Cloning of the RNase H genes from a metagenomic DNA library: identification of a new type 1 RNase H without a typical active-site motif.

AIMS: The study aimed to combine a metagenomics approach with complementary genetics to identify novel bacterial genes with orthologous functions, with the identification of novel RNase H genes as a test case. METHODS AND RESULTS: A metagenomic DNA library was prepared from leaf-and-branch compost and used to screen for the RNase H genes by their abilities to complement the temperature-sensitive growth phenotype of the rnhA mutant Escherichia coli strain MIC3001. Determination of the nucleotide sequences of the cloned DNA fragments allowed us to identify 12 different genes encoding type 1 RNases H. Eleven of them encode novel RNases H, which show 40-72% amino acid sequence identities to those available from database. One of them lacks a typical DEDD/E active-site motif, which is almost fully conserved in various RNases H. CONCLUSIONS: Functional screening of environmental DNA without cultivation of microbes is a useful procedure to isolate novel RNase H genes. SIGNIFICANCE AND IMPACT OF THE STUDY: One of the identified RNase H genes had no sequence similarity to a previously assumed conserved motif, suggesting multiple catalytic mechanisms exist. This test case illustrates that metagenomics combined with complementary genetics can identify novel genes that are orthologous without sequence similarity to those from cultivated bacteria.

Contribution of hydrophobic residues to the stability of human lysozyme: calorimetric studies and X-ray structural analysis of the five isoleucine to valine mutants.

In order to understand the contribution of hydrophobic residues to the conformational stability of human lysozyme, five Ile mutants (Ile --> Val) in the interior of the protein were constructed. The thermodynamic parameters characterizing the denaturation of these mutant proteins were determined by scanning calorimetry, and the three-dimensional structure of each mutant protein was solved at high resolution by X-ray crystallography. The thermodynamic analyses at 64.9 degrees C and at pH 2.7 revealed the following. (1) The stabilities of all the mutant proteins were decreased as compared with that of the wild-type protein. (2) The changes in the calorimetric enthalpies were larger than those in the Gibbs energies, and were compensated by entropy changes. (3) The destabilization mechanism of the mutant proteins differs, depending on the location of the mutation sites. X-ray analyses showed that the overall structures of all the mutant human lysozymes examined were identical to that of the wild-type protein, and only small structural rearrangements were observed locally around some of the mutation sites. The most striking change among the mutant proteins was found in the mutant protein, 159V, which contains a new water molecule in the cavity created by the mutation. The thermodynamic stabilities of the mutant proteins are discussed in light of the high-resolution X-ray structures of the wild-type and five mutant human lysozymes examined.

Synthesis and secretion of human nerve growth factor by Saccharomyces cerevisiae.

The DNA coding for human nerve growth factor (hNGF) was chemically synthesized and introduced into Saccharomyces cerevisiae. Expression and secretion of hNGF was obtained by use of the yeast phosphoglycerate kinase-encoding gene promoter and the pre-pro sequence of the yeast alpha-mating factor. Immunoblotting with antiserum raised against a protein A-hNGF fusion protein, allowed the detection of an immunoreactive material secreted into the culture medium. A preparation from the culture medium, partially purified by ion-exchange column chromatography, stimulated neurite outgrowth from rat pheochromocytoma PC12h cells.

Kinetic analyses of DNA-linked ribonucleases H with different sizes of DNA.

A series of DNA-linked ribonucleases H with DNA adducts varying in size and sequence, ranging from heptamer to nonamer, were constructed and examined for their ability to cleave the 12-base RNA (5'-CGGAGAUGACGG-3') site-specifically. The DNA-linked RNase H with the 9-base DNA (5'-GTCATCTCC-3') cleaved the 12-base RNA specifically at A6-U7. Kinetic studies revealed that the DNA-linked RNase H with the 8-base DNA (5'-TCATCTCC-3') cleaved it slightly more effectively than that with the 9-base DNA. Factors that may affect the specificity and catalytic efficiency of a DNA-linked RNase H are described.

Reconstitution of Escherichia coli RNase HI from the N-fragment with high helicity and the C-fragment with a disordered structure.

The Escherichia coli RNase HI variant with the Lys86-->Ala mutation is purified in two forms, as nicked and intact proteins. The nicked K86A protein, in which the N-fragment (Met1-Lys87) and the C-fragment (Arg88-Val155) remain associated, is enzymatically active. These N- and C-fragments were isolated and examined for reassociation. These peptides did not associate to form the nicked K86A protein at pH 3.0 in the absence of salt, but were associated, with a yield of 30-80%, when the pH was raised to 5.5 or when salt was added. Measurements of the CD spectra show that the alpha-helices are partially formed in the N-fragment at pH 3.0 in the absence of salt and are almost fully formed either at pH 5.5 or at pH 3.0 in the presence of 0.15 M NaCl. In contrast, the C-fragment remains almost fully disordered under these conditions. The N-fragment with this high (native-like) helicity shows the characteristics of a molten globule with respect to the content of the secondary and tertiary structures, the ability to bind a fluorescent probe (1-anilinonaphthalene-8-sulfonic acid), and the behavior on the thermal transition. These results suggest that the N-fragment contains an initial folding site, probably the alpha I-helix, and the completion of the folding in this site provides a surface that facilitates the folding of the C-fragment. This folding process may represent that of the intact RNase HI molecule.

Folding of human lysozyme in vivo by the formation of an alternative disulfide bond.

The mutant h-lysozyme, W64CC65A, with Trp64 and Cys65 replaced by Cys and Ala, respectively, was secreted by yeast and purified. Peptide mapping confirmed that W64CC65A contained a nonnative Cys64-Cys81 bond and three native disulfide bonds. The mutant had 2% of the lytic activity of the wild-type lysozyme. The midpoint concentration of the guanidine hydrochloride denaturation curve, the [D]1/2, was 2.7 M for W64CC65A at pH 3.0 and 25 degrees C, whereas the [D]1/2 for the wild-type h-lysozyme was 2.9 M. These results show that the W64CC65A protein is a compactly folded molecule. Our previous results, using the mutant C81A, indicate that Cys81 is not required for correct folding and activity, whereas Cys65 is indispensable (Taniyama, Y., Yamamoto, Y., Kuroki, R., and Kikuchi, M. (1990) J. Biol. Chem. 65, 7570-7575). Cys64 substituted for Cys65 in W64CC65A, even though the distance between the alpha-carbons at positions 64 and 81 in the wild-type h-lysozyme is not favorable for forming a disulfide bond. Unlike C81A, the mutant W64CC65/81A, which has the additional substitution of Ala for Cys81, did not fold. These results suggest that the absence of both the Cys64-Cys81 bond and the amino acid residue Trp64 caused the misfolding or destabilization of W64CC65/81A in vivo. It is proposed that the formation of the alternative bond, Cys64-Cys81 is important for the folding of W64CC65A in vivo.

Architecture of models for prebiotic synthesis of nucleic acids: template-directed synthesis of oligoguanylates using N-cyanoimidazole.

N-Cyanoimidazole is an efficient condensing agent for the polymerization of guanosine 5'-phosphate (pG) on a poly(C) template in an aqueous solution. At 0 degree C, up to about 30% of input pG was converted to a mixture of oligomers with a mean chain length of up to 7. The effect of divalent metal ions in the polymerization of pG on a poly(C) template was not so considerable as in that of oligo(A) on a poly(U) template. In the polymerization of pG, the moderate yields were obtaind in the presence of Co2+, Ni2+ and Cu2+.

Template-directed synthesis of oligoadenylates using cyanogen bromide and diiminosuccinonitrile.

Cyanogen bromide was found to be a highly effective reagent for the coupling of oligoadenylates on a polyuridylic acid template in aqueous solution. Imidazole and divalent metal ions were required for the reaction. Hexaadenylic acid was converted to (pA)12, (pA)18, (pA)24, (pA)30 and (pA)36 in the presence of 0.01 M Co2+ and imidazole. When the reaction was carried out at 25 degrees C for 3 days, the oligomers were obtained in a 34% overall yield.

Template-directed polymerization of oligoadenylates using cyanogen bromide.

Cyanogen bromide (BrCN) condensed oligoadenylates [oligo(A)] on a poly(uridylic acid) [poly(U)] template in an aqueous solution. Imidazole and divalent metal ions such as Mn2+, Co2+, Ni2+, Cu2+, Zn2+, Mg2+, and Fe2+ were required for the condensation. Chain length of oligo(A) and reaction temperature affected the coupling yield. Hexaadenylate [(pA)6] was converted to (pA)12, (pA)18, (pA)24, (pA)30, (pA)36, (pA)42, and (pA)48 in a 68% overall yield for 20 h at 25 degrees C. The coupling yield increased with increase in the poly(U) concentration. Five- to sevenfold molar excess of uridylyl residues of poly(U) to adenylyl residues of oligo(A) gave the best yield (68%). Metal ions affected the formation of linkage isomers of the phosphate bonds: The 2',5'- and 3',5'-phosphodiester bonds were predominant in the presence of Co2+, Zn2+, and Ni2+ and the 5',5'-pyrophosphate bond was predominant in the presence of Mn2+. In particular, Ni2+ gave the highest ratio of the 3',5'-phosphodiester bond (30%). N-Cyanoimidazole (1), N,N'-iminodiimidazole (2), and N-carboxamidoimidazole (3) were formed in a reaction of imidazole with BrCN in an aqueous solution. 1 and 2 had much the same condensing activity for the polymerization of adenylates as BrCN. A reaction pathway was proposed in which 1 and 2 are not only intermediates for the production of 3 but also the true condensing agent in the coupling reaction of oligo(A). Phosphorimidazolide derivative was detected in a reaction of 5'-AMP with either 1 or 2. The condensation would proceed by way of N-cyanoimidazole-phosphate adduct, the phosphorimidazolide derivative, or both.

Kinetic analysis of Escherichia coli ribonuclease HI using oligomeric DNA/RNA substrates suggests an alternative mechanism for the interaction between the enzyme and the substrate.

Escherichia coli ribonuclease HI mainly recognizes the DNA/RNA hybrid regions preceding the cleavage site. To understand the interaction between the enzyme and the substrate in more detail, the kinetic properties of the enzyme, as well as its variant with mutations in the basic protrusion, were studied using a series of oligomeric DNA/RNA hybrids as substrates. These substrates were prepared by hybridizing a 12-b RNA (5'-CGGAGAUGACGG-3') with DNA oligomers varying in size and sequence. The 12-b RNA hybridized to the complementary 12-b DNA was primarily cleaved at A9-C10. Since an increase in the length of the RNA between the cleavage site and the 5' end of the DNA/RNA hybrid, achieved using a longer DNA/RNA substrate, did not seriously affect the kinetic parameters of the enzyme, the 12-bp DNA/RNA hybrid seems to be large enough to contact the entire substrate-binding site of the enzyme. The kinetic data presented here suggest that the DNA residues complementary to the RNA residues located six or seven residues upstream from the cleavage site interact with the basic protrusion of the enzyme, regardless of whether or not it is hybridized to the RNA strand. Such an interaction is permitted only when the conformation of either the enzyme or the substrate, or both, is changed upon binding.

Involvement of two sulfur atoms of protein disulfide isomerase and one sulfur atom of the DsbA/PpfA protein in the oxidation of mutant human lysozyme.

Protein disulfide isomerase (PDI) and the DsbA/PpfA protein catalyze the oxidation of mutant human lysozyme, L79CC81A, which has two native disulfide bonds, Cys6-Cys128 and Cys30-Cys116, a non-native Cys79-Cys95, and 2 free cysteine residues at positions 65 and 77. Oxidation of L79CC81A (R-form) yielded two isomers, L79CC81A-a (A-form) with tandem-linked Cys65-Cys77 and Cys79-Cys95, and L79CC81A-b (B-form) with cross-linked Cys65-Cys79 and Cys77-Cys95 (Kanaya, E., Ishihara, K., Tsunasawa, S., Nokihara, K., and Kikuchi, M. (1993) Biochem. J. 292, 469-476). PDI mainly enhanced the formation of the A- form in the absence of oxidized glutathione (GSSG); however, as the concentration of GSSG increased, it markedly accelerated the formation of the B-form. In contrast, the DspA/PpfA protein mainly enhanced the formation of the A-form, regardless of the presence or absence of GSSG. These results and the presumed spatial locations of Cys65, Cys77, and Cys79-Cys95 in the R-form suggest that 1 of the half-cystine residues in the active site of PDI and the DsbA/PpfA protein can react with 1 of the 2 free Cys residues of the R-form. The dependence on GSSG of the B-form formation with PDI can be explained by the formation of two transient intermolecular disulfide bonds between PDI and the R-form and the attack of GSSG by the resultant thiolate anion of Cys79 or Cys95. The independence of the reaction with the DsbA/PpfA protein from GSSG can be explained by the formation of one transient intermolecular disulfide bond. The possible formation of the two transient intermolecular disulfide bonds involving two sulfur atoms of PDI and 2 cysteine or half-cystine residues of the substrate could explain the high isomerase activity of PDI.

Introduction of a non-native disulfide bridge to human lysozyme by cysteine scanning mutagenesis.

To examine whether the disulfide bridge between residues 65 and 81 can be replaced by a non-native disulfide bridge in the mutant h-lysozyme C77/95A and whether the formation of such a new disulfide bridge affects the folding of the protein, cysteine scanning mutagenesis has been performed within two discontinuous segments (residues 61-67 for the mutant C65/77/95A, and 74-84 for the mutant C77/81/95A). The position of the Cys residue at 65 or 81 was continuously shifted by site-directed mutagenesis. Of the mutants, only substitution of Cys for Trp64 allowed the secretion of mutant h-lysozyme(W64C) into the medium in a sufficient amount for analysis. After the purification, the mutant enzyme was obtained as two components (W64C-A and W64C-B). The only difference between A and B was that A had a peptide bond cleaved between Ala77 and His78. A non-native disulfide bridge between residues 64-81 was found in both components. Little difference was observed in CD spectra among wild-type and mutant enzymes. It is likely that the tertiary structure of the W64C mutant might be distorted at the location, because the directions of amino acid side chains at positions of 64 and 81 are shown to be opposite to each other in wild-type h-lysozyme by X-ray crystallographic analysis.

An esterase from Escherichia coli with a sequence similarity to hormone-sensitive lipase.

An esterase from Escherichia coli that is a member of the hormone-sensitive lipase (HSL) family was overproduced, purified and characterized. It is encoded by the ybaC gene and composed of 319 amino acid residues with an Mr of 36038. The enzymic activity was determined by using various p-nitrophenyl esters of fatty acids as a substrate at 25 degreesC and pH 7.1. The enzyme showed hydrolytic activity towards substrates with an acyl chain length of less than 8, whereas it showed little hydrolytic activity towards those with an acyl chain length of more than 10. In addition, it showed little hydrolytic activity towards trioleoylglycerol and cholesterol oleate. Determination of the kinetic parameters for the hydrolyses of the substrates from C2 to C8 indicates that C4 and C5 substrates are the most preferred. Close agreement between the Mr determined by SDS/PAGE (37000) and column chromatography (38000) suggests that the enzyme exists in a monomeric form. It is an acidic protein with a pI value of 4.1. The far-UV CD spectrum suggests that its helical content is 26.1%. Comparison of the amino acid sequence of this enzyme with those involved in the HSL family allows us to propose that Ser165, Asp262 and His292 constitute the catalytic triad of E. coli esterase.

DsbA-DsbB interaction through their active site cysteines. Evidence from an odd cysteine mutant of DsbA.

Formation of disulfide bonds in Escherichia coli envelope proteins is facilitated by the Dsb system, which is thought to consist of at least two components, a periplasmic soluble enzyme (DsbA) and a membrane-bound factor (DsbB). Although it is believed that DsbA directly oxidizes substrate cysteines and DsbB reoxidizes DsbA to allow multiple rounds of reactions, direct evidence for the DsbA-DsbB interaction has been lacking. We examined intracellular activities of mutant forms of DsbA, DsbA30S and DsbA33S, in which one of its active site cysteines (Cys30 or Cys33, respectively) has been replaced by serine. The DsbA33S protein was found to dominantly interfere with the disulfide bonds formation and to form intermolecular disulfide bonds with numerous other proteins when cells were grown in media containing low molecular weight disulfides such as GSSG. In the absence of added GSSG, DsbA33S protein remained specifically disulfide-bonded with DsbB. These in vivo results not only confirm the previous findings that Cys30 of DsbA is hyper-reactive in vitro but provide evidence that DsbA indeed interacts selectively with DsbB. We propose that the Cys30-mediated DsbA-DsbB complex represents an intermediate state of DsbA-DsbB recycling reaction that has been fixed because of the absence of Cys33 on DsbA.

Crystal structure of a mutant human lysozyme with a substituted disulfide bond.

The three-dimensional structure of a mutant human lysozyme, W64CC65A, in which a non-native disulfide bond Cys64--Cys81 is substituted for the Cys65--Cys81 of the wild type protein by replacing Trp64 and Cys65 with Cys and Ala, respectively, was determined by X-ray crystallography and refined to an R-value of 0.181, using 33,187 reflections at 1.87-A resolution. The refined model of the W64CC65A protein consisted of four molecules, which were related by two noncrystallographic twofold axes and a translation vector. Although no specific structural differences could be observed among these four molecules, the overall B-factors of each molecule were quite different. The overall structure of W64CC65A, especially in the alpha-helical domain, was found to be quite similar to that of the wild type protein. Moreover, the side-chain conformation of the newly formed Cys64--Cys81 bond was quite similar to that of the Cys65--Cys81 bond of the wild-type protein. However, in the beta-sheet domain, the main-chain atoms of the loop region from positions 66-75 could not be determined, and significant structural changes due to the formation of the non-native disulfide bond could be observed. From these results, it is clear that the loop region of the mutant protein does not fold with the specific folding as observed in the wild-type protein.

Poly(2-amino-8-methyldeoxyadenylic acid): contrasting effects in deoxy- and ribopolynucleotides of 2-amino and 8-methyl substituents.

Poly(2-amino-8-methyldeoxyadenylic acid) interacts readily with pyrimidine polynucleotides to form double helices only slightly less stable than those in which the purine polymer lacks the 8-Me group. In the ribo series, by contrast, complexes formed with poly(2-amino-8-methyladenylic acid) are very strongly destabilized by the 8-Me group, despite a larger stabilizing effect of the 2-NH2 group in the ribo series. These results are interpreted in terms of a smaller steric interference of the 8-Me group with 2'-CH2 than with 2'-CHOH, leading to a smaller population of syn structures in the deoxy chain and a consequent lower interference with homopolymer duplex formation. UV, circular dichroism (CD), and IR spectra of the new polymer and its complexes are reported and related to structural and energetic characteristics of the molecules. Since direct synthesis of 2-amino-8-methyldeoxyadenosine was not feasible, the corresponding riboside was prepared, the 3'- and 5'-positions were protected with a disilyloxy group, and a 2'-[(imidazol-1-yl)thiocarbonyl] group was introduced. Reduction with tributyltin hydride followed by deprotection gave the nucleoside, which was then converted to the triphosphate by standard methods. The homopolymer was prepared with terminal deoxynucleotidyl transferase.

Poly(8-bromodeoxyadenylic acid): properties of the polymer and contrast with the ribopolynucleotide analogue.

Introduction of the bulky 8-bromo substituent into adenine residues of polynucleotides has strikingly different consequences in the deoxy- and ribopolynucleotide series. Poly(r8BrA) was found in earlier studies to form a very stable double-helical self-structure but not to undergo interaction with potentially complementary polynucleotides. We find that poly(d8BrA), in contrast, does not form an ordered self-structure in 0.1 M Na+ but appears to exist as an electrostatically expanded rigid rod with unusual circular dichroism (CD) properties at very low ionic strength. The deoxy polymer, moreover, readily forms double helices with either deoxy or ribo pyrimidine polynucleotides, studied by UV, CD, and IR spectroscopy. These complexes are destabilized, relative to those formed by poly(dA), possibly because energy is needed to convert the purine residues from a more stable syn to an anti conformation, required for heteroduplex formation. The CD spectrum of (d8BrA)n X (dT)n is similar to that of B DNA. The deoxy-ribo hybrids (d8BrA)n X (rU)n and (d8BrA)n X (rBrU)n have CD spectra resembling those of A DNA or RNA. Unlike other deoxy-deoxy pairs (d8BrA)n X (dBrU)n, however, has a CD spectrum resembling RNA and other helices having the A form.

Stabilization and enhanced enzymatic activities of a mutant human lysozyme C77/95A with a cavity space by amino acid substitution.

Amino acid substitutions were examined to increase the stability of the mutant human lysozyme C77/95A by filling the cavity created by this mutation. To modulate the cavity with hydrophobic amino acids or by the formation of a hydrogen bond, five amino acid-substituted mutants, C77AC95L, C77AC95I, C77LC95A, C77IC95A and C77/95S, were designed and constructed based on computer graphics investigations for stabilizing the mutant protein. The values of the melting temperatures, Tm, at pH 3.0 of the two mutant proteins C77LC95A and C77/95S were increased by 2.9 degrees C and 2.3 degrees C, respectively, as compared to that of C77/95A. The C77IC95A and C77AC95L proteins showed almost the same stability as C77/95A. The increase in the stability of the proteins might be explained by the filling of the cavity space around positions 77 and 95 with the side residue of Leu77 in C77LC95A, and by the formation of a hydrogen bond between Ser77 and Ser95 in C77/95S. On the other hand, the substitution with isoleucine at 95 (C77AC95I) decreased the stability. The activities of the five mutant proteins against the synthetic substrate, p-nitrophenyl tetra-N-acetyl-beta-chitopentaoside, were higher than that of the wild-type human lysozyme, while the lytic activities against M. lysodeikticus were decreased in C77LC95A and C77IC95A, and increased in C77AC95L.

Characterization of the transcriptional activator CBF1 from Arabidopsis thaliana. Evidence for cold denaturation in regions outside of the DNA binding domain.

A transcriptional activator, CBF1, from Arabidopsis thaliana, which has the AP2 domain for DNA binding and regulates the cold acclimation response, was overexpressed in Escherichia coli, purified, and characterized. Analyses of the interaction between CBF1 and the C-repeat/dehydration-responsive element by fluorescence measurement showed that CBF1 binds to C-repeat/dehydration-responsive element as a monomer irrespective of the temperature. CD spectra of the intact and truncated CBF1 proteins (1-213, 41-213, 41-157, and 41-146) were measured to examine the temperature-dependent changes of the secondary structure of CBF1. The results suggested that the CBF1 protein has regions exhibiting reversible cold denaturation in the range between 30 and -5 degrees C and also has a region exhibiting thermal denaturation between 40 and 60 degrees C. This cold denaturation occurred in both the N-terminal and acidic regions. The thermal denaturation occurred in the region encompassing the AP2 domain. The difference between the retention time of CBF1 at 4 degrees C and that at 25 degrees C in gel filtration, and the decrease of the sedimentation coefficient, s20,w, caused by the temperature change from 25 to 3 degrees C, strongly suggested that the cold denaturation was accompanied by the extension of the molecule. The possible cold denaturation observed here might be a physiologically important structural response of CBF1 to cold stress.

Characterization of an artificial dimer of ribonuclease H using 1H NMR spectroscopy.

The protein fusion technique was applied in the synthesis of an artificial dimer of ribonuclease H (305 residues). 1H NMR spectroscopy was used to analyze the structure of this dimer. Spectral profiles and pKa values of the histidine residues obtained using 1H NMR indicate that the dimer retains the secondary and tertiary structures of the intact monomer. Selective spin-lattice relaxation measurements suggest that the two monomeric units in the dimer are in tight contact. Furthermore, the 2D 1H NMR and paramagnetic relaxation filter results show that the two monomers bind together through interactions between the N- and C-terminal sites of the linked regions.