The tetrameric structure of nucleotide-regulated pyrophosphatase and its modulation by deletion mutagenesis and ligand binding.

A quarter of prokaryotic Family II inorganic pyrophosphatases (PPases) contain a regulatory insert comprised of two cystathionine ß-synthase (CBS) domains and one DRTGG domain in addition to the two catalytic domains that form canonical Family II PPases. The CBS domain-containing PPases (CBS-PPases) are allosterically activated or inhibited by adenine nucleotides that cooperatively bind to the CBS domains. Here we use chemical cross-linking and analytical ultracentrifugation to show that CBS-PPases from Desulfitobacterium hafniense and four other bacterial species are active as 200-250-kDa homotetramers, which seems unprecedented among the four PPase families. The tetrameric structure is stabilized by Co2+, the essential cofactor, pyrophosphate, the substrate, and adenine nucleotides, including diadenosine tetraphosphate. The deletion variants of dhPPase containing only catalytic or regulatory domains are dimeric. Co2+ depletion by incubation with EDTA converts CBS-PPase into inactive tetrameric and dimeric forms. Dissociation of tetrameric CBS-PPase and its catalytic part by dilution renders them inactive. The structure of CBS-PPase tetramer was modelled from the structures of dimeric catalytic and regulatory parts. These findings signify the role of the unique oligomeric structure of CBS-PPase in its multifaced regulation.

Mu transpososome activity-profiling yields hyperactive MuA variants for highly efficient genetic and genome engineering.

The phage Mu DNA transposition system provides a versatile species non-specific tool for molecular biology, genetic engineering and genome modification applications. Mu transposition is catalyzed by MuA transposase, with DNA cleavage and integration reactions ultimately attaching the transposon DNA to target DNA. To improve the activity of the Mu DNA transposition machinery, we mutagenized MuA protein and screened for hyperactivity-causing substitutions using an in vivo assay. The individual activity-enhancing substitutions were mapped onto the MuA-DNA complex structure, containing a tetramer of MuA transposase, two Mu end segments and a target DNA. This analysis, combined with the varying effect of the mutations in different assays, implied that the mutations exert their effects in several ways, including optimizing protein-protein and protein-DNA contacts. Based on these insights, we engineered highly hyperactive versions of MuA, by combining several synergistically acting substitutions located in different subdomains of the protein. Purified hyperactive MuA variants are now ready for use as second-generation tools in a variety of Mu-based DNA transposition applications. These variants will also widen the scope of Mu-based gene transfer technologies toward medical applications such as human gene therapy. Moreover, the work provides a platform for further design of custom transposases.

Cooperativity in catalysis by canonical family II pyrophosphatases.

Bacterial family II pyrophosphatases (PPases) are homodimeric enzymes, with the active site located between two catalytic domains. Some family II PPases additionally contain regulatory cystathionine ß-synthase (CBS) domains and exhibit positive kinetic cooperativity, which is lost upon CBS domain removal. We report here that CBS domain-deficient family II PPases of Bacillus subtilis and Streptococcus gordonii also exhibit positive kinetic cooperativity, manifested as an up to a five-fold difference in the Michaelis constants for two active sites. An Asn79Ser replacement in S. gordonii PPase preserved its dimeric structure but abolished cooperativity. The results of our study indicated that kinetic cooperativity is an inherent property of all family II PPase types, is not induced by CBS domains, and is sensitive to minor structural changes. These findings may have inferences for other CBS-proteins, which include important enzymes and membrane transporters associated with hereditary diseases.

An asparagine residue mediates intramolecular communication in nucleotide-regulated pyrophosphatase.

Many prokaryotic soluble PPases (pyrophosphatases) contain a pair of regulatory adenine nucleotide-binding CBS (cystathionine ß-synthase) domains that act as 'internal inhibitors' whose effect is modulated by nucleotide binding. Although such regulatory domains are found in important enzymes and transporters, the underlying regulatory mechanism has only begun to come into focus. We reported previously that CBS domains bind nucleotides co-operatively and induce positive kinetic co-operativity (non-Michaelian behaviour) in CBS-PPases (CBS domain-containing PPases). In the present study, we demonstrate that a homodimeric ehPPase (Ethanoligenens harbinense PPase) containing an inherent mutation in an otherwise conserved asparagine residue in a loop near the active site exhibits non-co-operative hydrolysis kinetics. A similar N312S substitution in 'co-operative' dhPPase (Desulfitobacterium hafniense PPase) abolished kinetic co-operativity while causing only minor effects on nucleotide-binding affinity and co-operativity. However, the substitution reversed the effect of diadenosine tetraphosphate, abolishing kinetic co-operativity in wild-type dhPPase, but restoring it in the variant dhPPase. A reverse serine-to-asparagine replacement restored kinetic co-operativity in ehPPase. Molecular dynamics simulations revealed that the asparagine substitution resulted in a change in the hydrogen-bonding pattern around the asparagine residue and the subunit interface, allowing greater flexibility at the subunit interface without a marked effect on the overall structure. These findings identify this asparagine residue as lying at the 'crossroads' of information paths connecting catalytic and regulatory domains within a subunit and catalytic sites between subunits.

Cystathionine ß-Synthase (CBS) Domain-containing Pyrophosphatase as a Target for Diadenosine Polyphosphates in Bacteria.

Among numerous proteins containing pairs of regulatory cystathionine ß-synthase (CBS) domains, family II pyrophosphatases (CBS-PPases) are unique in that they generally contain an additional DRTGG domain between the CBS domains. Adenine nucleotides bind to the CBS domains in CBS-PPases in a positively cooperative manner, resulting in enzyme inhibition (AMP or ADP) or activation (ATP). Here we show that linear P(1),P(n)-diadenosine 5'-polyphosphates (ApnAs, where n is the number of phosphate residues) bind with nanomolar affinity to DRTGG domain-containing CBS-PPases of Desulfitobacterium hafniense, Clostridium novyi, and Clostridium perfringens and increase their activity up to 30-, 5-, and 7-fold, respectively. Ap4A, Ap5A, and Ap6A bound noncooperatively and with similarly high affinities to CBS-PPases, whereas Ap3A bound in a positively cooperative manner and with lower affinity, like mononucleotides. All ApnAs abolished kinetic cooperativity (non-Michaelian behavior) of CBS-PPases. The enthalpy change and binding stoichiometry, as determined by isothermal calorimetry, were ~10 kcal/mol nucleotide and 1 mol/mol enzyme dimer for Ap4A and Ap5A but 5.5 kcal/mol and 2 mol/mol for Ap3A, AMP, ADP, and ATP, suggesting different binding modes for the two nucleotide groups. In contrast, Eggerthella lenta and Moorella thermoacetica CBS-PPases, which contain no DRTGG domain, were not affected by ApnAs and showed no enthalpy change, indicating the importance of the DTRGG domain for ApnA binding. These findings suggest that ApnAs can control CBS-PPase activity and hence affect pyrophosphate level and biosynthetic activity in bacteria.

Cystathionine ß-synthase (CBS) domains confer multiple forms of Mg2+-dependent cooperativity to family II pyrophosphatases.

Regulated family II pyrophosphatases (CBS-PPases) contain a nucleotide-binding insert comprising a pair of cystathionine ß-synthase (CBS) domains, termed a Bateman module. By binding with high affinity to the CBS domains, AMP and ADP usually inhibit the enzyme, whereas ATP activates it. Here, we demonstrate that AMP, ADP, and ATP bind in a positively cooperative manner to CBS-PPases from four bacteria: Desulfitobacterium hafniense, Clostridium novyi, Clostridium perfringens, and Eggerthella lenta. Enzyme interaction with substrate as characterized by the Michaelis constant (Km) also exhibited positive catalytic cooperativity that decreased in magnitude upon nucleotide binding. The degree of both types of cooperativity increased with increasing concentration of the cofactor Mg(2+) except for the C. novyi PPase where Mg(2+) produced the opposite effect on kinetic cooperativity. Further exceptions from these general rules were ADP binding to C. novyi PPase and AMP binding to E. lenta PPase, neither of which had any effect on activity. A genetically engineered deletion variant of D. hafniense PPase lacking the regulatory insert was fully active but differed from the wild-type enzyme in that it was insensitive to nucleotides and bound substrate non-cooperatively and with a smaller Km value. These results indicate that the regulatory insert acts as an internal inhibitor and confers dual positive cooperativity to CBS domain-containing PPases, making them highly sensitive regulators of the PPi level in response to the changes in cell energy status that control adenine nucleotide distribution. These regulatory features may be common among other CBS domain-containing proteins.

Seminal vesicles and urinary bladder as sites of aromatization of androgens in men, evidenced by a CYP19A1-driven luciferase reporter mouse and human tissue specimens.

The human CYP19A1 gene is expressed in various tissues by the use of tissue-specific promoters, whereas the rodent cyp19a1 gene is expressed mainly in the gonads and brain. We generated a transgenic mouse model containing a >100-kb 5' region of human CYP19A1 gene connected to a luciferase reporter gene. The luciferase activity in mouse tissues mimicked the CYP19A1 gene expression pattern in humans. Interestingly, the reporter gene activity was 16 and 160 times higher in the urinary bladder and seminal vesicles, respectively, as compared with the activity in the testis. Accordingly, CYP19A1 gene and P450arom protein expression was detected in those human tissues. Moreover, the data revealed that the expression of CYP19A1 gene is driven by promoters PII, I.4, and I.3 in the seminal vesicles, and by promoters PII and I.4 in the urinary bladder. Furthermore, the reporter gene expression in the seminal vesicles was androgen dependent: Castration decreased the expression ∼20 times, and testosterone treatment restored it to the level of an intact mouse. This reporter mouse model facilitates studies of tissue-specific regulation of the human CYP19A1 gene, and our data provide evidence for seminal vesicles as important sites for estrogen production in males.

A single dose of enterolactone activates estrogen signaling and regulates expression of circadian clock genes in mice.

Enterolactone (EL) is an enterolignan produced by gut microbiota from dietary plant lignans. Epidemiological and experimental studies suggest that EL and plant lignans may reduce the risk of breast and prostate cancer as well as cardiovascular disease. These effects are thought to at least in part involve modulation of estrogen receptor activity. Surprisingly little is known about the in vivo estrogenicity of EL. In the present study, we investigated the target tissues of EL, the genes affected by EL treatment, and the response kinetics. Following a single dose of EL, luciferase was significantly induced in reproductive and nonreproductive tissues of male and female 3xERE-luciferase mice, indicating estrogen-like activity. Microarray analysis revealed that EL regulated the expression of only 1% of 17ß-estradiol target genes in the uterus. The majority of these genes were traditional estrogen target genes, but also members of the circadian signaling pathway were affected. Kinetic analyses showed that EL undergoes rapid phase II metabolism and is efficiently excreted. In vivo imaging demonstrated that the estrogen response followed similar, fast kinetics. We conclude that EL activates estrogen signaling in both male and female mice and that the transient responses may be due to the fast metabolism of the compound. Lastly, EL may represent a link among diet, gut microbiota, and circadian signaling.

Residue Network Involved in the Allosteric Regulation of Cystathionine ß-Synthase Domain-Containing Pyrophosphatase by Adenine Nucleotides.

Inorganic pyrophosphatase containing regulatory cystathionine ß-synthase (CBS) domains (CBS-PPase) is inhibited by adenosine monophosphate (AMP) and adenosine diphosphate and activated by adenosine triphosphate (ATP) and diadenosine polyphosphates; mononucleotide binding to CBS domains and substrate binding to catalytic domains are characterized by positive cooperativity. This behavior implies three pathways for regulatory signal transduction - between regulatory and active sites, between two active sites, and between two regulatory sites. Bioinformatics analysis pinpointed six charged or polar amino acid residues of Desulfitobacterium hafniense CBS-PPase as potentially important for enzyme regulation. Twelve mutant enzyme forms were produced, and their kinetics of pyrophosphate hydrolysis was measured in wide concentration ranges of the substrate and various adenine nucleotides. The parameters derived from this analysis included catalytic activity, Michaelis constants for two active sites, AMP-, ATP-, and diadenosine tetraphosphate-binding constants for two regulatory sites, and the degree of activation/inhibition for each nucleotide. Replacements of arginine 295 and asparagine 312 by alanine converted ATP from an activator to an inhibitor and markedly affected practically all the above parameters, indicating involvement of these residues in all the three regulatory signaling pathways. Replacements of asparagine 312 and arginine 334 abolished or reversed kinetic cooperativity in the absence of nucleotides but conferred it in the presence of diadenosine tetraphosphate, without effects on nucleotide-binding parameters. Modeling and molecular dynamics simulations revealed destabilization of the subunit interface as a result of asparagine 312 and arginine 334 replacements by alanine, explaining abolishment of kinetic cooperativity. These findings identify residues 295, 312, and 334 as crucial for CBS-PPase regulation via CBS domains.

A CBS domain-containing pyrophosphatase of Moorella thermoacetica is regulated by adenine nucleotides.

CBS (cystathionine beta-synthase) domains are found in proteins from all kingdoms of life, and point mutations in these domains are responsible for a variety of hereditary diseases in humans; however, the functions of CBS domains are not well understood. In the present study, we cloned, expressed in Escherichia coli, and characterized a family II PPase (inorganic pyrophosphatase) from Moorella thermoacetica (mtCBS-PPase) that has a pair of tandem 60-amino-acid CBS domains within its N-terminal domain. Because mtCBS-PPase is a dimer and requires transition metal ions (Co2+ or Mn2+) for activity, it resembles common family II PPases, which lack CBS domains. The mtCBS-PPase, however, has lower activity than common family II PPases, is potently inhibited by ADP and AMP, and is activated up to 1.6-fold by ATP. Inhibition by AMP is competitive, whereas inhibition by ADP and activation by ATP are both of mixed types. The nucleotides are effective at nanomolar (ADP) or micromolar concentrations (AMP and ATP) and appear to compete for the same site on the enzyme. The nucleotide-binding affinities are thus 100-10000-fold higher than for other CBS-domain-containing proteins. Interestingly, genes encoding CBS-PPase occur most frequently in bacteria that have a membrane-bound H+-translocating PPase with a comparable PP(i)-hydrolysing activity. Our results suggest that soluble nucleotide-regulated PPases act as amplifiers of metabolism in bacteria by enhancing or suppressing ATP production and biosynthetic reactions at high and low [ATP]/([AMP]+[ADP]) ratios respectively.

Inorganic pyrophosphatases of Family II-two decades after their discovery.

Inorganic pyrophosphatases (PPases) convert pyrophosphate (PPi ) to phosphate and are present in all cell types. Soluble PPases belong to three nonhomologous families, of which Family II is found in approximately a quarter of prokaryotic organisms, often pathogenic ones. Each subunit of dimeric canonical Family II PPases is formed by two domains connected by a flexible linker, with the active site located between the domains. These enzymes require both magnesium and a transition metal ion (manganese or cobalt) for maximal activity and are the most active (kcat ≈ 104 s-1 ) among all PPase types. Catalysis by Family II PPases requires four metal ions per substrate molecule, three of which form a unique trimetal center that coordinates the nucleophilic water and converts it to a reactive hydroxide ion. A quarter of Family II PPases contain an autoinhibitory regulatory insert formed by two cystathionine ß-synthase (CBS) domains and one DRTGG domain. Adenine nucleotide binding either activates or inhibits the CBS domain-containing PPases, thereby tuning their activity and, hence, PPi levels, in response to changes in cell energy status (ATP/ADP ratio).

Endometrial and endometriotic concentrations of estrone and estradiol are determined by local metabolism rather than circulating levels.

CONTEXT: Aberrant estrogen synthesis and metabolism have been suggested to increase local estradiol (E2) concentration in endometriosis and thus to promote the growth of the lesions. However, tissue estrogen concentrations within the endometrium and different types of endometriosis lesions have not been described. OBJECTIVE: The aim of the study was to evaluate local E2 and estrone (E1) concentrations in the endometrium and different types of endometriosis lesions, and to correlate them with the expression of estrogen-metabolizing enzymes. PATIENTS: Patients with endometriosis (n = 60) and healthy controls (n = 16) participated in the study. MAIN OUTCOME MEASURES: We measured serum and tissue concentrations of E2 and E1 as well as mRNA expression of the estrogen-metabolizing enzymes. RESULTS: Endometrial or endometriotic intratissue E2 concentrations did not reflect the corresponding serum levels. In the proliferative phase, endometrial E2 concentration was five to eight times higher than in the serum, whereas in the secretory phase the E2 concentration was about half of that in the serum. Accordingly, a markedly higher E2/E1 ratio was observed in the endometrium at the proliferative phase compared with the secretory phase. In the endometriosis lesions, E2 levels were predominating over those of E1 throughout the menstrual cycle. Among the hydroxysteroid (17ß) dehydrogenase (HSD17B) enzymes analyzed, HSD17B2 negatively correlated with the E2 concentration in the endometrium, and HSD17B6 was strongly expressed, especially in the deep lesions. CONCLUSIONS: Endometrial or endometriotic tissue E2 concentrations are actively regulated by local estrogen metabolism in the tissue. Thus, the inhibition of local E2 synthesis is a valid, novel approach to reduce local E2-dependent growth of endometriotic tissue.

Cd(2+)-induced aggregation of Escherichia coli pyrophosphatase.

We report here that Escherichia coli pyrophosphatase aggregates in the presence of millimolar Cd(2+). This highly cooperative process was specific to both the metal ion and the protein and could be reversed fully by decreasing the Cd(2+) concentration. Aggregation was enhanced by Mg(2+), the natural cofactor of pyrophosphatase, and Mn(2+). Mutations at the intersubunit metal-binding site had no effect, whereas mutation at Glu139, which is part of the peripheral metal-binding site found in pyrophosphatase crystals near the contact region between two enzyme molecules, suppressed aggregation. These findings indicate that aggregation is affected by Cd(2+) binding to the peripheral metal-binding site, probably by strengthening intermolecular Trp149-Trp149' stacking interactions.

Structural studies of metal ions in family II pyrophosphatases: the requirement for a Janus ion.

Family II inorganic pyrophosphatases (PPases) constitute a new evolutionary group of PPases, with a different fold and mechanism than the common family I enzyme; they are related to the "DHH" family of phosphoesterases. Biochemical studies have shown that Mn(2+) and Co(2+) preferentially activate family II PPases; Mg(2+) partially activates; and Zn(2+) can either activate or inhibit (Zyryanov et al., Biochemistry, 43, 14395-14402, accompanying paper in this issue). The three solved family II PPase structures did not explain the differences between the PPase families nor the metal ion differences described above. We therefore solved three new family II PPase structures: Bacillus subtilis PPase (Bs-PPase) dimer core bound to Mn(2+) at 1.3 A resolution, and, at 2.05 A resolution, metal-free Bs-PPase and Streptococcus gordonii (Sg-PPase) containing sulfate and Zn(2+). Comparison of the new and old structures of various family II PPases demonstrates why the family II enzyme prefers Mn(2+) or Co(2+), as an activator rather than Mg(2+). Both M1 and M2 undergo significant changes upon substrate binding, changing from five-coordinate to octahedral geometry. Mn(2+) and Co(2+), which readily adopt different coordination states and geometries, are thus favored. Combining our structures with biochemical data, we identified M2 as the high-affinity metal site. Zn(2+) activates in the M1 site, where octahedral geometry is not essential for catalysis, but inhibits in the M2 site, because it is unable to assume octahedral geometry but remains trigonal bipyramidal. Finally, we propose that Lys205-Gln81-Gln80 form a hydrophilic channel to speed product release from the active site.

Rates of elementary catalytic steps for different metal forms of the family II pyrophosphatase from Streptococcus gordonii.

Soluble inorganic pyrophosphatases (PPases) form two nonhomologous families, denoted I and II, that have similar active-site structures but different catalytic activities and metal cofactor specificities. Family II PPases, which are often found in pathogenic bacteria, are more active than family I PPases, and their best cofactor is Mn(2+) rather than Mg(2+), the preferred cofactor of family I PPases. Here, we present results of a detailed kinetic analysis of a family II PPase from Streptococcus gordonii (sgPPase), which was undertaken to elucidate the factors underlying the different properties of family I and II PPases. We measured rates of PP(i) hydrolysis, PP(i) synthesis, and P(i)/water oxygen exchange catalyzed by sgPPase with Mn(2+), Mg(2+), or Co(2+) in the high-affinity metal-binding site and Mg(2+) in the other sites, as well as the binding affinities for several active-site ligands (metal cofactors, fluoride, and P(i)). On the basis of these data, we deduced a minimal four-step kinetic scheme and evaluated microscopic rate constants for all eight relevant reaction steps. Comparison of these results with those obtained previously for the well-known family I PPase from Saccharomyces cerevisiae (Y-PPase) led to the following conclusions: (a) catalysis by sgPPase does not involve the enzyme-PP(i) complex isomerization known to occur in family I PPases; (b) the values of k(cat) for the magnesium forms of sgPPase and Y-PPase are similar because of similar rates of bound PP(i) hydrolysis and product release; (c) the marked acceleration of sgPPase catalysis in the presence of Mn(2+) and Co(2+) results from a combined effect of these ions on bound PP(i) hydrolysis and P(i) release; (d) sgPPase exhibits lower affinity for both PP(i) and P(i); and (e) sgPPase and Y-PPase exhibit similar values of k(cat)/K(m), which characterizes the PPase efficiency in vivo (i.e., at nonsaturating PP(i) concentrations).

Site-specific effects of zinc on the activity of family II pyrophosphatase.

Family II pyrophosphatases (PPases), recently found in bacteria and archaebacteria, are Mn(2+)-containing metalloenzymes with two metal-binding subsites (M1 and M2) in the active site. These PPases can use a number of other divalent metal ions as the cofactor but are inactive with Zn(2+), which is known to be a good cofactor for family I PPases. We report here that the Mg(2+)-bound form of the family II PPase from Streptococcus gordonii is nearly instantly activated by incubation with equimolar Zn(2+), but the activity thereafter decays on a time scale of minutes. The activation of the Mn(2+)-form by Zn(2+) was slower but persisted for hours, whereas activation was not observed with the Ca(2+)- and apo-forms. The bound Zn(2+) could be removed from PPase by prolonged EDTA treatment, with a complete recovery of activity. On the basis of the effect of Zn(2+) on PPase dimerization, the Zn(2+) binding constant appeared to be as low as 10(-12) M for S. gordonii PPase. Similar effects of Zn(2+) and EDTA were observed with the Mg(2+)- and apo-forms of Streptococcus mutans and Bacillus subtilis PPases. The effects of Zn(2+) on the apo- and Mg(2+)-forms of HQ97 and DE15 B. subtilis PPase variants (modified M2 subsite) but not of HQ9 variant (modified M1 subsite) were similar to that for the Mn(2+)-form of wild-type PPase. These findings can be explained by assuming that (a) the PPase tightly binds Mg(2+) and Mn(2+) at the M2 subsite; (b) the activation of the corresponding holoenzymes by Zn(2+) results from its binding to the M1 subsite; and (c) the subsequent inactivation of Mg(2+)-PPase results from Zn(2+) migration to the M2 subsite. The inability of Zn(2+) to activate apo-PPase suggests that Zn(2+) binds more tightly to M2 than to M1, allowing direct binding to M2. Zn(2+) is thus an efficient cofactor at subsite M1 but not at subsite M2.

Modulation of dimer stability in yeast pyrophosphatase by mutations at the subunit interface and ligand binding to the active site.

Yeast (Saccharomyces cerevisiae) pyrophosphatase (Y-PPase) is a tight homodimer with two active sites separated in space from the subunit interface. The present study addresses the effects of mutation of four amino acid residues at the subunit interface on dimer stability and catalytic activity. The W52S variant of Y-PPase is monomeric up to an enzyme concentration of 300 microm, whereas R51S, H87T, and W279S variants produce monomer only in dilute solutions at pH > or = 8.5, as revealed by sedimentation, gel electrophoresis, and activity measurements. Monomeric Y-PPase is considerably more sensitive to the SH reagents N-ethylmaleimide and p-hydroxymercurobenzosulfonate than the dimeric protein. Additionally, replacement of a single cysteine residue (Cys(83)), which is not part of the subunit interface or active site, with Ser resulted in insensitivity of the monomer to SH reagents and stabilization against spontaneous inactivation during storage. Active site ligands (Mg(2+) cofactor, P(i) product, and the PP(i) analog imidodiphosphate) stabilized the W279S dimer versus monomer predominantly by decreasing the rate of dimer to monomer conversion. The monomeric protein exhibited a markedly increased (5-9-fold) Michaelis constant, whereas k(cat) remained virtually unchanged, compared with dimer. These results indicate that dimerization of Y-PPase improves its substrate binding performance and, conversely, that active site adjustment through cofactor, product, or substrate binding strengthens intersubunit interactions. Both effects appear to be mediated by a conformational change involving the C-terminal segment that generally shields the Cys(83) residue in the dimer.