SummaryThe analysis of biological networks is increasingly important in the life sciences and in ... more SummaryThe analysis of biological networks is increasingly important in the life sciences and in particular in systems biology. Computer-based analysis tools are exploited for the investigation of these networks. However, to find relevant data sources can be a time-consuming task, access to information changes, frequently it is not obvious to a user which tools can be used in combination with specific data sources, and network data is often not available in a format directly usable by analysis tools.To assist in collecting information about biological network data sources, and to help in investigating possible combinations of data sources and analysis software, we created BiNCo-wiki. BiNCo-wiki is a wiki system that stores information related to biological networks and allows all users to add or modify this information in an easy way. The collaborative character of a wiki system supports easy sharing of information and allows the community-based extension of the information already ...
Fur binding sites were found upstream of around 20 of the regulated genes. Overall, adaptation to... more Fur binding sites were found upstream of around 20 of the regulated genes. Overall, adaptation to low iron conditions in C. difficile focused on an increase of iron import, a significant replacement of iron requiring metabolic pathways and the restructuring of the cell surface for protection during the complex adaptation phase and was only partly directly regulated by Fur.
In mass spectrometry-based untargeted metabolomics, rarely more than 30% of the compounds are ide... more In mass spectrometry-based untargeted metabolomics, rarely more than 30% of the compounds are identified. Without the true identity of these molecules it is impossible to draw conclusions about the biological mechanisms, pathway relationships and provenance of compounds. The only way at present to address this discrepancy is to use in silico fragmentation software to identify unknown compounds by comparing and ranking theoretical MS/MS fragmentations from target structures to experimental tandem mass spectra (MS/MS). We compared the performance of four publicly available in silico fragmentation algorithms (MetFragCL, CFM-ID, MAGMa+ and MS-FINDER) that participated in the 2016 CASMI challenge. We found that optimizing the use of metadata, weighting factors and the manner of combining different tools eventually defined the ultimate outcomes of each method. We comprehensively analysed how outcomes of different tools could be combined and reached a final success rate of 93% for the training data, and 87% for the challenge data, using a combination of MAGMa+, CFM-ID and compound importance information along with MS/MS matching. Matching MS/MS spectra against the MS/MS libraries without using any in silico tool yielded 60% correct hits, showing that the use of in silico methods is still important.
International journal of medical microbiology : IJMM, 2017
Clostridioides difficile (formerly Clostridium difficile) is a major nosocomial pathogen with an ... more Clostridioides difficile (formerly Clostridium difficile) is a major nosocomial pathogen with an increasing number of community-acquired infections causing symptoms from mild diarrhea to life-threatening colitis. The pathogenicity of C. difficile is considered to be mainly associated with the production of genome-encoded toxins A and B. In addition, some strains also encode and express the binary toxin CDT. However; a large number of non-toxigenic C. difficile strains have been isolated from the human gut and the environment. In this study, we characterized the growth behavior, motility and fermentation product formation of 17 different C. difficile isolates comprising five different major genomic clades and five different toxin inventories in relation to the C. difficile model strains 630Δerm and R20291. Within 33 determined fermentation products, we identified two yet undescribed products (5-methylhexanoate and 4-(methylthio)-butanoate) of C. difficile. Our data revealed major dif...
The heterotrophic marine bacterium Dinoroseobacter shibae utilizes aerobic respiration and anaero... more The heterotrophic marine bacterium Dinoroseobacter shibae utilizes aerobic respiration and anaerobic denitrification supplemented with aerobic anoxygenic photosynthesis for energy generation. The aerobic to anaerobic transition is controlled by four Fnr/Crp family regulators in a unique cascade-type regulatory network. FnrL is utilizing an oxygen-sensitive Fe-S cluster for oxygen sensing. Active FnrL is inducing most operons encoding the denitrification machinery and the corresponding heme biosynthesis. Activation of gene expression of the high oxygen affinity cbb3-type and repression of the low affinity aa3-type cytochrome c oxidase is mediated by FnrL. Five regulator genes including dnrE and dnrF are directly controlled by FnrL. Multiple genes of the universal stress protein (USP) and cold shock response are further FnrL targets. DnrD, most likely sensing NO via a heme cofactor, co-induces genes of denitrification, heme biosynthesis, and the regulator genes dnrE and dnrF. DnrE is ...
Microbiology and molecular biology reviews : MMBR, Mar 1, 2017
The advent of heme during evolution allowed organisms possessing this compound to safely and effi... more The advent of heme during evolution allowed organisms possessing this compound to safely and efficiently carry out a variety of chemical reactions that otherwise were difficult or impossible. While it was long assumed that a single heme biosynthetic pathway existed in nature, over the past decade, it has become clear that there are three distinct pathways among prokaryotes, although all three pathways utilize a common initial core of three enzymes to produce the intermediate uroporphyrinogen III. The most ancient pathway and the only one found in the Archaea converts siroheme to protoheme via an oxygen-independent four-enzyme-step process. Bacteria utilize the initial core pathway but then add one additional common step to produce coproporphyrinogen III. Following this step, Gram-positive organisms oxidize coproporphyrinogen III to coproporphyrin III, insert iron to make coproheme, and finally decarboxylate coproheme to protoheme, whereas Gram-negative bacteria first decarboxylate c...
Different biomolecules have been identified in bacterial pathogens that sense changes in temperat... more Different biomolecules have been identified in bacterial pathogens that sense changes in temperature and trigger expression of virulence programs upon host entry. However, the dynamics and quantitative outcome of this response in individual cells of a population, and how this influences pathogenicity are unknown. Here, we address these questions using a thermosensing virulence regulator of an intestinal pathogen (RovA of Yersinia pseudotuberculosis) as a model. We reveal that this regulator is part of a novel thermoresponsive bistable switch, which leads to high- and low-invasive subpopulations within a narrow temperature range. The temperature range in which bistability is observed is defined by the degradation and synthesis rate of the regulator, and is further adjustable via a nutrient-responsive regulator. The thermoresponsive switch is also characterized by a hysteretic behavior in which activation and deactivation occurred on vastly different time scales. Mathematical modeling...
The rate-limiting step in the biosynthesis of tetrapyrroles is the formation of 5-aminolevulinic ... more The rate-limiting step in the biosynthesis of tetrapyrroles is the formation of 5-aminolevulinic acid (ALA). In Pseudomonas aeruginosa ALA is synthesized via a two-step reaction from aminoacylated tRNA(Glu) by the action of glutamyl-tRNA reductase and glutamate-1-semialdehyde-2,1-amino mutase. To initiate an investigation of the regulation of the second step in ALA formation, the hemL gene was cloned from P. aeruginosa by complementation of an Escherichia coli hemL mutant. An open reading frame of 1284 bp encoding a protein of 427 amino acids with a calculated molecular mass of 45,404 Da was identified. The hemL gene was mapped to the SpeI fragment Z and the DpnI fragment J1 of the P. aeruginosa chromosome corresponding approximately to min 0.3-0.9. One transcription start site was located 280 bp upstream of the translational start site of the hemL gene. No classical sigma 70-dependent promoter was detected. Oxygen stress induced by the addition of H2O2 to the growth medium led to an approximately 3.5-fold increase in hemL expression as determined by mRNA dot blot assays. Anaerobic denitrifying growth led to a 2-fold stimulation of hemL transcription. Two additional open reading frames were detected downstream of the hemL gene. One open reading frame (orf1) of 549 bp encodes a protein of 182 amino acids with a calculated molecular mass of 19,638 Da.(ABSTRACT TRUNCATED AT 250 WORDS)
Rhodobacteraceae harbor a conspicuous wealth of extrachromosomal replicons (ECRs) and therefore t... more Rhodobacteraceae harbor a conspicuous wealth of extrachromosomal replicons (ECRs) and therefore the exchange of genetic material via horizontal transfer has been supposed to be a major evolutionary driving force. Many plasmids in this group encode type IV secretion systems (T4SS) that are expected to mediate transfer of proteins and/or DNA into host cells, but no experimental evidence of either has yet been provided. Dinoroseobacter shibae, a species of the Roseobacter group within the Rhodobacteraceae family, contains five ECRs that are crucial for anaerobic growth, survival under starvation and the pathogenicity of this model organism. Here we tagged two syntenous but compatible RepABC-type plasmids of 191 and 126-kb size, each encoding a T4SS, with antibiotic resistance genes and demonstrated their conjugational transfer into a distantly related Roseobacter species, namely Phaeobacter inhibens. Pulsed field gel electrophoresis showed transfer of those replicons into the recipient...
Pseudomonas aeruginosa produces and secretes several lipolytic enzymes, among them the lipases Li... more Pseudomonas aeruginosa produces and secretes several lipolytic enzymes, among them the lipases LipA and LipC. LipA is encoded within the lipA/lipH operon, together with its cognate foldase LipH, which was also found to be required for the functional expression of LipC. At present, the physiological function of LipC is unknown. We have cloned a synthetic operon consisting of the lipC structural gene and the foldase gene lipH obtained from the lipA/lipH operon and have constructed, in parallel, a lipC-deficient P. aeruginosa mutant. Inactivation of the lipC gene significantly impaired type IV pilus-dependent twitching and swarming motility, but also the flagella-mediated swimming motility of P. aeruginosa. Moreover, for the lipC mutant, we observed a significant decrease in the amount of extracellular rhamnolipids. Also, the P. aeruginosa lipC mutant showed a significantly altered biofilm architecture. Proteome analysis revealed the accumulation of the response regulator protein PhoP in the lipC mutant.
Uroporphyrinogen III decarboxylase catalyzes the fifth step in heme biosynthesis: the elimination... more Uroporphyrinogen III decarboxylase catalyzes the fifth step in heme biosynthesis: the elimination of carboxyl groups from the four acetate side chains of uroporphyrinogen-III to yield coproporphyrinogen-III. We have previously found that the rate-limiting step is substrate protonation, rather than decarboxylation itself, and that this protonation can be effected by a nearby arginine residue (Arg37). In this report, we have studied the reasons for the unusual choice of arginine as a general acid catalyst. Our density functional calculations show that, although substrate protonation by H 3 O + is both exergonic and very fast, in the presence of a protonated Arg37 substrate decarboxylation becomes rate-limiting and the substrate spontaneously breaks upon protonation. These results suggest that the active site must be shielded from solvent protons, and that therefore H 3 O + should be excluded from a role in both protonations present in this mechanism. A second Arg residue (Arg41) is uniquely positioned to act as donor of the second proton, with an activation barrier below 2 kcal mol-1. Additional site-directed mutagenesis experiments confirmed that no coproporphyrinogen is formed in the absence of any of these these Arg residues. This counter-intuitive use of two basic residues as general acids in two different proton donation steps by uroporphyrinogen decarboxylase may have arisen as an elegant solution to the problem of simultaneously binding the very negative uroporphyrinogen (which requires a positively charged active site), and selectively protonating it while preventing excessive carboxylate stabilization by positive charges.
After a shift of Bacillus subtilis from aerobic to anaerobic growth conditions, nitrate ammonific... more After a shift of Bacillus subtilis from aerobic to anaerobic growth conditions, nitrate ammonification and various fermentative processes replace oxygendependent respiration. Cell-free extracts prepared from wild-type B. subtilis and from mutants of the regulatory loci fnr and resDE grown under aerobic and various anaerobic conditions were compared by two-dimensional gel electrophoresis. Proteins involved in the adaptation process were identified by their N-terminal sequence. Induction of cytoplasmic lactate dehydrogenase (LctE) synthesis under anaerobic fermentative conditions was dependent on fnr and resDE. Anaerobic nitrate repression of LctE formation required fnrmediated expression of narGHJI, encoding respiratory nitrate reductase. Anaerobic induction of the flavohaemoglobin Hmp required resDE and nitrite. The general anaerobic induction of ywfI, encoding a protein of unknown function, was modulated by resDE and fnr. The ywfI gene shares its upstream region with the pta gene, encoding the fermentative enzyme acetyl-CoA : orthophosphate acetyltransferase. Anaerobic repression of the synthesis of a potential membrane-associated NADH dehydrogenase (YjlD, Ndh), and anaerobic induction of fructose-1,6-bisphosphate aldolase (FbaA) and dehydrolipoamide dehydrogenase (PhdD, Lpd) formation, did not require fnr or resDE participation. Synthesis of glycerol kinase (GlpK) was decreased under anaerobic conditions. Finally, the effect of anaerobic stress induced by the immediate shift from aerobic to strictly anaerobic conditions was analysed. The induction of various systems for the utilization of alternative carbon sources such as inositol (IolA, IolG, IolH, IolI), melibiose (MelA) and 6-phosphoα-glucosides (GlvA) indicated a catabolite-response-like stress reaction.
Glutamate 1-semialdehyde aminotransferase (glutamate 1-semialdehyde 2,l-aminomutase; EC 5.4.3.8; ... more Glutamate 1-semialdehyde aminotransferase (glutamate 1-semialdehyde 2,l-aminomutase; EC 5.4.3.8; GSA-AT) catalyzes the transfer of the amino group on carbon 2 of glutamate 1-semialdehyde (GSA) to the neighboring carbon 1 to form 6-aminolevulinic acid (ALA). To gain insight into the mechanism of this enzyme, possible intermediates were tested with purified enzyme and the reaction sequence was followed spectroscopically. While 4,5-dioxovaleric acid (DOVA) was efficiently converted to ALA by the pyridoxamine 5'-phosphate (PMP) form of the enzyme, 4,5-diaminovaleric acid (DAVA) was a substrate for the pyridoxal 5'-phosphate (PLP) form of GSA-AT. Thus, both substances are reaction intermediates. The purified enzyme showed an absorption spectrum with a peak around 338 nm. Addition of PLP led to increased absorption at 338 nm and a new peak around 438 nm. Incubation of the purified enzyme with PMP resulted in an additional absorption peak at 350 nm. The reaction of the PLP and PMP form of the enzyme with GSA allowed the detection of a series of peaks which varied in their intensities in a time-dependent manner. The most drastic changes to the spectrum that were observed during the reaction sequence were at 495 and 540 nm. Some of the detected absorption bands during GSA-AT catalysis were previously described for several other aminotransferases, indicating the relationship of the mechanisms. The reaction of the PMP form of the enzyme with DOVA resulted in a similar spectrum as described above, while the spectrum for the conversion of DAVA by the PLP form of the enzyme indicated a different mechanism. To understand the functional significance of a conserved lysyl residue found in all cloned GSA-ATs, lysine 265 of Escherichia coli GSA-AT was changed to arginine by oligonucleotidedirected mutagenesis. The mutant enzyme K265R was overexpressed, purified to apparent homogeneity, and analyzed for its structural, catalytic, and spectroscopic properties. The enzymatic activity of K265R was only 2% of the wild-type enzyme activity, while its dimeric structure was not influenced by the mutation. The enzyme activity was stimulated by the addition of exogenous amines such as ethanolamine and methylamine. The spectrum of purified K265R showed significant absorbance only at 280 nm, indicating the absence of bound cofactor. The addition of PLP to K265R in the presence of ethanolamine prompted the formation of a peak at 438 nm, which was subsequently converted into a new peak at 338 nm. Further addition of GSA led to the conversion of the 338-nm peak into a peak at 305 nm. The nature of the catalysis performed by the mutant enzyme is different from the activity of the wild-type enzyme. To test the loss of GSA-AT function in vivo, the hemL gene, encoding GSA-AT, was disrupted by insertion of the Tcr gene into its coding region. This E. coli strain has leaky ALA auxotrophy, indicating the presence of a compensatory pathway for ALA formation in E. coli. Transformation of the mutant strain with the wild-type gene restored normal growth, while transformation with the mutant gene encoding K265R resulted in drastically reduced growth but still differed from the control strain containing an empty plasmid.
SummaryThe analysis of biological networks is increasingly important in the life sciences and in ... more SummaryThe analysis of biological networks is increasingly important in the life sciences and in particular in systems biology. Computer-based analysis tools are exploited for the investigation of these networks. However, to find relevant data sources can be a time-consuming task, access to information changes, frequently it is not obvious to a user which tools can be used in combination with specific data sources, and network data is often not available in a format directly usable by analysis tools.To assist in collecting information about biological network data sources, and to help in investigating possible combinations of data sources and analysis software, we created BiNCo-wiki. BiNCo-wiki is a wiki system that stores information related to biological networks and allows all users to add or modify this information in an easy way. The collaborative character of a wiki system supports easy sharing of information and allows the community-based extension of the information already ...
Fur binding sites were found upstream of around 20 of the regulated genes. Overall, adaptation to... more Fur binding sites were found upstream of around 20 of the regulated genes. Overall, adaptation to low iron conditions in C. difficile focused on an increase of iron import, a significant replacement of iron requiring metabolic pathways and the restructuring of the cell surface for protection during the complex adaptation phase and was only partly directly regulated by Fur.
In mass spectrometry-based untargeted metabolomics, rarely more than 30% of the compounds are ide... more In mass spectrometry-based untargeted metabolomics, rarely more than 30% of the compounds are identified. Without the true identity of these molecules it is impossible to draw conclusions about the biological mechanisms, pathway relationships and provenance of compounds. The only way at present to address this discrepancy is to use in silico fragmentation software to identify unknown compounds by comparing and ranking theoretical MS/MS fragmentations from target structures to experimental tandem mass spectra (MS/MS). We compared the performance of four publicly available in silico fragmentation algorithms (MetFragCL, CFM-ID, MAGMa+ and MS-FINDER) that participated in the 2016 CASMI challenge. We found that optimizing the use of metadata, weighting factors and the manner of combining different tools eventually defined the ultimate outcomes of each method. We comprehensively analysed how outcomes of different tools could be combined and reached a final success rate of 93% for the training data, and 87% for the challenge data, using a combination of MAGMa+, CFM-ID and compound importance information along with MS/MS matching. Matching MS/MS spectra against the MS/MS libraries without using any in silico tool yielded 60% correct hits, showing that the use of in silico methods is still important.
International journal of medical microbiology : IJMM, 2017
Clostridioides difficile (formerly Clostridium difficile) is a major nosocomial pathogen with an ... more Clostridioides difficile (formerly Clostridium difficile) is a major nosocomial pathogen with an increasing number of community-acquired infections causing symptoms from mild diarrhea to life-threatening colitis. The pathogenicity of C. difficile is considered to be mainly associated with the production of genome-encoded toxins A and B. In addition, some strains also encode and express the binary toxin CDT. However; a large number of non-toxigenic C. difficile strains have been isolated from the human gut and the environment. In this study, we characterized the growth behavior, motility and fermentation product formation of 17 different C. difficile isolates comprising five different major genomic clades and five different toxin inventories in relation to the C. difficile model strains 630Δerm and R20291. Within 33 determined fermentation products, we identified two yet undescribed products (5-methylhexanoate and 4-(methylthio)-butanoate) of C. difficile. Our data revealed major dif...
The heterotrophic marine bacterium Dinoroseobacter shibae utilizes aerobic respiration and anaero... more The heterotrophic marine bacterium Dinoroseobacter shibae utilizes aerobic respiration and anaerobic denitrification supplemented with aerobic anoxygenic photosynthesis for energy generation. The aerobic to anaerobic transition is controlled by four Fnr/Crp family regulators in a unique cascade-type regulatory network. FnrL is utilizing an oxygen-sensitive Fe-S cluster for oxygen sensing. Active FnrL is inducing most operons encoding the denitrification machinery and the corresponding heme biosynthesis. Activation of gene expression of the high oxygen affinity cbb3-type and repression of the low affinity aa3-type cytochrome c oxidase is mediated by FnrL. Five regulator genes including dnrE and dnrF are directly controlled by FnrL. Multiple genes of the universal stress protein (USP) and cold shock response are further FnrL targets. DnrD, most likely sensing NO via a heme cofactor, co-induces genes of denitrification, heme biosynthesis, and the regulator genes dnrE and dnrF. DnrE is ...
Microbiology and molecular biology reviews : MMBR, Mar 1, 2017
The advent of heme during evolution allowed organisms possessing this compound to safely and effi... more The advent of heme during evolution allowed organisms possessing this compound to safely and efficiently carry out a variety of chemical reactions that otherwise were difficult or impossible. While it was long assumed that a single heme biosynthetic pathway existed in nature, over the past decade, it has become clear that there are three distinct pathways among prokaryotes, although all three pathways utilize a common initial core of three enzymes to produce the intermediate uroporphyrinogen III. The most ancient pathway and the only one found in the Archaea converts siroheme to protoheme via an oxygen-independent four-enzyme-step process. Bacteria utilize the initial core pathway but then add one additional common step to produce coproporphyrinogen III. Following this step, Gram-positive organisms oxidize coproporphyrinogen III to coproporphyrin III, insert iron to make coproheme, and finally decarboxylate coproheme to protoheme, whereas Gram-negative bacteria first decarboxylate c...
Different biomolecules have been identified in bacterial pathogens that sense changes in temperat... more Different biomolecules have been identified in bacterial pathogens that sense changes in temperature and trigger expression of virulence programs upon host entry. However, the dynamics and quantitative outcome of this response in individual cells of a population, and how this influences pathogenicity are unknown. Here, we address these questions using a thermosensing virulence regulator of an intestinal pathogen (RovA of Yersinia pseudotuberculosis) as a model. We reveal that this regulator is part of a novel thermoresponsive bistable switch, which leads to high- and low-invasive subpopulations within a narrow temperature range. The temperature range in which bistability is observed is defined by the degradation and synthesis rate of the regulator, and is further adjustable via a nutrient-responsive regulator. The thermoresponsive switch is also characterized by a hysteretic behavior in which activation and deactivation occurred on vastly different time scales. Mathematical modeling...
The rate-limiting step in the biosynthesis of tetrapyrroles is the formation of 5-aminolevulinic ... more The rate-limiting step in the biosynthesis of tetrapyrroles is the formation of 5-aminolevulinic acid (ALA). In Pseudomonas aeruginosa ALA is synthesized via a two-step reaction from aminoacylated tRNA(Glu) by the action of glutamyl-tRNA reductase and glutamate-1-semialdehyde-2,1-amino mutase. To initiate an investigation of the regulation of the second step in ALA formation, the hemL gene was cloned from P. aeruginosa by complementation of an Escherichia coli hemL mutant. An open reading frame of 1284 bp encoding a protein of 427 amino acids with a calculated molecular mass of 45,404 Da was identified. The hemL gene was mapped to the SpeI fragment Z and the DpnI fragment J1 of the P. aeruginosa chromosome corresponding approximately to min 0.3-0.9. One transcription start site was located 280 bp upstream of the translational start site of the hemL gene. No classical sigma 70-dependent promoter was detected. Oxygen stress induced by the addition of H2O2 to the growth medium led to an approximately 3.5-fold increase in hemL expression as determined by mRNA dot blot assays. Anaerobic denitrifying growth led to a 2-fold stimulation of hemL transcription. Two additional open reading frames were detected downstream of the hemL gene. One open reading frame (orf1) of 549 bp encodes a protein of 182 amino acids with a calculated molecular mass of 19,638 Da.(ABSTRACT TRUNCATED AT 250 WORDS)
Rhodobacteraceae harbor a conspicuous wealth of extrachromosomal replicons (ECRs) and therefore t... more Rhodobacteraceae harbor a conspicuous wealth of extrachromosomal replicons (ECRs) and therefore the exchange of genetic material via horizontal transfer has been supposed to be a major evolutionary driving force. Many plasmids in this group encode type IV secretion systems (T4SS) that are expected to mediate transfer of proteins and/or DNA into host cells, but no experimental evidence of either has yet been provided. Dinoroseobacter shibae, a species of the Roseobacter group within the Rhodobacteraceae family, contains five ECRs that are crucial for anaerobic growth, survival under starvation and the pathogenicity of this model organism. Here we tagged two syntenous but compatible RepABC-type plasmids of 191 and 126-kb size, each encoding a T4SS, with antibiotic resistance genes and demonstrated their conjugational transfer into a distantly related Roseobacter species, namely Phaeobacter inhibens. Pulsed field gel electrophoresis showed transfer of those replicons into the recipient...
Pseudomonas aeruginosa produces and secretes several lipolytic enzymes, among them the lipases Li... more Pseudomonas aeruginosa produces and secretes several lipolytic enzymes, among them the lipases LipA and LipC. LipA is encoded within the lipA/lipH operon, together with its cognate foldase LipH, which was also found to be required for the functional expression of LipC. At present, the physiological function of LipC is unknown. We have cloned a synthetic operon consisting of the lipC structural gene and the foldase gene lipH obtained from the lipA/lipH operon and have constructed, in parallel, a lipC-deficient P. aeruginosa mutant. Inactivation of the lipC gene significantly impaired type IV pilus-dependent twitching and swarming motility, but also the flagella-mediated swimming motility of P. aeruginosa. Moreover, for the lipC mutant, we observed a significant decrease in the amount of extracellular rhamnolipids. Also, the P. aeruginosa lipC mutant showed a significantly altered biofilm architecture. Proteome analysis revealed the accumulation of the response regulator protein PhoP in the lipC mutant.
Uroporphyrinogen III decarboxylase catalyzes the fifth step in heme biosynthesis: the elimination... more Uroporphyrinogen III decarboxylase catalyzes the fifth step in heme biosynthesis: the elimination of carboxyl groups from the four acetate side chains of uroporphyrinogen-III to yield coproporphyrinogen-III. We have previously found that the rate-limiting step is substrate protonation, rather than decarboxylation itself, and that this protonation can be effected by a nearby arginine residue (Arg37). In this report, we have studied the reasons for the unusual choice of arginine as a general acid catalyst. Our density functional calculations show that, although substrate protonation by H 3 O + is both exergonic and very fast, in the presence of a protonated Arg37 substrate decarboxylation becomes rate-limiting and the substrate spontaneously breaks upon protonation. These results suggest that the active site must be shielded from solvent protons, and that therefore H 3 O + should be excluded from a role in both protonations present in this mechanism. A second Arg residue (Arg41) is uniquely positioned to act as donor of the second proton, with an activation barrier below 2 kcal mol-1. Additional site-directed mutagenesis experiments confirmed that no coproporphyrinogen is formed in the absence of any of these these Arg residues. This counter-intuitive use of two basic residues as general acids in two different proton donation steps by uroporphyrinogen decarboxylase may have arisen as an elegant solution to the problem of simultaneously binding the very negative uroporphyrinogen (which requires a positively charged active site), and selectively protonating it while preventing excessive carboxylate stabilization by positive charges.
After a shift of Bacillus subtilis from aerobic to anaerobic growth conditions, nitrate ammonific... more After a shift of Bacillus subtilis from aerobic to anaerobic growth conditions, nitrate ammonification and various fermentative processes replace oxygendependent respiration. Cell-free extracts prepared from wild-type B. subtilis and from mutants of the regulatory loci fnr and resDE grown under aerobic and various anaerobic conditions were compared by two-dimensional gel electrophoresis. Proteins involved in the adaptation process were identified by their N-terminal sequence. Induction of cytoplasmic lactate dehydrogenase (LctE) synthesis under anaerobic fermentative conditions was dependent on fnr and resDE. Anaerobic nitrate repression of LctE formation required fnrmediated expression of narGHJI, encoding respiratory nitrate reductase. Anaerobic induction of the flavohaemoglobin Hmp required resDE and nitrite. The general anaerobic induction of ywfI, encoding a protein of unknown function, was modulated by resDE and fnr. The ywfI gene shares its upstream region with the pta gene, encoding the fermentative enzyme acetyl-CoA : orthophosphate acetyltransferase. Anaerobic repression of the synthesis of a potential membrane-associated NADH dehydrogenase (YjlD, Ndh), and anaerobic induction of fructose-1,6-bisphosphate aldolase (FbaA) and dehydrolipoamide dehydrogenase (PhdD, Lpd) formation, did not require fnr or resDE participation. Synthesis of glycerol kinase (GlpK) was decreased under anaerobic conditions. Finally, the effect of anaerobic stress induced by the immediate shift from aerobic to strictly anaerobic conditions was analysed. The induction of various systems for the utilization of alternative carbon sources such as inositol (IolA, IolG, IolH, IolI), melibiose (MelA) and 6-phosphoα-glucosides (GlvA) indicated a catabolite-response-like stress reaction.
Glutamate 1-semialdehyde aminotransferase (glutamate 1-semialdehyde 2,l-aminomutase; EC 5.4.3.8; ... more Glutamate 1-semialdehyde aminotransferase (glutamate 1-semialdehyde 2,l-aminomutase; EC 5.4.3.8; GSA-AT) catalyzes the transfer of the amino group on carbon 2 of glutamate 1-semialdehyde (GSA) to the neighboring carbon 1 to form 6-aminolevulinic acid (ALA). To gain insight into the mechanism of this enzyme, possible intermediates were tested with purified enzyme and the reaction sequence was followed spectroscopically. While 4,5-dioxovaleric acid (DOVA) was efficiently converted to ALA by the pyridoxamine 5'-phosphate (PMP) form of the enzyme, 4,5-diaminovaleric acid (DAVA) was a substrate for the pyridoxal 5'-phosphate (PLP) form of GSA-AT. Thus, both substances are reaction intermediates. The purified enzyme showed an absorption spectrum with a peak around 338 nm. Addition of PLP led to increased absorption at 338 nm and a new peak around 438 nm. Incubation of the purified enzyme with PMP resulted in an additional absorption peak at 350 nm. The reaction of the PLP and PMP form of the enzyme with GSA allowed the detection of a series of peaks which varied in their intensities in a time-dependent manner. The most drastic changes to the spectrum that were observed during the reaction sequence were at 495 and 540 nm. Some of the detected absorption bands during GSA-AT catalysis were previously described for several other aminotransferases, indicating the relationship of the mechanisms. The reaction of the PMP form of the enzyme with DOVA resulted in a similar spectrum as described above, while the spectrum for the conversion of DAVA by the PLP form of the enzyme indicated a different mechanism. To understand the functional significance of a conserved lysyl residue found in all cloned GSA-ATs, lysine 265 of Escherichia coli GSA-AT was changed to arginine by oligonucleotidedirected mutagenesis. The mutant enzyme K265R was overexpressed, purified to apparent homogeneity, and analyzed for its structural, catalytic, and spectroscopic properties. The enzymatic activity of K265R was only 2% of the wild-type enzyme activity, while its dimeric structure was not influenced by the mutation. The enzyme activity was stimulated by the addition of exogenous amines such as ethanolamine and methylamine. The spectrum of purified K265R showed significant absorbance only at 280 nm, indicating the absence of bound cofactor. The addition of PLP to K265R in the presence of ethanolamine prompted the formation of a peak at 438 nm, which was subsequently converted into a new peak at 338 nm. Further addition of GSA led to the conversion of the 338-nm peak into a peak at 305 nm. The nature of the catalysis performed by the mutant enzyme is different from the activity of the wild-type enzyme. To test the loss of GSA-AT function in vivo, the hemL gene, encoding GSA-AT, was disrupted by insertion of the Tcr gene into its coding region. This E. coli strain has leaky ALA auxotrophy, indicating the presence of a compensatory pathway for ALA formation in E. coli. Transformation of the mutant strain with the wild-type gene restored normal growth, while transformation with the mutant gene encoding K265R resulted in drastically reduced growth but still differed from the control strain containing an empty plasmid.
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Papers by Dieter Jahn