast, the T. acidophilum enzyme binds dATP at two separate sites, and its sequence contains putative effector-binding amino acids for a second site. The presence of a second site without apparent physiological function leads to the hypothesis that a functional site was present early during the evolution of ribonucleotide reductases, but that its function was lost from the T. acidophilum enzyme. The other two B12 enzymes lost not only the function, but also the structural basis for the site. Also a large subgroup (Ib) of class I enzymes, but none of the investigated class III enzymes, has lost this site. This is further indirect evidence that class II and I enzymes may have arisen by divergent evolution from class III enzymes.

Activation of the anaerobic ribonucleotide reductase from Escherichia coli. The essential role of the iron-sulfur center for S-adenosylmethionine reduction.

The anaerobic ribonucleotide reductase of Escherichia coli catalyzes the synthesis of the deoxyribonucleotides required for anaerobic DNA synthesis. The enzyme is an alpha2beta2 heterotetramer. In its active form, the large alpha2 subunit contains an oxygen-sensitive glycyl radical, whereas the beta2 small protein harbors a [4Fe-4S] cluster that joins its two polypeptide chains. Formation of the glycyl radical in the inactive enzyme requires S-adenosylmethionine (AdoMet), dithiothreitol, K+, and either an enzymatic (reduced flavodoxin) or chemical (dithionite or 5-deazaflavin plus light) reducing system. Here, we demonstrate that AdoMet is directly reduced by the Fe-S center of beta2 during the activation of the enzyme, resulting in methionine and glycyl radical formation. Direct binding experiments showed that AdoMet binds to beta2 with a Kd of 10 microM and a 1:1 stoichiometry. Binding was confirmed by EPR spectroscopy that demonstrated the formation of a complex between AdoMet and the [4Fe-4S] center of beta2. Dithiothreitol triggered the cleavage of AdoMet, leading to an EPR-silent form of beta2 and, in the case of alpha2beta2, to glycyl radical formation. In both instances, 3 methionines were formed per mol of protein. Our results indicate that the Fe-S center of beta2 is directly involved in the reductive cleavage of AdoMet and suggest a new biological function for an iron-sulfur center, i.e redox catalysis, as recently proposed by others (Staples, R. C., Ameyibor, E., Fu, W., Gardet-Salvi, L., Stritt-Etter, A. L., Schürmann, P., Knaff, D. B., and Johnson, M. K. (1996) Biochemistry 35, 11425-11434).

B12-dependent ribonucleotide reductases from deeply rooted eubacteria are structurally related to the aerobic enzyme from Escherichia coli.

The ribonucleotide reductases from three ancient eubacteria, the hyperthermophilic Thermotoga maritima (TM), the radioresistant Deinococcus radiodurans (DR), and the thermophilic photosynthetic Chloroflexus aurantiacus, were found to be coenzyme-B12 (class II) enzymes, similar to the earlier described reductases from the archaebacteria Thermoplasma acidophila and Pyrococcus furiosus. Reduction of CDP by the purified TM and DR enzymes requires adenosylcobalamin and DTT. dATP is a positive allosteric effector, but stimulation of the TM enzyme only occurs close to the temperature optimum of 80-90 degrees C. The TM and DR genes were cloned by PCR from peptide sequence information. The TM gene was sequenced completely and expressed in Escherichia coli. The deduced amino acid sequences of the two eubacterial enzymes are homologous to those of the archaebacteria. They can also be aligned to the sequence of the large protein of the aerobic E. coli ribonucleotide reductase that belongs to a different class (class I), which is not dependent on B12. Structure determinations of the E. coli reductase complexed with substrate and allosteric effectors earlier demonstrated a 10-stranded beta/alpha-barrel in the active site. From the conservation of substrate- and effector-binding residues we propose that the B12-dependent class II enzymes contain a similar barrel.

nrdD and nrdG genes are essential for strict anaerobic growth of Escherichia coli.

In Escherichia coli ribonucleotide reduction is catalyzed by two separate enzymes during aerobic and anaerobic growth. The aerobic enzyme is coded by the nrdAB genes, the anaerobic enzyme by nrdDG. We now show that knock-out mutants of either nrdD or nrdG cannot grow during strict anaerobiosis, achieved by inclusion of sodium sulfide in the medium. Interestingly, these mutants grow well under microaerophilic conditions by overproducing the aerobic enzyme. Under such conditions wild-type bacteria turn off nrdAB and switch on nrdDG.

Allosteric regulation of the third ribonucleotide reductase (NrdEF enzyme) from enterobacteriaceae.

Enterobacteriaceae contain genes for three separate ribonucleotide reductases: nrdAB code for a class Ia enzyme, active during aerobiosis, nrdDG for a class III enzyme, active during anaerobiosis, and nrdEF for a cryptic class Ib enzyme. The NrdEF enzyme provides the active reductase in other, widely different bacteria. Here, we describe the allosteric regulation of the Salmonella typhimurium NrdEF enzyme. It consists of two tightly bound homodimeric proteins, R1E and R2F. Nucleoside triphosphates (ATP, dATP, dGTP, and dTTP) regulate the substrate specificity by binding to a single site of the R1E protein (one nucleotide per polypeptide). Regulation is similar to that of the NrdAB enzyme, with one major exception: dATP stimulates reduction of CDP (and UDP) under conditions when dATP strongly inhibits all activity of the NrdAB enzyme. The nrdA-coded R1 protein contains a second binding site for dATP (and ATP) that controls general enzyme activity. All known R1E proteins lack the 50 N-terminal amino acids of R1, and we propose that the activity site is located in this area of the protein. The more sophisticated regulation of NrdAB enzymes of eukaryotes provides protection against the possibly harmful overproduction of dNTPs.

The anaerobic Escherichia coli ribonucleotide reductase. Subunit structure and iron sulfur center.

During anaerobic growth Escherichia coli uses a specific ribonucleoside triphosphate reductase for the production of deoxyribonucleoside triphosphates. The active species of this enzyme was previously found to be a large homodimer of 160 kDa (alpha 2) with a stable, oxygen-sensitive radical located at Gly-681 of the 80-kDa polypeptide chain. The radical is formed in an enzymatic reaction involving S-adenosylmethionine, NADPH, a reducing flavodoxin system and an additional 17.5-kDa polypeptide, previously called activase. Here, we demonstrate by EPR spectroscopy that this small protein contains a 4Fe-4S cluster that joins two peptides in a 35-kDa small homodimer (beta 2). A degraded form of this cluster may have been responsible for an EPR signal observed earlier in preparations of the large 160-kDa subunit that suggested the presence of a 3Fe-4S cluster in the reductase. These preparations were contaminated with a small amount of the small protein. The large and the small proteins form a tight complex. From sucrose gradient centrifugation, we determined a 1:1 stoichiometry of the two proteins in the complex. The anaerobic reductase thus has an alpha 2 beta 2 structure. We speculate that the small protein interacts with S-adenosylmethionine and forms a transient radical involved in the generation of the stable glycyl radical in the large protein that participates in the catalytic process.

The free radical of the anaerobic ribonucleotide reductase from Escherichia coli is at glycine 681.

The anaerobic ribonucleoside triphosphate reductase of Escherichia coli is an iron-sulfur protein carrying an oxygen-sensitive organic radical, which is essential for catalysis. The radical was tentatively proposed to be on glycine 681, based on a comparison with the glycyl radical-containing enzyme pyruvate formate-lyase. By EPR spectroscopy of selectively 2H- and 13C-labeled anaerobic ribonucleotide reductase, the radical was now unambiguously assigned to carbon-2 of a glycine residue. The large 1H hyperfine splitting (1.4 millitesla) was assigned to the alpha-proton. Site-directed mutagenesis was used to change glycine 681 into an alanine residue. In separate experiments, the two adjacent residues, cysteine 680 and tyrosine 682, were changed into serine and phenylalanine, respectively. All mutated proteins were retained on dATP-Sepharose, indicating that the mutant proteins had intact allosteric sites. They also contained amounts of iron comparable with the wild type reductase and showed the same iron-sulfur-related spectrum, suggesting that the mutant proteins were properly folded. Of the three mutant proteins only the G681A protein completely lacked the detectable glycyl radical as well as enzyme activity. Our results identify glycine 681 as the stable free radical site in E. coli anaerobic ribonucleotide reductase.

Formate is the hydrogen donor for the anaerobic ribonucleotide reductase from Escherichia coli.

During anaerobic growth Escherichia coli uses a specific ribonucleoside-triphosphate reductase (class III enzyme) for the production of deoxyribonucleoside triphosphates. In its active form, the enzyme contains an iron-sulfur center and an oxygen-sensitive glycyl radical (Gly-681). The radical is generated in the inactive protein from S-adenosylmethionine by an auxiliary enzyme system present in E. coli. By modification of the previous purification procedure, we now prepared a glycyl radical-containing reductase, active in the absence of the auxiliary reducing enzyme system. This reductase uses formate as hydrogen donor in the reaction. During catalysis, formate is stoichiometrically oxidized to CO2, and isotope from [3H]formate appears in water. Thus E. coli uses completely different hydrogen donors for the reduction of ribonucleotides during anaerobic and aerobic growth. The aerobic class I reductase employs redox-active thiols from thioredoxin or glutaredoxin to this purpose. The present results strengthen speculations that class III enzymes arose early during the evolution of DNA.

The mechanism of the anaerobic Escherichia coli ribonucleotide reductase investigated with nuclear magnetic resonance spectroscopy.

During the reduction of ribonucleotides with [3H]formate by the class III anaerobic ribonucleotide reductase from Escherichia coli tritium appears in water and not in the product deoxyribonucleotide. In D2O, deuterium replaces the OH-group at carbon-2' with retention of configuration. In addition we find 1-2% deuterium in the 3'-position demonstrating a small exchange of this hydrogen with the protons of water during catalysis. Class I and II enzymes catalyze identical reactions. Members of the three classes of reductases apparently use the same chemical mechanism in spite of having completely different protein structures.

Generation of the glycyl radical of the anaerobic Escherichia coli ribonucleotide reductase requires a specific activating enzyme.

The anaerobic ribonucleotide reductase from Escherichia coli contains a glycyl radical as part of its polypeptide structure. The radical is generated by an enzyme system present in E. coli. The reductase is coded for by the nrdD gene located at 96 min. Immediately downstream, we now find an open reading frame with the potential to code for a 17.5-kDa protein with sequence homology to a protein required for the generation of the glycyl radical of pyruvate formate lyase. The protein corresponding to this open reading frame is required for the generation of the glycyl radical of the anaerobic reductase and binds tightly to the reductase. The "activase" contains iron, required for activity. The general requirements for generation of a glycyl radical are identical for the reductase and pyruvate formate lyase. For the reductase, the requirement of an iron-containing activase suggests the possibility that the iron-sulfur cluster of the enzyme is not involved in radical generation but may participate directly in the reduction of the ribonucleotide.

Allosteric control of the substrate specificity of the anaerobic ribonucleotide reductase from Escherichia coli.

The reduction of ribonucleotides is catalyzed by different enzymes in aerobic and anaerobic Escherichia coli, each with a different primary and quaternary structure. Here, we describe the allosteric regulation of the substrate specificity of the anaerobic ribonucleoside triphosphate reductase. The enzyme reduced ribonucleotides at a low basal rate. Reduction was stimulated up to 10-fold by an appropriate modulator (dGTP for ATP reduction, ATP for CTP and UTP reduction, and dTTP for GTP reduction). dGTP and dTTP inhibited the reduction of the "incorrect" substrate; dATP inhibited reduction of all four. From kinetic, effector binding, and competition experiments we conclude that the enzyme has two classes of sites, one that binds ATP and dATP and regulates pyrimidine ribonucleotide reduction ("pyrimidine site"), the other that binds dATP, dGTP, and dTTP and regulates purine ribonucleotide reduction ("purine site"). This model differs slightly from the model for the aerobic reductase, but the physiological consequences remain the same and explain how a single enzyme can provide a balanced supply of the four dNTPs. The similarity of a highly sophisticated control mechanism for the aerobic and anaerobic enzymes suggests that both arose by divergent evolution from a common ancestor, in spite of their different structures.

Interactions of 2'-modified azido- and haloanalogs of deoxycytidine 5'-triphosphate with the anaerobic ribonucleotide reductase of Escherichia coli.

The anaerobic Escherichia coli ribonucleotide reductase (class III reductase) responsible for the synthesis of the deoxyribonucleotides required for anaerobic DNA replication contains an oxygen-sensitive glycyl radical (Gly-681) suggesting involvement of radical chemistry in catalysis. The amino acid sequence of this enzyme completely differs from that of earlier described aerobic class I (prototype, aerobic E. coli) and class II (prototype, Lactobacillus leichmanii) reductases that use radical chemistry but employ other means for radical generation. Here, we study the interaction between the anaerobic E. coli reductase with the 5'-triphosphates of 2'-chloro-2'-deoxycytidine, 2'-fluoro-2'-deoxycytidine, and 2'-azido-2'-deoxycytidine (N3CTP), which are mechanism-based inhibitors of class I and II reductases and, on interaction with these enzymes, decompose to base, inorganic di(tri)phosphate and 2'-methylene-3(2H)-furanone. Also, with the anaerobic E. coli reductase, the 2'-substituted nucleotides act as mechanism-based inhibitors and decompose. N3CTP scavenges the glycyl radical of the enzyme similar to the interaction of N3CDP with the tyrosyl radical of class I enzymes. However, we found no evidence for a new transient radical species as is the case with class I enzymes. Our results suggest that the chemistry at the nucleotide level for the reduction of ribose by class III enzymes is similar to the chemistry employed by class I and II enzymes.