Rainer Merkl | University of Regensburg (original) (raw)

Papers by Rainer Merkl

Research paper thumbnail of Cytochrome P450 monooxygenases consisting of thermophilic bacteria

Research paper thumbnail of Faculty of 1000 evaluation for The evolutionary history of protein fold families and proteomes confirms that the archaeal ancestor is more ancient than the ancestors of other superkingdoms

F1000 - Post-publication peer review of the biomedical literature, 2012

Research paper thumbnail of Faculty of 1000 evaluation for Assessing computational methods for predicting protein stability upon mutation: good on average but not in the details

F1000 - Post-publication peer review of the biomedical literature, 2009

Research paper thumbnail of Faculty of 1000 evaluation for Practically useful: what the Rosetta protein modeling suite can do for you

F1000 - Post-publication peer review of the biomedical literature, 2010

Research paper thumbnail of Faculty of 1000 evaluation for Sequence space and the ongoing expansion of the protein universe

F1000 - Post-publication peer review of the biomedical literature, 2010

Research paper thumbnail of Faculty of 1000 evaluation for Discovery of new enzymes and metabolic pathways by using structure and genome context

F1000 - Post-publication peer review of the biomedical literature, 2013

Research paper thumbnail of Faculty of 1000 evaluation for FINDSITE: a threading-based approach to ligand homology modeling

F1000 - Post-publication peer review of the biomedical literature, 2009

Research paper thumbnail of Faculty of 1000 evaluation for An integrated semiconductor device enabling non-optical genome sequencing

F1000 - Post-publication peer review of the biomedical literature, 2011

Research paper thumbnail of Faculty of 1000 evaluation for Principles for designing ideal protein structures

F1000 - Post-publication peer review of the biomedical literature, 2013

Research paper thumbnail of Faculty of 1000 evaluation for Towards Robot Scientists for autonomous scientific discovery

F1000 - Post-publication peer review of the biomedical literature, 2011

Research paper thumbnail of Faculty of 1000 evaluation for Discriminative modeling of context-specific amino acid substitution probabilities

F1000 - Post-publication peer review of the biomedical literature, 2012

Research paper thumbnail of Faculty of 1000 evaluation for Surface sites for engineering allosteric control in proteins

F1000 - Post-publication peer review of the biomedical literature, 2008

Research paper thumbnail of Faculty of 1000 evaluation for Parallel adaptations to high temperatures in the Archaean eon

F1000 - Post-publication peer review of the biomedical literature, 2009

Research paper thumbnail of Ancestral Tryptophan Synthase Reveals Functional Sophistication of Primordial Enzyme Complexes

Cell chemical biology, Jan 23, 2016

Modern enzyme complexes are characterized by a high catalytic efficiency and allosteric communica... more Modern enzyme complexes are characterized by a high catalytic efficiency and allosteric communication between the constituting protein subunits. We were interested in whether primordial enzyme complexes from extinct species displayed a similar degree of functional sophistication. To this end, we used ancestral sequence reconstruction to resurrect the α and β subunits of the tryptophan synthase (TS) complex from the last bacterial common ancestor (LBCA), which presumably existed more than 3.4 billion years ago. We show that the LBCA TS subunits are thermostable and exhibit high catalytic activity. Moreover, they form a complex with αββα stoichiometry whose crystal structure is similar to that of modern TS. Kinetic analysis revealed that the reaction intermediate indole is channeled from the α to the β subunits and suggests that allosteric communication already occurred in LBCA TS.

Research paper thumbnail of A comparative categorization of protein function encoded in bacterial or archeal genomic islands

Journal of Molecular Evolution, Feb 1, 2006

Genomes of prokaryotes harbor genomic islands (GIs), which are frequently acquired via horizontal... more Genomes of prokaryotes harbor genomic islands (GIs), which are frequently acquired via horizontal gene transfer (HGT). Here I present an analysis of GIs with respect to gene-encoded functions. GIs were identified by statistical analysis of codon usage and clustering. Genes classified as putatively alien (pA) or putatively native (pN) were categorized according to the COG database. Among pA and pN genes, the distribution of COG functions and classes were studied for different groupings of prokaryotes. Groups were formed according to taxonomical relation or habitats. In all groups, genes related to class L (replication, recombination, and repair) were statistically significantly overrepresented in GIs. GIs of bacteria and archaea showed a distinct pattern of preferences. In archeal GIs, genes belonging to COG class M (cell wall/membrane/envelope biogenesis) or Q (secondary metabolites biosynthesis, transport, and catabolism) were more frequent. In bacterial GIs, genes of classes U (intracellular trafficking, secretion, and vesicular transport), N (cell motility), and V (defense mechanisms) were predominant. Underrepresentation was strongest for genes belonging to class J (translation, ribosomal structure, and biogenesis). Among single COG functions overrepresented in GIs were transferases and transporters. In both superkingdoms, HGT enhances genomic content by meeting demands that are independent of the studied habitats. These findings are in agreement with the complexity theory, which predicts the preferential import of operational genes. However, only specific subsets of operational genes were enriched in GIs. Modification of the cell envelope, cell motility, secretion, and protection of cellular DNA are major issues in HGT.

Research paper thumbnail of Long-Term Persistence of Bi-functionality Contributes to the Robustness of Microbial Life through Exaptation

PLOS Genetics, 2016

Modern enzymes are highly optimized biocatalysts that process their substrates with extreme effic... more Modern enzymes are highly optimized biocatalysts that process their substrates with extreme efficiency. Many enzymes catalyze more than one reaction; however, the persistence of such ambiguities, their consequences and evolutionary causes are largely unknown. As a paradigmatic case, we study the history of bi-functionality for a time span of approximately two billion years for the sugar isomerase HisA from histidine biosynthesis. To look back in time, we computationally reconstructed and experimentally characterized three HisA predecessors. We show that these ancient enzymes catalyze not only the HisA reaction but also the isomerization of a similar substrate, which is commonly processed by the isomerase TrpF in tryptophan biosynthesis. Moreover, we found that three modern-day HisA enzymes from Proteobacteria and Thermotogae also possess low TrpF activity. We conclude that this bi-functionality was conserved for at least two billion years, most likely without any evolutionary pressure. Although not actively selected for, this trait can become advantageous in the case of a gene loss. Such exaptation is exemplified by the Actinobacteria that have lost the trpF gene but possess the bi-functional HisA homolog PriA, which adopts the roles of both HisA and TrpF. Our findings demonstrate that bi-functionality can perpetuate in the absence of selection for very long time-spans.

Research paper thumbnail of Versatile Controlling System for Preparation Techniques in Electron-Microscopy

An universal software for controlling complex preparation protocols in electron microscopy was de... more An universal software for controlling complex preparation protocols in electron microscopy was developed. Its versatility will be demonstrated on the preparation technique of freeze-substitution. It controls all steps of the preparation protocol from the beginning, i.e., cryofixation, to the end, i.e., polymerisation.

Research paper thumbnail of Conversion of Anthranilate Synthase into Isochorismate Synthase: Implications for the Evolution of Chorismate-Utilizing Enzymes

Angewandte Chemie International Edition, 2015

Chorismate-utilizing enzymes play a vital role in the biosynthesis of metabolites in plants as we... more Chorismate-utilizing enzymes play a vital role in the biosynthesis of metabolites in plants as well as free-living and infectious microorganisms. Among these enzymes are the homologous primary metabolic anthranilate synthase (AS) and secondary metabolic isochorismate synthase (ICS). Both catalyze mechanistically related reactions by using ammonia and water as nucleophiles, respectively. We report that the nucleophile specificity of AS can be extended from ammonia to water by just two amino acid exchanges in a channel leading to the active site. The observed ICS/AS bifunctionality demonstrates that a secondary metabolic enzyme can readily evolve from a primary metabolic enzyme without requiring an initial gene duplication event. In a general sense, these findings add to our understanding how nature has used the structurally predetermined features of enzyme superfamilies to evolve new reactions.

Research paper thumbnail of Umwandlung einer Anthranilatsynthase in eine Isochorismatsynthase: Implikationen für die Evolution von Chorismat-umsetzenden Enzymen

Angewandte Chemie, 2015

ABSTRACT Chorismat-umsetzende Enzyme spielen in Pflanzen sowie frei lebenden und infektiösen Mikr... more ABSTRACT Chorismat-umsetzende Enzyme spielen in Pflanzen sowie frei lebenden und infektiösen Mikroorganismen eine entscheidende Rolle bei der Biosynthese zahlreicher Metabolite. Zu diesen Enzymen zählen die primärmetabolische Anthranilatsynthase (AS) und die homologe, sekundärmetabolische Isochorismatsynthase (ICS). Beide Enzyme katalysieren mechanistisch ähnliche Reaktionen, wobei sie Ammoniak bzw. Wasser als Nukleophil nutzen. Wir zeigen hier, dass die Nukleophilspezifität der AS durch nur zwei Aminosäureaustausche im Zugangskanal zum aktiven Zentrum von Ammoniak auf Wasser erweitert werden kann. Die resultierende Bifunktionalität dieser AS/ICS-Variante verdeutlicht, dass die Evolution eines sekundärmetabolischen Enzyms von einem primärmetabolischen aus durch wenige Mutationen erfolgen kann und nicht zwingend eine initiale Genduplikation voraussetzt. Allgemein betrachtet, tragen unsere Erkenntnisse zum Verständnis bei, wie in der Natur innerhalb von Enzym-Superfamilien neue Reaktionen evolvieren.

Research paper thumbnail of Two-step glycosylation of the contact site A protein of Dictyostelium discoideum and transport of an incompletely glycosylated form to the cell surface

Journal of Biological Chemistry

Two different types of oligosaccharides, designated type 1 and 2 carbohydrate residues, are prese... more Two different types of oligosaccharides, designated type 1 and 2 carbohydrate residues, are present on the contact site A molecule, an 80-kDa glycoprotein involved in the formation of EDTA-stable cell adhesion during cell aggregation in Dictyostelium discoideum. The first precursor detected by pulse-chase labeling with [35S]methionine was a 68-kDa glycoprotein carrying type 1 carbohydrate. Conversion of the precursor into the 80-kDa form occurred simultaneously with the addition of type 2 carbohydrate. Tunicamycin inhibited type 1 glycosylation more efficiently than type 2 glycosylation. The first precursor detected in tunicamycin-treated cells by pulse-chase labeling was a 53-kDa protein lacking both carbohydrates, which was converted through addition of type 2 carbohydrate into a 66-kDa final product. Labeling of intact cells indicated that this 66-kDa glycoprotein is transported to the cell surface. Prolonged treatment with tunicamycin resulted in the accumulation within the cell...

Research paper thumbnail of Cytochrome P450 monooxygenases consisting of thermophilic bacteria

Research paper thumbnail of Faculty of 1000 evaluation for The evolutionary history of protein fold families and proteomes confirms that the archaeal ancestor is more ancient than the ancestors of other superkingdoms

F1000 - Post-publication peer review of the biomedical literature, 2012

Research paper thumbnail of Faculty of 1000 evaluation for Assessing computational methods for predicting protein stability upon mutation: good on average but not in the details

F1000 - Post-publication peer review of the biomedical literature, 2009

Research paper thumbnail of Faculty of 1000 evaluation for Practically useful: what the Rosetta protein modeling suite can do for you

F1000 - Post-publication peer review of the biomedical literature, 2010

Research paper thumbnail of Faculty of 1000 evaluation for Sequence space and the ongoing expansion of the protein universe

F1000 - Post-publication peer review of the biomedical literature, 2010

Research paper thumbnail of Faculty of 1000 evaluation for Discovery of new enzymes and metabolic pathways by using structure and genome context

F1000 - Post-publication peer review of the biomedical literature, 2013

Research paper thumbnail of Faculty of 1000 evaluation for FINDSITE: a threading-based approach to ligand homology modeling

F1000 - Post-publication peer review of the biomedical literature, 2009

Research paper thumbnail of Faculty of 1000 evaluation for An integrated semiconductor device enabling non-optical genome sequencing

F1000 - Post-publication peer review of the biomedical literature, 2011

Research paper thumbnail of Faculty of 1000 evaluation for Principles for designing ideal protein structures

F1000 - Post-publication peer review of the biomedical literature, 2013

Research paper thumbnail of Faculty of 1000 evaluation for Towards Robot Scientists for autonomous scientific discovery

F1000 - Post-publication peer review of the biomedical literature, 2011

Research paper thumbnail of Faculty of 1000 evaluation for Discriminative modeling of context-specific amino acid substitution probabilities

F1000 - Post-publication peer review of the biomedical literature, 2012

Research paper thumbnail of Faculty of 1000 evaluation for Surface sites for engineering allosteric control in proteins

F1000 - Post-publication peer review of the biomedical literature, 2008

Research paper thumbnail of Faculty of 1000 evaluation for Parallel adaptations to high temperatures in the Archaean eon

F1000 - Post-publication peer review of the biomedical literature, 2009

Research paper thumbnail of Ancestral Tryptophan Synthase Reveals Functional Sophistication of Primordial Enzyme Complexes

Cell chemical biology, Jan 23, 2016

Modern enzyme complexes are characterized by a high catalytic efficiency and allosteric communica... more Modern enzyme complexes are characterized by a high catalytic efficiency and allosteric communication between the constituting protein subunits. We were interested in whether primordial enzyme complexes from extinct species displayed a similar degree of functional sophistication. To this end, we used ancestral sequence reconstruction to resurrect the α and β subunits of the tryptophan synthase (TS) complex from the last bacterial common ancestor (LBCA), which presumably existed more than 3.4 billion years ago. We show that the LBCA TS subunits are thermostable and exhibit high catalytic activity. Moreover, they form a complex with αββα stoichiometry whose crystal structure is similar to that of modern TS. Kinetic analysis revealed that the reaction intermediate indole is channeled from the α to the β subunits and suggests that allosteric communication already occurred in LBCA TS.

Research paper thumbnail of A comparative categorization of protein function encoded in bacterial or archeal genomic islands

Journal of Molecular Evolution, Feb 1, 2006

Genomes of prokaryotes harbor genomic islands (GIs), which are frequently acquired via horizontal... more Genomes of prokaryotes harbor genomic islands (GIs), which are frequently acquired via horizontal gene transfer (HGT). Here I present an analysis of GIs with respect to gene-encoded functions. GIs were identified by statistical analysis of codon usage and clustering. Genes classified as putatively alien (pA) or putatively native (pN) were categorized according to the COG database. Among pA and pN genes, the distribution of COG functions and classes were studied for different groupings of prokaryotes. Groups were formed according to taxonomical relation or habitats. In all groups, genes related to class L (replication, recombination, and repair) were statistically significantly overrepresented in GIs. GIs of bacteria and archaea showed a distinct pattern of preferences. In archeal GIs, genes belonging to COG class M (cell wall/membrane/envelope biogenesis) or Q (secondary metabolites biosynthesis, transport, and catabolism) were more frequent. In bacterial GIs, genes of classes U (intracellular trafficking, secretion, and vesicular transport), N (cell motility), and V (defense mechanisms) were predominant. Underrepresentation was strongest for genes belonging to class J (translation, ribosomal structure, and biogenesis). Among single COG functions overrepresented in GIs were transferases and transporters. In both superkingdoms, HGT enhances genomic content by meeting demands that are independent of the studied habitats. These findings are in agreement with the complexity theory, which predicts the preferential import of operational genes. However, only specific subsets of operational genes were enriched in GIs. Modification of the cell envelope, cell motility, secretion, and protection of cellular DNA are major issues in HGT.

Research paper thumbnail of Long-Term Persistence of Bi-functionality Contributes to the Robustness of Microbial Life through Exaptation

PLOS Genetics, 2016

Modern enzymes are highly optimized biocatalysts that process their substrates with extreme effic... more Modern enzymes are highly optimized biocatalysts that process their substrates with extreme efficiency. Many enzymes catalyze more than one reaction; however, the persistence of such ambiguities, their consequences and evolutionary causes are largely unknown. As a paradigmatic case, we study the history of bi-functionality for a time span of approximately two billion years for the sugar isomerase HisA from histidine biosynthesis. To look back in time, we computationally reconstructed and experimentally characterized three HisA predecessors. We show that these ancient enzymes catalyze not only the HisA reaction but also the isomerization of a similar substrate, which is commonly processed by the isomerase TrpF in tryptophan biosynthesis. Moreover, we found that three modern-day HisA enzymes from Proteobacteria and Thermotogae also possess low TrpF activity. We conclude that this bi-functionality was conserved for at least two billion years, most likely without any evolutionary pressure. Although not actively selected for, this trait can become advantageous in the case of a gene loss. Such exaptation is exemplified by the Actinobacteria that have lost the trpF gene but possess the bi-functional HisA homolog PriA, which adopts the roles of both HisA and TrpF. Our findings demonstrate that bi-functionality can perpetuate in the absence of selection for very long time-spans.

Research paper thumbnail of Versatile Controlling System for Preparation Techniques in Electron-Microscopy

An universal software for controlling complex preparation protocols in electron microscopy was de... more An universal software for controlling complex preparation protocols in electron microscopy was developed. Its versatility will be demonstrated on the preparation technique of freeze-substitution. It controls all steps of the preparation protocol from the beginning, i.e., cryofixation, to the end, i.e., polymerisation.

Research paper thumbnail of Conversion of Anthranilate Synthase into Isochorismate Synthase: Implications for the Evolution of Chorismate-Utilizing Enzymes

Angewandte Chemie International Edition, 2015

Chorismate-utilizing enzymes play a vital role in the biosynthesis of metabolites in plants as we... more Chorismate-utilizing enzymes play a vital role in the biosynthesis of metabolites in plants as well as free-living and infectious microorganisms. Among these enzymes are the homologous primary metabolic anthranilate synthase (AS) and secondary metabolic isochorismate synthase (ICS). Both catalyze mechanistically related reactions by using ammonia and water as nucleophiles, respectively. We report that the nucleophile specificity of AS can be extended from ammonia to water by just two amino acid exchanges in a channel leading to the active site. The observed ICS/AS bifunctionality demonstrates that a secondary metabolic enzyme can readily evolve from a primary metabolic enzyme without requiring an initial gene duplication event. In a general sense, these findings add to our understanding how nature has used the structurally predetermined features of enzyme superfamilies to evolve new reactions.

Research paper thumbnail of Umwandlung einer Anthranilatsynthase in eine Isochorismatsynthase: Implikationen für die Evolution von Chorismat-umsetzenden Enzymen

Angewandte Chemie, 2015

ABSTRACT Chorismat-umsetzende Enzyme spielen in Pflanzen sowie frei lebenden und infektiösen Mikr... more ABSTRACT Chorismat-umsetzende Enzyme spielen in Pflanzen sowie frei lebenden und infektiösen Mikroorganismen eine entscheidende Rolle bei der Biosynthese zahlreicher Metabolite. Zu diesen Enzymen zählen die primärmetabolische Anthranilatsynthase (AS) und die homologe, sekundärmetabolische Isochorismatsynthase (ICS). Beide Enzyme katalysieren mechanistisch ähnliche Reaktionen, wobei sie Ammoniak bzw. Wasser als Nukleophil nutzen. Wir zeigen hier, dass die Nukleophilspezifität der AS durch nur zwei Aminosäureaustausche im Zugangskanal zum aktiven Zentrum von Ammoniak auf Wasser erweitert werden kann. Die resultierende Bifunktionalität dieser AS/ICS-Variante verdeutlicht, dass die Evolution eines sekundärmetabolischen Enzyms von einem primärmetabolischen aus durch wenige Mutationen erfolgen kann und nicht zwingend eine initiale Genduplikation voraussetzt. Allgemein betrachtet, tragen unsere Erkenntnisse zum Verständnis bei, wie in der Natur innerhalb von Enzym-Superfamilien neue Reaktionen evolvieren.

Research paper thumbnail of Two-step glycosylation of the contact site A protein of Dictyostelium discoideum and transport of an incompletely glycosylated form to the cell surface

Journal of Biological Chemistry

Two different types of oligosaccharides, designated type 1 and 2 carbohydrate residues, are prese... more Two different types of oligosaccharides, designated type 1 and 2 carbohydrate residues, are present on the contact site A molecule, an 80-kDa glycoprotein involved in the formation of EDTA-stable cell adhesion during cell aggregation in Dictyostelium discoideum. The first precursor detected by pulse-chase labeling with [35S]methionine was a 68-kDa glycoprotein carrying type 1 carbohydrate. Conversion of the precursor into the 80-kDa form occurred simultaneously with the addition of type 2 carbohydrate. Tunicamycin inhibited type 1 glycosylation more efficiently than type 2 glycosylation. The first precursor detected in tunicamycin-treated cells by pulse-chase labeling was a 53-kDa protein lacking both carbohydrates, which was converted through addition of type 2 carbohydrate into a 66-kDa final product. Labeling of intact cells indicated that this 66-kDa glycoprotein is transported to the cell surface. Prolonged treatment with tunicamycin resulted in the accumulation within the cell...