X-ray structural analysis of magnesium-containing Serratia marcescens endonuclease]

The three-dimensional crystal structure of the DNA/RNA nonspecific endonuclease from Serratia marcescens was refined at the resolution of 1.07 A to R factor of 12.4% and Rfree factor of 15.3% using the anisotropic approximation. The structure includes 3924 non-hydrogen atoms, 715 protein-bound water molecules, and a Mg2+ ion in each binding site of each subunit of the nuclease homodimeric globular molecule. The 3D topological model of the enzyme was revealed, the inner symmetry of the monomers in its N- and C-termini was found, and the local environment of the magnesium cofactor in the nuclease active site was defined. Mg2+ ion was found to be bound to the Asn119 residue and surrounded by five associated water molecules that form an octahedral configuration. The coordination distances for the water molecules and the O delta 1 atom of Asn119 were shown to be within a range of 2.01-2.11 A. The thermal factors for the magnesium ion in subunits are 7.08 and 4.60 A2, and the average thermal factors for the surrounding water molecules are 11.14 and 10.30 A2, respectively. The region of the nuclease subunit interactions was localized, and the alternative side chain conformations were defined for 51 amino acid residues of the nuclease dimer.     

Cleavage of lambda DNA by a site-specific endonuclease from Serratia marcescens.

Three sites recognized by SmaI endonuclease, purified from Serratia marcescens SB, have been located on lambda DNA at 0.406, 0.656, and 0.825 fractional lengths from the left end of the DNA molecule.     

Solvent participation in Serratia marcescens endonuclease complexes

The monomer and dimer of the bacterium Serratia marcescens endonuclease (SMnase) are each catalytically active and the two subunits of the dimer function independently of each other. Specific interfacial waters may play a role in stability, complex formation, and functionality. We performed molecular dynamics simulations of both a subunit of SMnase and its model built complex with DNA and analyzed the relation of the hydration sites to the catalytic mechanism. It was found that the binding of DNA has little influence on the global hydration properties of the protein, including occupancy and water residence time distributions. DNA and protein recognition in our model mainly involves direct contacts by hydrogen bond and hydrophobic interactions. Water-mediated contacts exist, but are less common. Three interior water clusters were identified for SMnase. One cluster around the active site in the monomer-DNA complex shows relatively strong interactions between hydration sites as well as between the sites and the biomolecules. The simulated cluster properties agreed well with experimental data. The magnesium ion shows ligand exchange. Although Mg2+ keeps six ligands during the entire simulation, upon the binding of DNA, Asn119 loses its coordination with Mg2+, while one nonbridging oxygen of the phosphate of a DNA residue and two oxygen atoms of the side chain of Glu127 become the ligands. Waters in a nearby cluster exchange and participate in the resolvation of groups in the presence of DNA. Water thus not only participates in the cleavage of DNA but also can stabilize the transition state and the leaving groups in our model.     

Effect of sodium dodecylbenzenesulfonate and antifoaming agents on the endonuclease activity of Serratia marcescens

The effect of sodium dodecylbenzene sulfonate (sulfonol) and certain froth breakers on the activity of endonuclease was studied in the cultural broth of Serratia marcescens in order to find out whether sulfonol could be used for limiting the infection. Sulfonol was found to have no effect on the cultural growth; it increased the activity of endonuclease in the cultural broth, and the peak of the activity appeared earlier than in the control medium. Propanol B-400 was shown to be the best froth breaker.     

Serratia marcescens endonuclease. Properties of the enzyme

Some physico-chemical properties of endonuclease (EC 3.1.4.9) from Serratia marcescens were studied and the amino acid composition of the enzyme was determined. The protein molecule was shown to contain one SH-group and one S-S-bond, which renders it different from the well studied nuclease (EC 3.1.4.7) from Staph. pyogenes. The conditions for reconstitution of the S-S-bond by dithioerythritol for quantitative estimation of cysteine residues of the endonuclease molecule were selected. The N-terminal amino acid was found to be threonine. The UV spectra for the enzyme are typical for proteins; A 0,1% 1cm,280nm is 1.46, epsilon 25 degrees 280nm,pH7,4 is 47292 M-1 cm-1. The sedimentation coefficient in phosphate buffer sW, 20 degrees is 3.4 S, pI is 6.5 and 7.5.     

Isolation and characteristics of intracellular nuclease isoforms from Serratia marcescens]

Two enzyme forms were isolated from the commercial preparation of extracellular endonuclease of Serratia marcescens strain B10 M1. The chromatographic and electrophoretic properties, isoelectric points and N-terminal amino acid residues are different for both enzymes. At the final step of the purification procedure including ion-exchange chromatography on phospho- and DEAE-cellulose columns the yields of nucleases Sm1 and Sm2 were 13% and 25%, respectively. No significant differences were found in the specific activities of nucleases Sm1 and Sm2 (3.6 x 10(6) and 4.0 x 10(6) un. act./mg of protein). A comparative analysis of tryptic nuclease hydrolysate peptides was carried out. The amino acid sequences of some polypeptide segments of the proteins were determined. The structural similarity of the enzyme was established and the amino terminal regions of the proteins were identified. The localization of the disulfide bonds in the molecules of the both nucleases was determined. The similarity of nucleases Sm1 and Sm2 strain B10 M1 to S. marcescens endonucleases obtained from other strains was demonstrated.     

Induction of Yellow Pigmentation in Serratia marcescens

The appearance of yellow pigmentation in nonpigmented strains of Serratia sp. has been demonstrated to be due to the production of a muconic acid, 2-hydroxy-5-carboxymethylmuconic acid semialdehyde. The 3,4-dihydroxyphenylacetate 2,3-dioxygenase responsible for the synthesis of this muconic acid was induced in all strains tested. Another muconic acid, the β-cis-cis-carboxymuconic acid, could also be synthesized from 3,4-dihydroxybenzoate, but this product was not colored. Mutants that were unable to grow on tyrosine and produced yellow pigment were isolated from nonpigmented strains. These mutants had properties similar to those of the yellow-pigmented strains. The ability to produce pigment may be more widespread among Serratia marcescens strains than is currently known.     

Stereochemistry of cleavage of internucleotide bonds by Serratia marcescens endonuclease.

Endonuclease from Serratia marcescens hydrolyzes internucleotide phosphorothioate linkages of R(P) configuration with inversion of configuration at P-atom. This observation supports a reported architecture of the active site, with 3'-bridging and pro-S(P) non-bridging oxygen atoms of the scissile phosphate group involved in direct contact with hydrated magnesium cation, while His-89 activates a water molecule which attacks the phosphorus atom according to a one-step in-line mechanism. The presence of a phosphorothioate bond of S(P) configuration downstream to that one being cleaved reduces the rate of hydrolysis. This suggests participation of the pro-S(P) oxygen atom of that phosphate bond in the mechanism of action of the enzyme, which was not detected in published crystallographic analyses.     

Effect of mitomycin on the biosynthesis of extracellular endonuclease by Serratia marcescens

The effect of mitomycin C on extracellular endonuclease activity of Serratia marcescens was studied. It was shown that in a concentration of 0.02-1.0 micrograms/ml, mitomycin C markedly increased biosynthesis of the endonuclease by growing and washed cells, the cell productivity being increased 80-100 times. The highest increase in the cell productivity was observed when mitomycin C was added to the cells at the end of the growth exponential phase. The increase in the activity of the extracellular endonuclease was due to the de novo synthesis of the enzyme since it was inhibited by chloramphenicol.     

Effects of dimerization of Serratia marcescens endonuclease on water dynamics

The dynamics and structure of Serratia marcescens endonuclease and its neighboring solvent are investigated by molecular dynamics (MD). Comparisons are made with structural and biochemical experiments. The dimer form is physiologic and functions more processively than the monomer. We previously found a channel formed by connected clusters of waters from the active site to the dimer interface. Here, we show that dimerization clearly changes correlations in the water structure and dynamics in the active site not seen in the monomer. Our results indicate that water at the active sites of the dimer is less affected compared with bulk solvent than in the monomer where it has much slower characteristic relaxation times. Given that water is a required participant in the reaction, this gives a clear advantage to dimerization in the absence of an apparent ability to use both active sites simultaneously.     

Regulation of carbamylphosphate synthesis in Serratia marcescens.

Serratia marcescens HY possessed a single carbamylphosphate synthase (CPSase) which was subject to cumulative repression by arginine and a pyrimidine. CPSase did not appear to be a part of a multifunctional enzyme complex as is the case for other enzymes of pyrimidine biosynthesis in this organism. CPSase was purified to homogeneity. The molecular weight of the enzyme was estimated to be 167,000 by sucrose density gradient ultracentrifugation. The double-reciprocal plot for magnesium adenosine triphosphate was linear, yielding a Km value of 2.5 mM. The enzyme utilized either glutamine (Km, 0.1 mM) or NH3 (Km, 10.5 mM) as a nitrogen donor in the reaction. CPSase activity was subject to activation by ornithine and feedback inhibition by uridine monophosphate, as is the case for other enteric bacteria. Carbamate kinase activity, detected in crude extracts of S. marcescens, was shown to be due to a constitutive acetate kinase. The absence of carbamate kinase from S. marcescens HY is consistent with the inability of this organism to utilize arginine as a source of energy under anaerobic conditions.     

Structure of chromatin subunits: an endonuclease Serratia marcescens study.

Electrophoretic properties of chromatin subunits--nucleosomes--obtained by treatment of chromatin with the Serratia marcescens endonuclease have been studied. Double-stranded breaks of DNA between adjacent nucleosomes do not necessarily lead to their disjunction. Fragmentation of the DNA within the nucleosomes may proceed simultaneously with the breakdown of the DNA between the nucleosomes at early stages of the endonuclease digestion. Electrophoretic mobility and chromatographic properties of mononucleosomes, their dimers and trimers with internally degraded DNA was not changed. It has been deduced that the integrity of chromatin particles with internally fragmented DNA is supported by histone interaction inside and between the nucleosomes.     

Isolation of Serratia marcescens mutants superproducers of endonuclease by exposure to nitrosomethylurea in a synchronized culture

The work was aimed at studying the capability of chemical mutagens, such as hydroxylamine (HA), dimethyl sulfate (DMS) and nitrosomethylurea (NMU), to produce mutants of Serratia marcescens with an elevated synthesis of endonuclease. HA and DMS did not induce these mutations at tested concentrations. NMU caused such mutations resulting in a sharp rise of endonuclease production. The mutagen was added to the medium at different periods of synchronized DNA replication to generate a selective effect on certain genes located in the replication sites. A 15-minute period 40 min after the beginning of the lag-phase was shown to be most sensitive to NMU treatment for producing mutants with a high yield of endonuclease. A mutant of S. marcescens was obtained, which synthesized endonuclease 40-100 times as effective as the parent strain did. The ability of S. marcescens endonuclease to hydrolyze RNA and DNA and to inhibit the reproduction of RNA- and DNA-containing viruses is of practical importance.     

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).     

Oxidation of vanillin by Serratia marcescens

A Serratia marcescens strain quantitatively converted vanillin (0·1%, w/v) to vanillic acid, which exerted an inhibitory effect on bacterial growth. Vanillic acid producing activity was found in cell-free extracts of cultures grown in media with and without vanillin, when glucose was the substrate.     

Induction of pigmentation in nonproliferating cells of Serratia marcescens by addition of single amino acids

Addition of casein hydrolysate to suspensions of washed, nonpigmented, nonproliferating Serratia marcescens incubating at 27 C induced biosynthesis of prodigiosin. Four amino acids of casein hydrolysate, dl-aspartic acid, l-glutamic acid, l-proline, and l-alanine caused formation of pigment when added individually. dl-Ornithine also was effective. Optimal concentrations for maximal pigmentation were 5 to 10 mg/ml; at these high concentrations, d-serine also induced biosynthesis of some prodigiosin. dl-Alanine and -ornithine were as effective as the l-iosomers, but l-glutamic acid and l-proline gave better responses than their racemic mixtures. Kinetics of prodigiosin biosynthesis after addition of dl-alanine (20 mg/ml) were similar to those of cells suspended in 0.2% casein hydrolysate. The other amino acids were less effective. Addition of 5 mg of dl-alanine or casein hydrolysate per ml to minimal medium increased by 30% the amount of prodigiosin formed by growing cells after incubation for 7 days at 27 C. Cultures grown for 7 days at 27 C in 0.2% casein hydrolsate formed more prodigiosin than did suspensions of nonproliferating cells containing individual amino acids or casein hydrolysate. However, more pigment was produced by cells suspended in l-alanine (5 mg/ml) or l-proline (10 mg/ml) than when suspended in 0.4% natural or synthetic casein hydrolysate. Filtrates from suspensions of nonproliferating cells forming pigment in l-proline induced more rapid formation of prodigiosin, but filtrates from suspensions in dl-alanine did not. The data supported the hypothesis that pyrrole groups of prodigiosin may be synthesized from 5-carbon amino acids such as proline, ornithine, aspartic, and glutamic acids, but the role of alanine is unknown.     

Oxidation of Aromatic Aldehydes by Serratia marcescens

Cell suspensions of Serratia marcescens catalyzed the oxidation of aromatic aldehydes into the corresponding acids in high yield under mild conditions.