The small subunit of M. AquI is responsible for sequence-specific DNA recognition and binding in the absence of the catalytic domain

AquI DNA methyltransferase (M. AquI) catalyzes the transfer of a methyl group from S-adenosyl-L-methionine to the C5 position of the outermost deoxycytidine base in the DNA sequence 5'-CCCGGG-3'. M. AquI is a heterodimer in which the polypeptide chain is separated at the junction between the two equivalent structural domains in the related enzyme M. HhaI. Recently, we reported the subcloning, overexpression, and purification of the subunits (alpha and beta) of M. AquI separately. Here we describe the DNA binding properties of M. AquI. The results presented here indicate that the beta subunit alone contains all of the information for sequence-specific DNA recognition and binding. The first step in the sequence-specific recognition of DNA by M. AquI involves the formation of binary complex with the target recognition domain in conjunction with conserved sequence motifs IX and X, found in all known C5 DNA methyltransferases, contained in the beta subunit. The alpha subunit enhances the binding of the beta subunit to DNA specifically and nonspecifically. It is likely that the addition of the alpha subunit to the beta subunit stabilizes the conformation of the beta subunit and thereby enhances its affinity for DNA indirectly. Addition of S-adenosyl-L-methionine and its analogues S-adenosyl-L-homocysteine and sinefungin enhances binding, but only in the presence of the alpha subunit. These compounds did not have any effect on DNA binding by the beta subunit alone. Using a 30-mer oligodeoxynucleotide substrate containing 5-fluorodeoxycytidine (5-FdC), it was found that the beta subunit alone did not form a covalent complex with its specific sequence in the absence or presence of S-adenosyl-L-methionine. However, the addition of the alpha subunit to the beta subunit led to the formation of a covalent complex with specific DNA sequence containing 5-FdC.     

Functional studies of the small subunit of EcoHK31I DNA methyltransferase

EcoHK31I DNA methyltransferase recognizes the sequence 5'-YGGCCR-3' and adds a methyl group to the fifth position of the internal cytosine to protect the DNA from cleavage by its cognate endonuclease. M.EcoHK31I is composed of polypeptides alpha and beta. Polypeptide beta only contains the conserved IX motif of the C5-MTase family, and provides a unique example to show that this motif alone may be dislocated to another polypeptide. By electromobility shift assay, we found that the alpha/beta complex recognizes specific oligonucleotide substrates. Polypeptide alpha formed aggregates with DNA, while polypeptide beta alone did not bind DNA. Therefore, polypeptide beta assists in the proper binding of polypeptide alpha to DNA substrate. The complex of polypeptide alpha and a polypeptide beta variant with an N-terminal deletion of 41 amino acids showed a 16-fold reduction in methylation activity. Further deletion resulted in an inactive methyltransferase. The dissociation equilibrium constant (Kd) of the alpha/beta complex was 56.4 nM, while the Kd value for the alpha/deltaN46-polypeptide beta complex was increased approximately 95-fold, caused by a drastic decrease in dissociate rate constant (kd) and an increase in the association rate constant (ka). This indicates that the N-terminal region of polypeptide beta takes part in subunit interaction, while the C-terminal region is involved in DNA binding.     

Studies on the subunits of Escherichia coli coenzyme A transferase. Reconstitution of an active enzyme.

The alpha and beta subunits of the acetyl-CoA:acetoacetate-CoA transferase were purified by isoelectric focusing of the enzyme in the presence of 6 M urea. The purified beta subunit, in which the active center of the enzyme is located, exhibits low catalytic activity (2% of the specific activity of the native enzyme) which is stimulated 5-6-fold in the presence of an equimolar concentration of alpha subunit. The presence of the substrate,acetoacetyl-CoA, is required to recover the catalytic activity of the beta subunit and mixtures containing purified alpha and beta subunits. When the enzyme is dissociation in the presence of 6 M urea and the subunits are not fractioned, removal of the urea by dialysis results in the recovery of 88-98% of enzymic activity and the native alpha2beta2 subunit structure. However, analysis of this renatured enzyme by immunochemical techniques shows that the enzyme does not refold to a completely native conformation. This renatured enzyme exhibits an immunological reactivity more closely resembling the isolated alpha subunit. The results indicate that the alpha subunit serves as a structural subunit, or possible a maturation subunit, imposing a conformation on the beta subunit that is catalytically more competent.     

Cloning, expression, and purification of a thermostable nonhomodimeric restriction enzyme, BslI

BslI is a thermostable type II restriction endonuclease with interrupted recognition sequence CCNNNNN/NNGG (/, cleavage position). The BslI restriction-modification system from Bacillus species was cloned and expressed in Escherichia coli. The system is encoded by three genes: the 2,739-bp BslI methylase gene (bslIM), the bslIRalpha gene, and the bslIRbeta gene. The alpha and beta subunits of BslI can be expressed independently in E. coli in the absence of BslI methylase (M.BslI) protection. BslI endonuclease activity can be reconstituted in vitro by mixing the two subunits together. Gel filtration chromatography and native polyacrylamide gel electrophoresis indicated that BslI forms heterodimers (alphabeta), heterotetramers (alpha(2)beta(2)), and possibly oligomers in solution. Two beta subunits can be cross-linked by a chemical cross-linking agent, indicating formation of heterotetramer BslI complex (alpha(2)beta(2)). In DNA mobility shift assays, neither subunit alone can bind DNA. DNA mobility shift activity was detected after mixing the two subunits together. Because of the symmetric recognition sequence of the BslI endonuclease, we propose that its active form is alpha(2)beta(2). M.BslI contains nine conserved motifs of N-4 cytosine DNA methylases within the beta group of aminomethyltransferase. Synthetic duplex deoxyoligonucleotides containing cytosine hemimethylated or fully methylated at N-4 in BslI sites in the first or second cytosine are resistant to BslI digestion. C-5 methylation of the second cytosine on both strands within the recognition sequence also renders the site refractory to BslI digestion. Two putative zinc fingers are found in the alpha subunit of BslI endonuclease.     

Dissociation of alpha beta DNA polymerase of avian myeloblastosis virus by dimethyl sulfoxide.

The alpha beta DNA polymerase of avian myeloblastosis virus was treated with dimethyl sulfoxide to dissociate the enzyme subunits. The dimethyl sulfoxide treated enzymes were passed over phosphocellulose to purify and characterize the dissociated subunits as well as to remove the dimethyl sulfoxide. RNA-directed DNA polymerase, RNase H, and nucleic acid-binding activity were monitored, as well as the subunit structure (on sodium dodecyl sulfate-polyacrylamide gels) of the various enzyme species obtained. With 30% dimethyl sulfoxide, the majority of DNA polymerase and RNase H activities as well as the alpha subunit were displaced from the alpha beta DNA polymerase position on phosphocellulose (0.23 M potassium phosphate) to the alpha DNA polymerase position (0.1 M). The association of DNA polymerase and RNase H activities with the alpha subunit suggests that alpha is the enzymatically active subunit in alpha beta. In addition to alpha DNA polymerase, a minor polymerase species eluted from phosphocellulose at 0.4 M potassium phosphate. The dissociated beta subunit eluted from phosphocellulose at a wide range of salt concentrations (0.28 to 0.5 M potassium phosphate). The dissociated beta subunit bound 3H-labeled murine leukemia virus RNA and [3H]poly(dT)-poly(dA) approximately 20-fold more avidly than alpha DNA polymerase alone. In contrast to the results with the alpha subunit, there was no correlation between DNA polymerase and RNase H activity profiles and the elution profile of the beta subunit from phosphocellulose. These observations suggest the beta subunit is either enzymatically inactive or possesses limited DNA polymerase and RNase H activity when compared with the alpha subunit.     

Carboxyl-terminal processing may be essential for production of active NiFe hydrogenase in Azotobacter vinelandii.

The NiFe hydrogenase from Azotobacter vinelandii is a membrane-bound alpha beta heterodimer that can oxidize H2 to protons and electrons and thereby provide energy. Genes encoding the alpha and beta subunits, hoxG and hoxK respectively, followed by thirteen contiguous accessory genes potentially involved in H2 oxidation, have been previously sequenced. Mutations in some of these accessory genes give rise to inactive enzyme containing an alpha subunit with decreased electrophoretic mobility. Mass spectral analysis of the subunits demonstrated that the alpha subunit had a molecular weight 1,663 Da less than that predicted from hoxG. Since the N-terminal sequence of the purified alpha subunit matches the sequence predicted from hoxG we suggest this difference is due to removal of the C-terminus of the alpha subunit which may be an important step linked to metal insertion, localization, and formation of active hydrogenase.     

Substrate-specific selenoprotein B of glycine reductase from Eubacterium acidaminophilum. Biochemical and molecular analysis.

The substrate-specific selenoprotein B of glycine reductase (PBglycine) from Eubacterium acidaminophilum was purified and characterized. The enzyme consisted of three different subunits with molecular masses of about 22 (alpha), 25 (beta) and 47 kDa (gamma), probably in an alpha 2 beta 2 gamma 2 composition. PBglycine purified from cells grown in the presence of [75Se]selenite was labeled in the 47-kDa subunit. The 22-kDa and 47-kDa subunits both reacted with fluorescein thiosemicarbazide, indicating the presence of a carbonyl compound. This carbonyl residue prevented N-terminal sequencing of the 22-kDa (alpha) subunit, but it could be removed for Edman degradation by incubation with o-phenylenediamine. A DNA fragment was isolated and sequenced which encoded beta and alpha subunits of PBglycine (grdE), followed by a gene encoding selenoprotein A (grdA2) and the gamma subunit of PBglycine (grdB2). The cloned DNA fragment represented a second GrdB-encoding gene slightly different from a previously identified partial grdBl-containing fragment. Both grdB genes contained an in-frame UGA codon which confirmed the observed selenium content of the 47-kDa (gamma) subunit. Peptide sequence analyses suggest that grdE encodes a proprotein which is cleaved into the previously sequenced N-terminal 25-kDa (beta) subunit and a 22-kDa (alpha) subunit of PBglycine. Cleavage most probably occurred at an -Asn-Cys- site concomitantly with the generation of the blocking carbonyl moiety from cysteine at the alpha subunit.     

Domain structure and subunit interactions in the type I DNA methyltransferase M.EcoR124I.

The type IC DNA methyltransferase M.EcoR124I is a trimeric enzyme of 162 kDa consisting of two modification subunits, HsdM, and a single specificity subunit, HsdS. Studies have been largely restricted to the HsdM subunit or to the intact methyltransferase since the HsdS subunit is insoluble when over-expressed independently of HsdM. Two soluble fragments of the HsdS subunit have been cloned, expressed and purified; a 25 kDa N-terminal fragment (S3) comprising the N-terminal target recognition domain together with the central conserved domain, and a 8.6 kDa fragment (S11) comprising the central conserved domain alone. Analytical ultracentrifugation shows that the S3 subunit exists principally as a dimer of 50 kDa. Gel retardation and competition assays show that both S3 and S11 are able to bind to HsdM, each with a subunit stoichiometry of 1:1. The tetrameric complex (S3/HsdM)(2) is required for effective DNA binding. Cooperative binding is observed and at low enzyme concentration, the multisubunit complex dissociates, leading to a loss of DNA binding activity. The (S3/HsdM)(2) complex is able to bind to both the EcoR124I DNA recognition sequence GAAN(6)RTCG and a symmetrical DNA sequence GAAN(7)TTC, but has a 30-fold higher affinity binding for the latter DNA sequence. Exonuclease III footprinting of the (S3/HsdM)(2) -DNA complex indicates that 29 nucleotides are protected on each strand, corresponding to a region 8 bp on both the 3' and 5' sides of the recognition sequence bound by the (S3/HsdM)(2) complex.     

Purification and properties of the membrane-associated coenzyme F420-reducing hydrogenase from Methanobacterium formicicum.

The membrane-associated coenzyme F420-reducing hydrogenase of Methanobacterium formicicum was purified 87-fold to electrophoretic homogeneity. The enzyme contained alpha, beta, and gamma subunits (molecular weights of 43,000, 36,700, and 28,800, respectively) and formed aggregates (molecular weight, 1,020,000) of a coenzyme F420-active alpha 1 beta 1 gamma 1 trimer (molecular weight, 109,000). The hydrogenase contained 1 mol of flavin adenine dinucleotide (FAD), 1 mol of nickel, 12 to 14 mol of iron, and 11 mol of acid-labile sulfide per mol of the 109,000-molecular-weight species, but no selenium. The isoelectric point was 5.6. The amino acid sequence I-N3-P-N2-R-N1-EGH-N6-V (where N is any amino acid) was conserved in the N-termini of the alpha subunits of the F420-hydrogenases from M. formicicum and Methanobacterium thermoautotrophicum and of the largest subunits of nickel-containing hydrogenases from Desulfovibrio baculatus, Desulfovibrio gigas, and Rhodobacter capsulatus. The purified F420-hydrogenase required reductive reactivation before assay. FAD dissociated from the enzyme during reactivation unless potassium salts were present, yielding deflavoenzyme that was unable to reduce coenzyme F420. Maximal coenzyme F420-reducing activity was obtained at 55 degrees C and pH 7.0 to 7.5, and with 0.2 to 0.8 M KCl in the reaction mixture. The enzyme catalyzed H2 production at a rate threefold lower than that for H2 uptake and reduced coenzyme F420, methyl viologen, flavins, and 7,8-didemethyl-8-hydroxy-5-deazariboflavin. Specific antiserum inhibited the coenzyme F420-dependent but not the methyl viologen-dependent activity of the purified enzyme.     

Methyltransferase and subunit association domains of vaccinia virus mRNA capping enzyme.

RNA triphosphatase, RNA guanylyltransferase, and RNA (guanine-N7-)-methyltransferase activities are associated with the vaccinia virus mRNA capping enzyme, a heterodimeric protein containing polypeptides of M(r) 95,000 and 31,000. Although the RNA triphosphatase and RNA guanylyltransferase domains have been localized to a M(r) 59,000 fragment of the capping enzyme large subunit, the location of the methyltransferase domain within the protein and the catalytic role of individual subunits in methyl group transfer remain unclear. In the present work, through the study of methyltransferase activity of truncated forms of capping enzyme translated in vitro in a rabbit reticulocyte lysate, we have localized the methyltransferase domain to a complex consisting of the small subunit and the carboxyl-terminal portion of the large subunit. The M(r) 31,000 subunit translated alone was not sufficient for methyltransferase activity. This requirement for both subunits may explain the tight physical association of the two polypeptides in vivo. We have recreated the association of the large and small enzyme subunits in vitro through the translation of synthetic mRNAs encoding the two polypeptides. Study of the ability of deleted versions of the large subunit to bind the small subunit, as detected by co-immunoprecipitation, defined a 347-amino acid carboxyl-terminal region of the large subunit that was sufficient for heterodimerization. Colocalization within the large subunit of the methyltransferase and subunit association domains suggests that dimerization of the subunits may be required for methyltransferase activity.     

Purification and characterisation of a novel DNA methyltransferase,M.AhdI

We have cloned the M and S genes of the restriction-modification (R-M) system AhdI and have purified the resulting methyltransferase to homogeneity. M.AhdI is found to form a 170 kDa tetrameric enzyme having a subunit stoichiometry M₂S₂ (where the M and S subunits are responsible for methylation and DNA sequence specificity, respectively). Sedimentation equilibrium experiments show that the tetrameric enzyme dissociates to form a heterodimer at low concentration, with K_d ≈ 2 µM. The intact (tetrameric) enzyme binds specifically to a 30 bp DNA duplex containing the AhdI recognition sequence GACN₅GTC with high affinity (K_d ≈ 50 nM), but at low enzyme concentration the DNA binding activity is governed by the dissociation of the tetramer into dimers, leading to a sigmoidal DNA binding curve. In contrast, only non-specific binding is observed if the duplex lacks the recognition sequence. Methylation activity of the purified enzyme was assessed by its ability to prevent restriction by the cognate endonuclease. The subunit structure of the M.AhdI methyltransferase resembles that of type I MTases, in contrast to the R.AhdI endonuclease which is typical of type II systems. AhdI appears to be a novel R-M system with properties intermediate between simple type II systems and more complex type I systems, and may represent an intermediate in the evolution of R-M systems.     

Isolation and characterization of recombinant human casein kinase II subunits alpha and beta from bacteria

cDNA encoding the casein kinase II (CKII) subunits alpha and beta of human origin were expressed in Escherichia coli using expression vector pT7-7. Significant expression was obtained with E. coli BL21(DE3). The CKII subunits accounted for approximately 30% of the bacterial protein; however, most of the expressed proteins were produced in an insoluble form. The recombinant CKII alpha subunit was purified by DEAE-cellulose chromatography, followed by phosphocellulose and heparin-agarose chromatography. The recombinant CKII beta subunit was extracted from the insoluble pellet and purified in a single step on phosphocellulose. From 10 g bacterial cells, the yield of soluble protein was 12 mg alpha subunit and 5 mg beta subunit. SDS/PAGE analysis of the purified recombinant proteins indicated molecular masses of 42 kDa and 26 kDa for the alpha and beta subunits, respectively, in agreement with the molecular masses determined for the subunits of the native enzyme. The recombinant alpha subunit exhibited protein kinase activity which was greatest in the absence of monovalent ions. With increasing amounts of salt, alpha subunit kinase activity declined rapidly. Addition of the beta subunit led to maximum stimulation at a 1:1 ratio of both subunits. Using a synthetic peptide (RRRDDDSDDD) as a substrate, the maximum protein kinase stimulation observed was fourfold under the conditions used. The Km of the reconstituted enzyme for the synthetic peptide (80 microM) was comparable to the mammalian enzyme (40-60 microM), whereas the alpha subunit alone had a Km of 240 microM. After sucrose density gradient analysis, the reconstituted holoenzyme sedimented at the same position as the mammalian CKII holoenzyme.     

Coexpression of both alpha and beta subunits is required for assembly of regulated casein kinase II

Casein kinase II is an ubiquitous serine-threonine kinase whose functional significance and regulation in the living cell are not clearly understood. The native enzyme has an oligomeric structure made of two different (alpha and beta) subunits with an alpha 2 beta 2 stoichiometry. To facilitate the study of the structure-activity relationship of the kinase, we have expressed its isolated subunits in a baculovirus-directed insect cell expression system. The resulting isolated recombinant alpha subunit exhibited a protein kinase catalytic activity, in agreement with previous observations [Cochet, C., & Chambaz, E. M. (1983) J. Biol. Chem. 258, 1403-1406]. Coinfection of insect cells with recombinant viruses encoding the two kinase subunits resulted in the biosynthesis of a functional enzyme. Active recombinant oligomeric kinase was purified to near homogeneity with a yield of about 5 mg of enzymatic protein per liter, showing that, in coinfected host cells, synthesis was followed, at least in part, by recombination of the two subunits with an alpha 2 beta 2 stoichiometry. The catalytic properties of the recombinant enzyme appeared highly similar to those previously observed for casein kinase II purified from bovine tissue. Access to the isolated subunits and to their alpha 2 beta 2 association disclosed that the beta subunit is required for optimal catalytic activity of the kinase. In addition, the beta subunit is suggested to play an essential role in the regulated activity of the native casein kinase II. This is clearly illustrated by the observation of the effect of spermine which requires the presence of the beta subunit to stimulate the kinase catalytic activity which is borne by the alpha subunit.(ABSTRACT TRUNCATED AT 250 WORDS)     

The genes encoding the two carboxyltransferase subunits of Escherichia coli acetyl-CoA carboxylase.

We report characterization of the component proteins and molecular cloning of the genes encoding the two subunits of the carboxyltransferase component of the Escherichia coli acetyl-CoA carboxylase. Peptide mapping of the purified enzyme component indicates that the carboxyltransferase component is a complex of two nonidentical subunits, a 35-kDa alpha subunit and a 33-kDa beta subunit. The alpha subunit gene encodes a protein of 319 residues and is located immediately downstream of the polC gene (min 4.3 of the E. coli genetic map). The deduced amino acid composition, molecular mass, and amino acid sequence match those determined for the purified alpha subunit. Six sequenced internal peptides also match the deduced sequence. The amino-terminal sequence of the beta subunit was found within a previously identified open reading frame of unknown function called dedB and usg (min 50 of the E. coli genetic map) which encodes a protein of 304 residues. Comparative peptide mapping also indicates that the dedB/usg gene encodes the beta subunit. Moreover, the deduced molecular mass and amino acid composition of the dedB/usg-encoded protein closely match those determined for the beta subunit. The deduced amino acid sequences of alpha and beta subunits show marked sequence similarities to the COOH-terminal half and the NH2-terminal halves, respectively, of the rat propionyl-CoA carboxylase, a biotin-dependent carboxylase that catalyzes a similar carboxyltransferase reaction reaction. Several conserved regions which may function as CoA-binding sites are noted.     

Functional properties of recombinant Azospirillum brasilense glutamate synthase, a complex iron-sulfur flavoprotein.

Azospirillum brasilense glutamate synthase is a complex iron-sulfur flavoprotein that catalyses the NADPH-dependent reductive transfer of glutamine amide group to the C(2) carbon of 2-oxoglutarate to yield L-glutamate. Its catalytically active alphabeta protomer is composed of two dissimilar subunits (alpha subunit, 164.2 kDa; beta subunit, 52.3 kDa) and contains one FAD (at Site 1, the pyridine nucleotide site within the beta subunit), one FMN (at Site 2, the 2-oxoglutarate/L-glutamate site in the alpha subunit) and three different iron-sulfur clusters (one 3Fe-4S center on the alpha subunit and two 4Fe-4S clusters of unknown location). A plasmid harboring the gltD and gltB genes, the genes encoding the glutamate synthase beta and alpha subunits, respectively, each one under the control of the T7/lac promoter of pET11a was found to be suitable for the overproduction of glutamate synthase holoenzyme in Escherichia coli BL21(DE3) cells. Recombinant A. brasilense glutamate synthase could be purified to homogeneity from overproducing E. coli cells by ion exchange chromatography, gel filtration and affinity chromatography on a 2',5' ADP-Sepharose 4B column. The purified enzyme was indistinguishable from that prepared from Azospirillum cells with respect to cofactor content, N-terminal sequence of the subunits, aggregation state, kinetic and spectroscopic properties. The study of the recombinant holoenzyme allowed us to establish that the tendency of glutamate synthase to form a stable (alphabeta)4 tetramer at high protein concentrations is a property unique to the holoenzyme, as the isolated beta subunit does not oligomerize, while the isolated glutamate synthase alpha subunit only forms dimers at high protein concentrations. Furthermore, the steady-state kinetic analysis of the glutamate synthase reaction was extended to the study of the effect of adenosine-containing nucleotides. Compounds such as cAMP, AMP, ADP and ATP have no effect on the enzyme activity, while the 2'-phosphorylated analogs of AMP and NADP(H) analogs act as inhibitors of the reaction, competitive with NADPH. Thus, it can be ruled out that glutamate synthase reaction is subjected to allosteric modulation by adenosine containing (di)nucleotides, which may bind to the putative ADP-binding site at the C-terminus of the alpha subunit. At the same time, the strict requirement of a 2'-phosphate group in the pyridine nucleotide for binding to glutamate synthase (GltS) was established. Finally, by comparing the inhibition constants exhibited by a series of NADP+ analogs, the contribution to the binding energy of the various parts of the pyridine nucleotide has been determined along with the effect of substituents on the 3 position of the pyridine ring. With the exception of thio-NADP+, which binds the tightest to GltS, it appears that the size of the substituent is the factor that affects the most the interaction between the NADP(H) analog and the enzyme.     

Rhodopsin and the retinal G-protein distinguish among G-protein beta gamma subunit forms

The beta gamma subunits of G-proteins are composed of closely related beta 35 and beta 36 subunits tightly associated with diverse 6-10 kDa gamma subunits. We have developed a reconstitution assay using rhodopsin-catalyzed guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) binding to resolved alpha subunit of the retinal G-protein transducin (Gt alpha) to quantitate the activity of beta gamma proteins. Rhodopsin facilitates the exchange of GTP gamma S for GDP bound to Gt alpha beta gamma with a 60-fold higher apparent affinity than for Gt alpha alone. At limiting rhodopsin, G-protein-derived beta gamma subunits catalytically enhance the rate of GTP gamma S binding to resolved Gt alpha. The isolated beta gamma subunit of retinal G-protein (beta 1, gamma 1 genes) facilitates rhodopsin-catalyzed GTP gamma S exchange on Gt alpha in a concentration-dependent manner (K0.5 = 254 +/- 21 nM). Purified human placental beta 35 gamma, composed of beta 2 gene product and gamma-placenta protein (Evans, T., Fawzi, A., Fraser, E.D., Brown, L.M., and Northup, J.K. (1987) J. Biol. Chem. 262, 176-181), substitutes for Gt beta gamma reconstitution of rhodopsin with Gt alpha. However, human placental beta 35 gamma facilitates rhodopsin-catalyzed GTP gamma S exchange on Gt alpha with a higher apparent affinity than Gt beta gamma (K0.5 = 76 +/- 54 nM). As an alternative assay for these interactions, we have examined pertussis toxin-catalyzed ADP-ribosylation of the Gt alpha subunit which is markedly enhanced in rate by beta gamma subunits. Quantitative analyses of rates of pertussis modification reveal no differences in apparent affinity between Gt beta gamma and human placental beta 35 gamma (K0.5 values of 49 +/- 29 and 70 +/- 24 nM, respectively). Thus, the Gt alpha subunit alone does not distinguish among the beta gamma subunit forms. These results clearly show a high degree of functional homology among the beta 35 and beta 36 subunits of G-proteins for interaction with Gt alpha and rhodopsin, and establish a simple functional assay for the beta gamma subunits of G-proteins. Our data also suggest a specificity of recognition of beta gamma subunit forms which is dependent both on Gt alpha and rhodopsin. These results may indicate that the recently uncovered diversity in the expression of beta gamma subunit forms may complement the diversity of G alpha subunits in providing for specific receptor recognition of G-proteins.     

Removal of a frameshift between the hsdM and hsdS genes of the EcoKI Type IA DNA restriction and modification system produces a new type of system and links the different families of Type I systems

The EcoKI DNA methyltransferase is a trimeric protein comprised of two modification subunits (M) and one sequence specificity subunit (S). This enzyme forms the core of the EcoKI restriction/modification (RM) enzyme. The 3′ end of the gene encoding the M subunit overlaps by 1 nt the start of the gene for the S subunit. Translation from the two different open reading frames is translationally coupled. Mutagenesis to remove the frameshift and fuse the two subunits together produces a functional RM enzyme in vivo with the same properties as the natural EcoKI system. The fusion protein can be purified and forms an active restriction enzyme upon addition of restriction subunits and of additional M subunit. The Type I RM systems are grouped into families, IA to IE, defined by complementation, hybridization and sequence similarity. The fusion protein forms an evolutionary intermediate form lying between the Type IA family of RM enzymes and the Type IB family of RM enzymes which have the frameshift located at a different part of the gene sequence.     

Nitrile hydratase of Rhodococcus sp. N-774. Purification and amino acid sequences

The nitrile hydratase of Rhodococcus sp. N-774 was purified and crystallized. The enzyme is composed of two different subunits (molecular masses: subunit alpha, 28,500 Da; subunit beta, 29,000 Da). The amino-terminal amino acid sequence of each subunit was determined. There is no sequence homology between the two subunits, suggesting that the peptides originate from different cistrons. The activity of the purified enzyme did not decrease during incubation in the dark, whereas it gradually decreased in intact cells.     

DNA-methyltransferase SsoII as a bifunctional protein: Features of the interaction with the promoter region of SsoII restriction-modification genes

DNA duplexes bearing an aldehyde group at the 2'-position of the sugar moiety were used for affinity modification of (cytosine-5)-DNA methyltransferase SsoII. It is shown that lysine residues of M.SsoII N-terminal region are located in proximity to DNA sugar-phosphate backbone of a regulatory sequence of promoter region of SsoII restriction-modification enzyme coding genes. The ability of the two M.SsoII subunits to interact with DNA regulatory sequence has been demonstrated by affinity modification using DNA duplexes with two 2'-aldehyde groups. Changes in nucleotide sequence of one half of the regulatory region prevented cross-linking of the second M.SsoII subunit. The results on sequential affinity modification of M.SsoII by two types of modified DNA ligands (i.e. by 2'-aldehyde-containing and phosphoryldisulfide-containing) have demonstrated the possibility of covalent attachment of the protein to two different DNA recognition sites: regulatory sequence and methylation site.