Polydispersity of Serratia marcescens nuclease at optimal pH values]

Treatment with dimethyl suberimidate, a cross-linking bifunctional agent, showed that Sm1 and Sm2 nucleases of Serratia marcescens B10M1 are polydisperse in solution and consist of monomers and dimers at the level of pH optimal for the enzyme activity. The data suggest that nucleases from the strain B10M1 and any other strain are polydisperse at pH optimum if their amino acid sequences are identical.     

The isolation of nucleases Sm1 and Sm2 in preparative amounts and a study of their antigenicity]

Two enzyme forms of endonuclease (Sm 1 and Sm 2) strain B10M1 in 60 and 100 mg respectively have been isolated from the culture fluid Serratia marcescens. The chromatographic and electrophoretic properties and N-terminal amino acid residues are different for both enzymes. The purification procedure consists of dialysis and ion-exchange chromatography on DEAE- and phosphocellulose. The yield of nucleases Sm1 and Sm2 are 14% and 28% respectively. The antigenic differences of nucleases Sm1 and Sm2 have been found by cross immunoenzyme analysis.     

Isolation of isoforms and crystallization of endonucleases of Serratia marcescens

Two isoforms of an extracellular endonuclease, nuclease Sm1 and nuclease Sm2, were isolated from the culture filtrate of Serratia marcescens strain B10 M1 by the ligand-exchange chromatography on iminodiacetate-agarose in Cu2(+)-form, and chromatography on phosphocellulose and DEAE-Toyopearl 650S. The pI for nucleases Sm1 and Sm2 were found to be 7.1 and 6.7, respectively. The amino acid analysis and N-terminal amino acid sequencing of the proteins showed a significant degree of homology between the enzymes. The secondary structure of nuclease Sm2 was calculated. Crystals of nuclease Sm2 were obtained with the space group P2(1)2(1)2(1), a 69.0; b 106.7; c 74.8 A.     

Two isoforms of Serratia marcescens nuclease. Crystallization and preliminary X-ray investigation of the enzyme.

Two isoforms of an extracellular endonuclease, nucleases Sm1 and Sm2, were purified from culture fluid of Serratia marcescens strain BIO MI by ligand-exchange chromatography on phosphocellulose and DEAE-Toyopearl 650S. The pI-values for nucleases Sm1 and Sm2 were found to be 7.1 and 6.7, respectively. The amino acid analysis and N-terminal amino acid sequencing of the proteins showed a significant degree of homology between the enzymes. The nuclease Sm1 has been crystallized from ammonium sulfate solution by the vapour diffusion technique. The crystals belong to the space group P2(1)2(1)2(1) with unit cell constants a = 69.0, b = 106.7, c = 74.8 A, contain two molecules in an asymmetric unit, packing density Vm = 2.3 A/Da, and diffract to at least 1.5 A resolution. The Pt- and UO2-derivatives of the protein were obtained. Preliminary X-ray investigation of nuclease Sm2 crystals was carried out.     

Purification and Partial Characterization of the B Subunit of Serratia marcescens Tryptophan Synthetase

A trpE mutant of Serratia marcescens (E-7) was isolated, and the multimeric enzyme tryptophan synthetase (EC 4.2.1.20) was purified to homogeneity from derepressed cells. The A and B subunits were resolved, and the B subunit was partially characterized and compared with the Escherichia coli B subunit as part of a comparative evolution study of the trpB cistron of the trp operon in the Enterobacteriaceae. The S. marcescens B subunit is a dimer (beta(2)), and its molecular weight was estimated to be 89,000. The separate subunits (beta monomers) had molecular weights of approximately 43,000. The B subunit required pyridoxal phosphate for catalytic activity and had an apparent K(m) of 9 x 10(-6) M. The N terminus of the B subunit was unavailable for reaction with terminal amine reagents (blocked), whereas carboxypeptidase digestion released a C-terminal isoleucine. Using S. marcescens B antiserum in agar immunodiffusion gave an almost complete reaction of identity between the B subunits of S. marcescens and E. coli. The antiserum was used in microcomplement fixation, allowing for a comparison of the overall antigenic surface structure of the two B subunits. The index of dissimilarity for the heterologous E. coli enzyme compared with the homologous S. marcescens enzyme was 2.4, indicating extensive similarity of the two proteins at their surfaces. Comparative antiserum neutralization of B-subunit enzyme activity showed the E. coli enzyme to cross-react 85% as well as the S. marcescens enzyme. With regard to the biochemical and immunochemical parameters used in this study, the S. marcescens and E. coli B subunits were either identical or very similar. These findings support the idea that the trpB cistron of the trp operon is a relatively conserved gene in the Enterobacteriaceae.     

Comparative studies of genes encoding thermostable L-2-halo acid dehalogenase from Pseudomonas sp. strain YL, other dehalogenases, and two related hypothetical proteins from Escherichia coli.

We have determined the nucleotide sequence of the gene encoding thermostable L-2-halo acid dehalogenase (L-DEX) from the 2-chloroacrylate-utilizable bacterium Pseudomonas sp. strain YL. The open reading frame consists of 696 nucleotides corresponding to 232 amino acid residues. The protein molecular weight was estimated to be 26,179, which was in good agreement with the subunit molecular weight of the enzyme. The gene was efficiently expressed in the recombinant Escherichia coli cells: the amount of L-DEX corresponds to about 49% of the total soluble proteins. The predicted amino acid sequence showed a high level of similarity to those of L-DEXs from other bacterial strains and haloacetate dehalogenase H-2 from Moraxella sp. strain B (38 to 57% identity) but a very low level of similarity to those of haloacetate dehalogenase H-1 from Moraxella sp. strain B (10%) and haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 (12%). By searching the protein amino acid sequence database, we found two E. coli hypothetical proteins similar to the Pseudomonas sp. strain YL L-DEX (21 to 22%).     

Properties of D-arabinose isomerase purified from two strains of Escherichia coli

d-Arabinose isomerase (EC 5.3.1.3) has been isolated from l-fucose-induced cultures of Escherichia coli K-12 and d-arabinose-induced cultures of E. coli B/r. Both enzymes were homogeneous in an ultracentrifuge and migrated as single bands upon disc electrophoresis in acrylamide gels. The s(20,w) was 14.5 x 10(-13) sec for the E. coli K-12 enzyme and 14.3 x 10(-13) sec for the E. coli B/r enzyme. The molecular weight, determined by high-speed sedimentation equilibrium, was 3.55 +/- 0.06 x 10(5) for the E. coli K-12 enzyme and 3.42 +/- 0.04 x 10(5) for the enzyme isolated from E. coli B/r. Both enzyme preparations were active wth l-fucose or d-arabinose as substrates and showed no activity on any of the other aldopentoses or aldohexoses tested. With the E. coli K-12 enzyme, the K(m) was 2.8 x 10(-1)m for d-arabinose and 4.5 x 10(-2)m for l-fucose; with the E. coli B/r enzyme, the K(m) was 1.7 x 10(-1)m for d-arabinose and 4.2 x 10(-2)m for l-fucose. Both enzymes were inhibited by several of the polyalcohols tested, ribitol, l-arabitol, and dulcitol being the strongest. Both enzymes exhibited a broad plateau of optimal catalytic activity in the alkaline range. Both enzymes were stimulated by the presence of Mn(2+) or Co(2+) ions, but were strongly inhibited by the presence of Cd(2+) ions. Both enzymes were precipitated by antisera prepared against either enzyme preparation. The amino acid composition for both proteins has been determined; a striking similarity has been detected. Both enzymes could be dissociated, by protonation at pH 2 or by dialysis against buffer containing 8 m urea, into subunits that were homogeneous in an ultracentrifuge and migrated as single bands on disc electrophoresis in acrylamide gels containing urea. The molecular weight of the subunit, determined by high-speed sedimentation equilibrium, was 9.09 +/- 0.2 x 10(4) for the enzyme from E. coli K-12 and 8.46 +/- 0.1 x 10(4) for the enzyme from E. coli B/r. On the basis of biophysical studies, both isomerases appear to be oligomeric proteins consisting of four identical subunits.     

Identification of catalytically relevant amino acids of the extracellular Serratia marcescens endonuclease by alignment-guided mutagenesis.

By sequence alignment of the extracellular Serratia marcescens nuclease with three related nucleases we have identified seven charged amino acid residues which are conserved in all four sequences. Six of these residues together with four other partially conserved His or Asp residues were changed to alanine by site-directed PCR-mediated mutagenesis using a variant of the nuclease gene in which the coding sequence of the signal peptide was replaced by the coding sequence for an N-terminal affinity tag [Met(His)6GlySer]. Four of the mutant proteins showed almost no reduction in nuclease activity but five displayed a 10- to 1000-fold reduction in activity and one (His110Ala) was inactive. Based upon these results it is suggested that the S.marcescens nuclease employs a mechanism in which His110 acts in concert with a Mg2+ ion and three carboxylates (Asp107, Glu148 and Glu232) as well as one or two basic amino acid residues (Arg108, Arg152).     

Site-directed mutagenesis studies to probe the role of specific residues in the external loop (L3) of OmpF and OmpC porins in susceptibility of Serratia marcescens to antibiotics

Serratia marcescens is a nosocomial bacterium with natural resistance to a broad spectrum of antibiotics, making treatment challenging. One factor contributing to this natural antibiotic resistance is reduced outer membrane permeability, controlled in part by OmpF and OmpC porin proteins. To investigate the direct role of these porins in the diffusion of antibiotics across the outer membrane, we have created an ompF-ompC porin-deficient strain of S. marcescens. A considerable similarity between the S. marcescens porins and those from other members of Enterobacteriaceae was detected by sequence alignment, with the exception of a change in a conserved region of the third external loop (L3) of the S. marcescens OmpC protein. Serratia marcescens OmpC has aspartic acid instead of glycine in position 112, methionine instead of aspartic acid in position 114, and glutamine in position 124, while in S. marcescens OmpF this is a glycine at position 124. To investigate the role of amino acid positions 112, 114, and 124 and how the observed changes within OmpC porin may play a part in pore permeability, 2 OmpC sites were altered in the Enterobacteriaceae consensus (D112G and M114D) through site-directed mutagenesis. Also, Q124G in OmpC, G124Q in OmpF, and double mutants of these amino acid residues were constructed. Antibiotic accumulation assays and minimal inhibitory concentrations of the strains harboring the mutated porins were performed, while liposome swelling experiments were performed on purified porins. Our results demonstrate that the amino acid at position 114 is not responsible for either antibiotic size or ionic selection, the amino acid at position 112 is responsible for size selection only, and position 124 is involved in both size and ionic selection.     

The lipA gene of Serratia marcescens which encodes an extracellular lipase having no N-terminal signal peptide.

The lipA gene encoding an extracellular lipase was cloned from the wild-type strain of Serratia marcescens Sr41. Nucleotide sequencing showed a major open reading frame encoding a 64.9-kDa protein of 613 amino acid residues; the deduced amino acid sequence contains a lipase consensus sequence, GXSXG. The lipase had 66 and 56% homologies with the lipases of Pseudomonas fluorescens B52 and P. fluorescens SIK W1, respectively, but did not show any overall homology with lipases from other origins. The Escherichia coli cells carrying the S. marcescens lipA gene did not secrete the lipase into the medium. The S. marcescens lipase had no conventional N-terminal signal sequence but was also not subjected to any processing at both the N-terminal and C-terminal regions. A specific short region similar to the regions of secretory proteins having no N-terminal signal peptide was observed in the amino acid sequence. Expression of the lipA gene in S. marcescens was affected by the carbon source and the addition of Tween 80.     

Identification and characterization of heparan sulfate-binding proteins from human lung carcinoma cells

The heparan sulfate proteoglycan/heparin-binding proteins of the human lung carcinoma cell line LX-1 have been identified, partially purified, and characterized. Analysis of the binding of [3H]heparin to membranes isolated from LX-1 cells indicated the presence of two classes of binding sites, with Kd values of approximately 2 x 10(-10) and 4 x 10(-8) M and corresponding Bmax values of 1 x 10(5) and 2 x 10(7) binding sites/cell. Binding was also observed with isolated heparan sulfate chains and with intact heparan sulfate proteoglycan isolated from two different cell types. With each ligand, binding was inhibited by addition of unlabeled heparin. The binding proteins were extracted from LX-1 cell membranes in detergent solution, and two size classes of binding proteins were identified by overlaying transblots of electrophoretically separated proteins with radioactive ligands. These two classes of binding proteins were shown to contain doublets with estimated molecular masses of approximately 16 kDa (HSBP1A and HSBP1B) and approximately 32 kDa (HSBP2A and HSBP2B). The proteins were partially purified by heparin-Sepharose chromatography and shown to bind heparin and heparan sulfate proteoglycan. By amino acid composition, N-terminal amino acid sequence, and reactivity with antibody, HSBP1A was shown to be very similar to histone 2B; HSBP1B may also be related to histone 2A. HSBP2A and HSBP2B, however, did not react with antibodies to the major histones and had compositions different from one another and from HSBP1.     

Use of the Rhodopseudomonas palustris genome sequence to identify a single amino acid that contributes to the activity of a coenzyme A ligase with chlorinated substrates

Rhodopseudomonas palustris strain RCB100 degrades 3-chlorobenzoate (3-CBA) anaerobically. We purified from this strain a coenzyme A ligase that is active with 3-CBA and determined its N-terminal amino acid sequence to be identical to that of a cyclohexanecarboxylate-CoA ligase encoded by aliA from the R. palustris strain (CGA009) that has been sequenced. Strain CGA009 differs from strain RCB100 in that it does not use 3-CBA as a sole carbon source. The aliA gene from the 3-CBA degrading strain differed by a single nucleotide from the aliA gene from strain CGA009, causing the substitution of a serine for a threonine at position 208. Both AliA enzymes, purified as His-tagged fusion proteins, had comparable activities with cyclohexanecarboxylate. However, AliA from the 3-CBA degrading strain was 10-fold more active with 3-CBA (kcat/Km of 4.3 x 10(4) M(-1) s(-1)) than the enzyme from the sequenced strain (kcat/Km 0.32 x 10(4) M(-1) s(-1)). The CGA009 enzyme was not sufficiently active with 3-CBA to complement an RCB100 aliA mutant for growth on this compound. Here, whole genome sequence information enabled us to identify a single nucleotide among 5.4 million nucleotides that contributes to the substrate preference of a coenzyme A ligase.     

Chloroplast leucyl-tRNA synthetase from Euglena gracilis. Purification, kinetic analysis, and structural characterization

Euglena gracilis chloroplast leucyl-tRNA synthetase was purified to homogeneity by a series of steps including ammonium sulfate precipitation and chromatography on hydroxylapatite, DEAE-cellulose, Sepharose 6B, phosphocellulose, and Blue Dextran-Sepharose. The purified enzyme exhibits a specific activity of 1233 units/mg of protein, which is one of the highest specific activities obtained for an aminoacyl-tRNA synthetase prepared from plant cells. The enzyme has an apparent Km value of 8 x 10(-6) M for L-leucine, 1.3 x 10(-4) M for ATP, and 1.3 x 10(-6) M for tRNALeu. Chloroplast leucyl-tRNA synthetase appears to be a monomeric enzyme with a molecular weight of 100 000. The amino acid composition of chloroplast leucyl-tRNA synthetase has been determined. It is the first reported for a chloroplast aminoacyl-tRNA synthetase, and it reveals a relatively large proportion of apolar residues, as in the case of prokaryotic aminoacyl-tRNA synthetases.     

Identification and sequence analysis of the soxB gene essential for sulfur oxidation of Paracoccus denitrificans GB17.

The coding region for lithotrophic sulfur oxidation (Sox) in Paracoccus denitrificans GB17 was identified by isolation of a transposon Tn5-mob mutant with a Sox- phenotype (strain TP19). The corresponding wild-type region was cloned previously (G. Mittenhuber, K. Sonomoto, M. Egert, and C. G. Friedrich, J. Bacteriol. 173:7340-7344, 1991). Sequence analysis of a 2.5-kb subclone that complemented strain TP19 revealed that Tn5-mob was inserted into a coding region for a 553-amino-acid polypeptide named SoxB. This polypeptide had an M(r) of 60.573, including a possible signal peptide. The function of the SoxB protein of P. denitrificans GB17 appeared to be identical to that of enzyme B of the thiosulfate-oxidizing enzyme system of Thiobacillus versutus. The amino acid compositions of the two proteins were identical, and the amino acid sequences of three internal peptides of enzyme B as determined by Edman degradation were identical to corresponding sequences of the deduced SoxB protein of P. denitrificans GB17.     

Purification and characterization of urease from dehusked pigeonpea (Cajanus cajan L) seeds

Urease has been purified from the dehusked seeds of pigeonpea (Cajanus cajan L.) to apparent electrophoretic homogeneity with approximately 200 fold purification, with a specific activity of 6.24 x10(3) U mg(-1) protein. The enzyme was purified by the sequence of steps, namely, first acetone fractionation, acid step, a second acetone fractionation followed by gel filtration and anion-exchange chromatographies. Single band was observed in both native- and SDS-PAGE. The molecular mass estimated for the native enzyme was 540 kDa whereas subunit values of 90 kDa were determined. Hence, urease is a hexamer of identical subunits. Nickel was observed in the purified enzyme from atomic absorption spectroscopy with approximately 2 nickel ions per enzyme subunit. Both jack bean and soybean ureases are serologically related to pigeonpea urease. The amino acid composition of pigeonpea urease shows high acidic amino acid content. The N-terminal sequence of pigeonpea urease, determined up to the 20th residue, was homologous to that of jack bean and soybean seed ureases. The optimum pH was 7.3 in the pH range 5.0-8.5. Pigeonpea urease shows K(m) for urea of 3.0+/-0.2 mM in 0.05 M Tris-acetate buffer, pH 7.3, at 37 degrees C. The turnover number, k(cat), was observed to be 6.2 x 10(4) s(-1) and k(cat)/K(m) was 2.1 x 10(7) M(-1) s(-1). Pigeonpea urease shows high specificity for its primary substrate urea.     

Comparison of the Amino Acid Sequence and Phylogenetic Analysis of the Peplomer,Integral Membrane and Nucleocapsid Proteins of Feline,Canine and Porcine Coronaviruses

Complete nucleotide sequences were determined by cDNA cloning of peplomer (S), integral membrane (M) and nucleocapsid (N) genes of feline infectious peritonitis virus (FIPV) type I strain KU-2, UCD1 and Black, and feline enteric coronavirus (FECV) type II strain 79–1683. Only M and N genes were analyzed in strain KU-2 and strain 79–1683, which still had unknown nucleotide sequences. Deduced amino acid sequences of S, M and N proteins were compared in a total of 7 strains of coronaviruses, which included FIPV type II strain 79–1146, canine coronavirus (CCV) strain Insavc-1 and transmissible gastroenteritis virus of swine (TGEV) strain Purdue. Comparison of deduced amino acid sequences of M and N proteins revealed that both M and N proteins had an identity of at least 90% between FIPV type I and type II. The phylogenetic tree of the M and N protein-deduced amino acid sequences showed that FIPV type I and type II form a group with FECV type II, and that these viruses were evolutionarily distant from CCV and TGEV. On the other hand, when the S protein-deduced amino acid sequences was compared, identity of only about 45% was found between FIPV type I and type II. The phylogenetic tree of the S protein-deduced amino acid sequences indicated that three strains of FIPV type I form a group, and that it is a very long distance from the FIPV type II, FECV type II, CCV and TGEV groups.     

Characterization of a purified beta-fructofuranosidase from Bifidobacterium infantis ATCC 15697

AIMS: To characterize the beta-fructofuranosidase of Bifidobacterium infantis ATCC 15697 and to compare it with other bacterial beta-fructofuranosidases. METHODS AND RESULTS: The beta-fructofuranosidase of B. infantis ATCC 15697 was purified 46.8 times over the crude extract by anion exchange chromatography, ultrafiltration and gel filtration. The sequence of 15 amino acid residues of the NH2 terminal was determined. This enzyme was a monomeric protein (Mr 70 kDa) with beta-fructofuranosidase and invertase activities. The isoelectric point was pH 4.3, the optimum pH 6.0 and pKas (4.5 and 7.2) of two active groups were obtained. The activities were inhibited by Hg2+ and p-chloromercuribenzoic acid (pCMB). The optimal temperature was 37 degrees C and activities were unstable at 55 degrees C. beta-fructofuranosidase activity was more efficient than that of invertase with Vm/Km ratios of 0.65 and 0.025 x 10-3 l min(-1) mg(-1), respectively. The enzyme catalyses the hydrolysis of fructo-oligosaccharides, sucrose and inulin at relative velocities of 100, 10 and 6, respectively. CONCLUSIONS: The enzyme of B. infantis ATCC 15697 is an exo-inulinase which has beta-fructofuranosidase and invertase activities. This protein was different from the beta-fructofuranosidase of another strain of B. infantis (B. infantis JCM no. 7007). SIGNIFICANCE AND IMPACT OF THE STUDY: A better knowledge of bacterial beta-fructofuranosidases, especially from bifidobacteria, has been gained.     

Guinea-pig liver leukotriene A4 hydrolase. Purification, characterization and structural properties

Leukotriene A4 hydrolase from perfused guinea-pig liver was purified 1200-fold to near homogeneity with a yield of about 20%. Apparent values of Km and Vmax at 37 degrees C (27 microM and 68 mumol x mg-1 x min-1), turnover number, and activation energy for the conversion of leukotriene A4 into leukotriene B4 were estimated from kinetic data obtained at -10 degrees C, 0 degree C and +10 degrees C (Arrhenius plots). Physical properties including Mr (67,000-71,000), pH optimum, isoelectric point and Stokes' radius were determined. The amino acid composition and N-terminal amino acid sequence were established after carboxymethylation of the enzyme. Unlike liver cytosolic epoxide hydrolase, the purified enzyme did not catalyze the conversion of leukotriene A4 into (5S,6R)-5,6-dihydroxy-7,9-trans-11,14-cis-icosatetraenoic acid.     

Amino acids in anthers of Milo and in cytoplasmic genetic male sterile sorghums (Sorghum bicolor L. Moench) of Indian origin

Summary Amino acid composition of proteins from anthers of milo and Indian origin male steriles were determined. Comparison of amino acid between A and B lines showed lower contents of histidine, threonine, glutamic acid, glycine, leucine and phenylalanine and higher contents of alanine, serine, proline and tyrosine in line A compared to line B. Alanine content in anthers of A lines was more than two fold higher than that in the anthers from B lines. Marked differences in amino acid composition of anthers of A and B lines are suggestive of their involvement in male sterility. Cytoplasmic male steriles of Indian origin M35-1A and M31-2A showed greater similarity but differed from milo, VZM2A and B.     

Isolation and characterization of nucleases from a clinical isolate of Serratia marcescens kums 3958

Two new extracellular nucleases, nucleases SM1 and SM2, were purified from the culture fluid of S. marcescens kums 3958, a fresh clinical isolate. The purification was carried out by the following steps; ammonium sulfate precipitation, and DEAE-cellulose and Sephadex G-100 column chromatography. At the final step, nucleases SM1 and SM2 were purified about 3,700- and 1,000-fold, respectively. They were free from phosphomonoesterase and phosphodiesterase activities. The pIs were 8.1 and 7.5 for nucleases SM1 and SM2, respectively. The molecular weight was estimated to be 35,000 for both enzymes by SDS-polyacrylamide disc gel electrophoresis. The results of amino acid analyses showed that both the threonine and serine contents were higher in nuclease SM2 than in SM1. Furthermore, nuclease SM1 was more stable than nuclease SM2 at 4 degrees C. The other properties of the two enzymes were similar; pH optimum (8.0), Mg2+ or Mn2+ for activation, and inhibition by chemical reagents such as EDTA and pyrophosphate. No significant difference was found in base specificity between nucleases SM1 and SM2. Both enzymes specifically degraded double-stranded homopolymers, especially poly(I). poly(C), as well as yeast RNA and calf thymus DNA. They hardly degraded, however, single-stranded homopolymers such as poly(dA), poly(G), and poly(U).