C-terminal hybrid mutant of Bacillus pumilus cyanide dihydratase dramatically enhances thermal stability and pH tolerance by reinforcing oligomerization.

AIMS: To investigate the impact of the highly variable C-terminal domain of cyanide dihydratase, a member of the nitrilase superfamily, on its activity and stability. METHODS AND RESULTS: Generating and analysing the thermal stability and pH tolerance of chimeric cyanide dihydratase proteins has provided a platform to investigate domains within the C-terminus and their effect on quaternary structure of the protein. The protein oligomerization state was inferred from native protein size by gel exclusion chromatography. CONCLUSIONS: Our data indicates that the influence of the cyanide dihydratase C-terminus on thermal stability stems from its participation in oligomerization at the major C-surface interface. The formation of this surface is crucial for the activity and stability of CynD. Gel filtration chromatography of an N-terminal deletion mutant, CynDpum ∆303, revealed a defect in oligomerization, and another mutant R67C was suppressed by introduction of a heterologous C-terminus as a chimeric protein. This indicates that the C-terminus from Pseudomonas stutzeri stabilizes CynD by supporting oligomerization between dimers at the C-surface. The chimeric protein CynDpum-stut exhibited full activity at pH 9, a pH where the parent enzyme is nearly inactive, and retained 40% of its activity at pH 9·5 making it a unique pH tolerant mutant. SIGNIFICANCE AND IMPACT OF THE STUDY: The study characterized a chimeric protein with remarkable thermal stability and tolerance to alkaline conditions, features essential for practical application as industrial cyanide solutions are maintained as highly alkaline solutions to prevent formation of hydrogen cyanide gas.

Microbial nitrilases: versatile, spiral forming, industrial enzymes.

The nitrilases are enzymes that convert nitriles to the corresponding acid and ammonia. They are members of a superfamily, which includes amidases and occur in both prokaryotes and eukaryotes. The superfamily is characterized by having a homodimeric building block with a alpha beta beta alpha-alpha beta beta alpha sandwich fold and an active site containing four positionally conserved residues: cys, glu, glu and lys. Their high chemical specificity and frequent enantioselectivity makes them attractive biocatalysts for the production of fine chemicals and pharmaceutical intermediates. Nitrilases are also used in the treatment of toxic industrial effluent and cyanide remediation. The superfamily enzymes have been visualized as dimers, tetramers, hexamers, octamers, tetradecamers, octadecamers and variable length helices, but all nitrilase oligomers have the same basic dimer interface. Moreover, in the case of the octamers, tetradecamers, octadecamers and the helices, common principles of subunit association apply. While the range of industrially interesting reactions catalysed by this enzyme class continues to increase, research efforts are still hampered by the lack of a high resolution microbial nitrilase structure which can provide insights into their specificity, enantioselectivity and the mechanism of catalysis. This review provides an overview of the current progress in elucidation of structure and function in this enzyme class and emphasizes insights that may lead to further biotechnological applications.

Helical structure of unidirectionally shadowed metal replicas of cyanide hydratase from Gloeocercospora sorghi.

The helical filaments of the cyanide hydratase from Gloeocercospora sorghi have been reconstructed in three dimensions from freeze dried, unidirectionally shadowed specimens using iterative real-space helical reconstruction. The average power spectrum of all selected images has three clear reflections on different layer lines. The reconstruction is complicated by the fact that three possible indexing schemes are possible and reconstructions using the starting symmetries based on each of these indexing schemes converge on three-dimensional volumes which appear plausible. Because only one side is visible in shadowed specimens, it is necessary to examine the phases from a single filament by cryo-electron microscopy in order to make an unequivocal assignment of the symmetry. Because of the novel nature of the reconstruction method used here, conventional cryo-EM methods were also used to determine a second reconstruction, allowing us to make comparisons between the two. The filament is shown to have a left-handed one-start helix with D(1) symmetry, 5.46 dimers per turn and a pitch of 7.15nm. The reconstruction suggests the presence of an interaction across the groove not previously seen in nitrilase helical fibres.

Oligomeric structure of nitrilases: effect of mutating interfacial residues on activity.

Nitrilases are important industrial enzymes that convert nitriles into their corresponding acids or, occasionally, amides. Atomic resolution structures of four members of the nitrilase superfamily have been determined, but these differ from microbial nitrilases in that they do not form typical large homo-oligomeric complexes. At least two nitrilases, the cyanide dihydratases from Pseudomonas stutzeri AK61 and Bacillus pumilus C1, form unusual spiral structures of 14 and 18 subunits, respectively. Evidence suggests that the formation of the spiral structure is essential for activity. Sequence analysis reveals that the nitrilases differ from the nonspiral-forming homologs by two insertions of between 12 and 14 amino acids and a C-terminal extension of up to 35 amino acids. The insertions are positioned at an intermolecular interface in the spiral and probably contribute to its formation. The other interfaces responsible for the formation and/or stabilization of the spirals can also be identified. Comparative structure modeling enables identification of the residues involved in these interacting surfaces, which are remote from the active site. Mutation of these interacting residues usually leads to loss of activity. The effect of the mutations on activity in most cases can be rationalized in terms of a possible effect on spiral formation.

The cyanide degrading nitrilase from Pseudomonas stutzeri AK61 is a two-fold symmetric, 14-subunit spiral.

The quaternary structure of the cyanide dihydratase from Pseudomonas stutzeri AK61 was determined by negative stain electron microscopy and three-dimensional reconstruction using the single particle technique. The structure is a spiral comprising 14 subunits with 2-fold symmetry. Interactions across the groove cause a decrease in the radius of the spiral at the ends and the resulting steric hindrance prevents the addition of further subunits. Similarity to two members of the nitrilase superfamily, the Nit domain of NitFhit and N-carbamyl-D-amino acid amidohydrolase, enabled the construction of a partial atomic model that could be unambiguously fitted to the stain envelope. The model suggests that interactions involving two significant insertions in the sequence relative to these structures leads to the left-handed spiral assembly.

Study of the mechanism of action of p-chloromercuribenzoate on endonuclease from the bacterium Serratia marcescens.

The mechanism of action of p-chloromercuribenzoate (PCMB) on Serratia marcescens nuclease was investigated. The analysis showed that PCMB forms complexes with DNA. Binding of C7H5O2Hg+ to DNA changes the secondary structure of the DNA. These changes alter the enzymatic activity of S. marcescens nuclease, which was previously found to be sensitive to the secondary structure of the substrates. The nuclease activity was either suppressed or stimulated in the presence of PCMB depending on the C7H5O2Hg+ to nucleotide equivalent ratio. Binding of C7H5O2Hg+ to DNA did not form an abortive enzyme-substrate complex. Binding of Mg2+ to the C7H5O2Hg-DNA complex caused appropriate changes in secondary structure of the substrate. Since Mg2+ and C7H5O2Hg+, though differing in the type of metal cation, are similar in their mechanisms of influence on enzymatic activity of S. marcescens nuclease, the identity of other metal-containing effectors in their mechanism of action on Serratia marcescens nuclease is assumed.

Characterization of the alanine racemases from two mycobacteria.

D-Alanine is a necessary precursor in the biosynthesis of the bacterial peptidoglycan. The naturally occurring L-alanine isomer is racemized to its D-form through the action of a class of enzymes called alanine racemases. These enzymes are ubiquitous among prokaryotes, and with very few exceptions are absent in eukaryotes, making them a logical target for the development of novel antibiotics. The alanine racemase gene from both Mycobacterium tuberculosis and M. avium was amplified by PCR and cloned in Escherichia coli. Overexpression of the proteins in the E. coli BL21 system, both as native and as His-tagged recombinant products, has been achieved. The proteins have been purified to electrophoretic homogeneity and analyzed biochemically. A D-alanine requiring double knock-out mutant of E. coli (alr, dadX) was constructed and the cloned genes were able to complement its deficiencies.

Characterization of the alanine racemases from Pseudomonas aeruginosa PAO1.

Alanine racemases are ubiquitous, almost uniquely prokaryotic enzymes catalyzing the racemization between l- and d-alanine. The requirement for d-alanine as a necessary component of the bacterial cell wall makes this class of enzymes a logical target for the development of novel antibiotics. In an effort to better understand the structure and mechanism of these enzymes, we have cloned the two independent alanine racemases from Pseudomonas aeruginosa, an important opportunistic bacterial pathogen of humans and animals. The dadX(PA) and alr(PA) genes have been sequenced, overexpressed, and their activity was demonstrated by complementing d-alanine auxotrophs of Escherichia coli. Both gene products were purified to electrophoretic homogeneity, the enzymes were characterized biochemically, and preliminary crystals were obtained.

Nup124p is a nuclear pore factor of Schizosaccharomyces pombe that is important for nuclear import and activity of retrotransposon Tf1.

The long terminal repeat (LTR)-containing retrotransposon Tf1 propagates within the fission yeast Schizosaccharomyces pombe as the result of several mechanisms that are typical of both retrotransposons and retroviruses. To identify host factors that contribute to the transposition process, we mutagenized cultures of S. pombe and screened them for strains that were unable to support Tf1 transposition. One such strain contained a mutation in a gene we named nup124. The product of this gene contains 11 FXFG repeats and is a component of the nuclear pore complex. In addition to the reduced levels of Tf1 transposition, the nup124-1 allele caused a significant reduction in the nuclear localization of Tf1 Gag. Surprisingly, the mutation in nup124-1 did not cause any reduction in the growth rate, the nuclear localization of specific nuclear localization signal-containing proteins, or the cytoplasmic localization of poly(A) mRNA. A two-hybrid analysis and an in vitro precipitation assay both identified an interaction between Tf1 Gag and the N terminus of Nup124p. These results provide evidence for an unusual mechanism of nuclear import that relies on a direct interaction between a nuclear pore factor and Tf1 Gag.

[Polydispersity of Serratia marcescens nuclease at optimal pH values]. / Polidispersnost' nukleazy Serratia marcescens pri optimal'nom znachenii pH.

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.

A new member of the Sin3 family of corepressors is essential for cell viability and required for retroelement propagation in fission yeast.

Tf1 is a long terminal repeat (LTR)-containing retrotransposon that propagates within the fission yeast Schizosaccharomyces pombe. LTR-retrotransposons possess significant similarity to retroviruses and therefore serve as retrovirus models. To determine what features of the host cell are important for the proliferation of this class of retroelements, we screened for mutations in host genes that reduced the transposition activity of Tf1. We report here the isolation and characterization of pst1(+), a gene required for Tf1 transposition. The predicted amino acid sequence of Pst1p possessed high sequence homology with the Sin3 family of proteins, known for their interaction with histone deacetylases. However, unlike the SIN3 gene of Saccharomyces cerevisiae, pst1(+) is essential for cell viability. Immunofluorescence microscopy indicated that Pst1p was localized in the nucleus. Consistent with the critical role previously reported for Sin3 proteins in the histone acetylation process, we found that the growth of the strain with the pst1-1 allele was supersensitive to the specific histone deacetylase inhibitor trichostatin A. However, our analysis of strains with the pst1-1 mutation was unable to detect any changes in the acetylation of specific lysines of histones H3 and H4 as measured in bulk chromatin. Interestingly, the pst1-1 mutant strain produced wild-type levels of Tf1-encoded proteins and cDNA, indicating that the defect in transposition occurred after reverse transcription. The results of immunofluorescence microscopy showed that the nuclear localization of the Tf1 capsid protein was disrupted in the strain with the pst1-1 mutation, indicating an important role of pst1(+) in modulating the nuclear import of Tf1 virus-like particles.

Microbial denitrogenation of fossil fuels.

The microbial degradation of nitrogen compounds from fossil fuels is important because of the contribution these contaminants make to the formation of nitrogen oxides (NOx) and hence to air pollution and acid rain. They also contribute to catalyst poisoning during the refining of crude oil, thus reducing process yields. We review the current status of microbial degradation of aromatic nitrogen compounds and discuss the potential of microbial processes to alleviate these problems.

Serratia marcescens and its extracellular nuclease.

Serratia marcescens produces an endonuclease with extraordinarily high specific activity that is released into the surrounding medium. This enzyme has been the focus of studies on gene regulation, protein secretion, endonuclease action, and protein structure; it has also been found to have many applications in biotechnology. Here we briefly review these different facets of research regarding the Serratia nuclease and summarize the current state of knowledge about this enzyme.

Nuclease overexpression mutants of Serratia marcescens.

A family of mutants overexpressing the Serratia marcescens extracellular nuclease has been known for decades. A number of these alleles are characterized here at the molecular level, and the mutant genes are identified, yielding a likely model for their phenotype. The known mutations exert their effect indirectly on nucA expression by elevating the basal SOS response of the cell. Mutations have been found in xerC and uvrD, both of which result in partial SOS induction. A classic nucsu allele, that of strain W1050, is also likely to be in xerC.

The Serratia marcescens NucE protein functions as a holin in Escherichia coli.

The recently discovered nucC locus of Serratia marcescens encodes the cryptic prophage genes nucE, nucD, and nucC. NucC is required for expression of the S. marcescens nuclease and functions as a transcriptional activator of the nuclease gene, nucA. NucE and NucD are dispensable for nuclease expression but were proposed to allow for secretion of the nuclease by Escherichia coli. Here, we show (i) that the NucE protein is membrane bound, (ii) that it can complement the lambda S holin, (iii) that it can be triggered by potassium cyanide, (iv) that it is detrimental to cell viability, and (v) that the concomitant expression of nucE and nucD results in cell lysis. Apparently NucE and NucD function as a holin and an endolysin, respectively. This suggests that their roles in nuclease secretion by E. coli are indirect, possibly through directed cell lysis.

Disruption of polyamine modulation by a single amino acid substitution on the L3 loop of the OmpC porin channel.

Structural studies have demonstrated that the extracellular L3 loop of porin constricts the channel and suggest that this loop might be involved in channel selectivity and gating. We previously showed that positively charged polyamines can induce changes in porin gating kinetics by stabilization of closed states. Here we report the effects of the mutation of two different aspartate residues of Escherichia coli OmpC porin on the polyamine sensitivity of the channel. Aspartate 105 or aspartate 118 on the L3 loop was replaced by glutamine by site-directed mutagenesis. The gating activity of the wild-type and mutant channels were studied by patch-clamp of liposomes containing reconstituted outer membrane fractions, in the absence or the presence of either polyamine spermine or cadaverine. Porin channels with a D118Q mutation, at the root of L3, still showed some, albeit milder, sensitivity to polyamine modulation. On the other hand, the D105Q mutation, at the tip of L3, abolished the increase in closing frequency which is typically observed in the presence of polyamines. We conclude that aspartate 105 primarily, but not aspartate 118, plays an important role in mediating the polyamine-induced changes in gating kinetics that result in the inhibition of the OmpC channel.

Secretion of nuclease across the outer membrane of Serratia marcescens and its energy requirements.

Extracellular secretion of Serratia marcescens nuclease occurs as a two-step process via a periplasmic intermediate. Unlike other extracellular proteins secreted by gram-negative bacteria by the general secretory pathway, nuclease accumulates in the periplasm in its active form for an unusually long time before its export into the growth medium. The energy requirements for extracellular secretion of nuclease from the periplasm were investigated. Our results suggest that the second step of secretion across the outer membrane is dependent upon the external pH; acidic pH effectively but reversibly blocks extracellular secretion. However, electrochemical proton gradient, and possibly ATP hydrolysis, are not required for this step. We suggest that nuclease uses a novel mechanism for the second step of secretion in S. marcescens.

Isoforms of Serratia marcescens nuclease. The role of Mg2+ in the hydrolysis mechanism.

Structural and functional differences between isoforms Sm1 and Sm2, a lack of influence of free Mg2+ on the isoform structures, formation of DNA-magnesium complex serving with great probability as a real substrate for the nuclease has been summarized on the basis of experimental data. Mg2+ forming a complex with phosphate groups of DNA are supposed to further increase the electrophilicity of the phosphorus atoms besides causing a conformational change of the substrate.

Two-step secretion of the Serratia marcescens extracellular nuclease.

The extracellular nuclease of Serratia marcescens is one of a wide variety of enzymes secreted into the growth medium. Its appearance occurs late in the growth of a culture, and its gene, nucA, is transcriptionally regulated in a complex fashion by growth phase and other factors. Pulse-labeling studies reveal that extracellular secretion of nuclease occurs as a two-step process. In the first step, nuclease is rapidly translocated across the cytoplasmic membrane into the periplasm, where it accumulates as a mature active nuclease. A precursor protein, nuclease still carrying its signal sequence, was detected in the presence of carbonyl cyanide m-chlorophenylhydrazone or sodium azide, suggesting that this initial translocation and signal processing step involves an energy-dependent and Sec-dependent pathway in S. marcescens. The second step of secretion across the outer membrane is a slow process requiring between 30 to 120 min, depending on growth conditions.

Inhibition of Serratia marcescens nuclease secretion by a truncated nuclease peptide.

The production of extracellular nuclease (Nuc) from the Serratia marcescens nucA chromosomal locus is inhibited in cells producing the N-terminal portion of Nuc from a multicopy plasmid. This inhibition in trans is not at the level of nucA expression, but rather at the level of secretion of the Nuc protein. Production of the periplasmic protein beta-lactamase (Bla) does not inhibit Nuc production unless fused to the nucA signal peptide and expressed from nucAp. Inhibition by either the truncated Nuc peptide (delta Nuc) or a Bla fusion protein is promoter specific and observed when expressed from nucAp; little inhibition is observed when the same protein is expressed from the lacZpo promoter-operator. This promoter specificity is also true for the secretion of Nuc itself.