Martina Geier - Academia.edu (original) (raw)

Papers by Martina Geier

Figure S1. 1H NMR of trifluoperazine metabolite; Figure S2. 2D HSQC NMR of trifluoperazine metabo... more Figure S1. 1H NMR of trifluoperazine metabolite; Figure S2. 2D HSQC NMR of trifluoperazine metabolite; Figure S3. 2D HMBC NMR of trifluoperazine metabolite; Figure S4. 1H NMR of benzydamine metabolite and benzydamine; Figure S5. Comparison of DEPT NMR of benzydamine metabolite with 13C NMR of parent benzydamine.

Chimica Oggi-chemistry Today, 2013

for a correctly incorporated heme-cofactor and the need for the presence of their electron transf... more for a correctly incorporated heme-cofactor and the need for the presence of their electron transfer partners. If employed as whole-cell biocatalysts, the host systems should also exhibit features advantageous for process relevant parameters such as scalability and down-stream processing. During the recent years the methylotrophic yeast Pichia pastoris has emerged from a tool for single-cell protein production to a highly successful expression host for many proteins which are diffi cult to express in more simple hosts such as E. coli (4). Its success can be attributed to several factors including the ease of genetic manipulation, the presence of the strong and controllable AOX1 promoter allowing high intraand extracellular recombinant protein levels as well as the presence of a typical eukaryotic post-translational protein modifi cation machinery. Furthermore, P. pastoris can be grown on cheap media to cell densities yielding up to 130 gCDW/liter (4), thus allowing the simple product...

Biotechnology Advances, 2020

Competitive sustainable production in industry demands new and better biocatalysts, optimized bio... more Competitive sustainable production in industry demands new and better biocatalysts, optimized bioprocesses and cost-effective product recovery. Our review sheds light on the progress made for the individual steps towards these goals, starting with the discovery of new enzymes and their corresponding genes. The enzymes are subsequently engineered to improve their performance, combined in reaction cascades to expand the reaction scope and integrated in whole cells to provide an optimal environment for the bioconversion. Strain engineering using synthetic biology methods tunes the host for production, reaction design optimizes the reaction conditions and downstream processing ensures the efficient recovery of commercially viable products. Selected examples illustrate how modified enzymes can revolutionize future-oriented applications ranging from the bioproduction of bulk-, specialty-and fine chemicals, active pharmaceutical ingredients and carbohydrates, over the conversion of the greenhouse-gas CO 2 into valuable products and biocontrol in agriculture, to recycling of synthetic polymers and recovery of precious metals.

Nature Communications, 2018

The original HTML version of this Article included an incomplete 'Description of Additional Suppl... more The original HTML version of this Article included an incomplete 'Description of Additional Supplementary Files' which lacked legends for each file. The HTML version of the Article has been updated to include a corrected version of the 'Description of Additional Supplementary Files'.

Nature Communications, 2018

Numerous synthetic biology endeavors require well-tuned co-expression of functional components fo... more Numerous synthetic biology endeavors require well-tuned co-expression of functional components for success. Classically, monodirectional promoters (MDPs) have been used for such applications, but MDPs are limited in terms of multi-gene co-expression capabilities. Consequently, there is a pressing need for new tools with improved flexibility in terms of genetic circuit design, metabolic pathway assembly, and optimization. Here, motivated by nature’s use of bidirectional promoters (BDPs) as a solution for efficient gene co-expression, we generate a library of 168 synthetic BDPs in the yeast Komagataella phaffii (syn. Pichia pastoris), leveraging naturally occurring BDPs as a parts repository. This library of synthetic BDPs allows for rapid screening of diverse expression profiles and ratios to optimize gene co-expression, including for metabolic pathways (taxadiene, β-carotene). The modular design strategies applied for creating the BDP library could be relevant in other eukaryotic ho...

Angewandte Chemie, 2018

Schema 1. CYP2C9 katalysiert die Hydroxylierungvon Diclofenac (1). Schema 2. AOX-katalysierte Oxi... more Schema 1. CYP2C9 katalysiert die Hydroxylierungvon Diclofenac (1). Schema 2. AOX-katalysierte Oxidation von Phenanthridin (3). Schema 3. AOX-katalysierte Oxidation von Famciclovir (5).

Biotechnology and bioengineering, Jan 27, 2017

Carbon source regulated promoters are well-studied standard tools for controlling gene expression... more Carbon source regulated promoters are well-studied standard tools for controlling gene expression. Acquiring control over the natural regulation of promoters is important for metabolic engineering and synthetic biology applications. In the commonly used protein production host Komagataella phaffii (Pichia pastoris), methanol-inducible promoters are used because of their tight regulation and exceptional strength. Yet, induction with toxic and flammable methanol can be a considerable safety risk and cannot be applied in many existing fermentation plants. Here we studied new regulatory circuits based on the most frequently used alcohol oxidase 1 promoter (PAOX1 ), which is tightly repressed in presence of repressing carbon sources and strongly induced by methanol. We compared different overexpression strategies for putative carbon source dependent regulators identified by a homology search in related yeasts and previously published literature in order to convert existing methanol depen...

AIMS Bioengineering, 2015

With the emergence of synthetic biology and the vast knowledge about individual biocatalytic reac... more With the emergence of synthetic biology and the vast knowledge about individual biocatalytic reactions, the challenge nowadays is to implement whole natural or synthetic pathways into microorganisms. For this purpose balanced enzyme activities throughout the pathway need to be achieved in addition to simple functional gene expression to avoid bottlenecks and to obtain high titers of the desired product. As the optimization of pathways in a specific biological context is often hard to achieve by rational design, combinatorial approaches have been developed to address this issue. Here, current strategies and proof of concepts for combinatorial pathway assembly in yeasts are reviewed. By exploiting its ability to join multiple DNA fragments in a very efficient and easy manner, the yeast Saccharomyces cerevisiae does not only constitute an attractive host for heterologous pathway expression, but also for assembling pathways by recombination in vivo.

Journal of Biotechnology, 2016

Strains of the species Komagataella phaffii are the most frequently used "Pichia pastoris" strain... more Strains of the species Komagataella phaffii are the most frequently used "Pichia pastoris" strains employed for recombinant protein production as well as studies on peroxisome biogenesis, autophagy and secretory pathway analyses. Genome sequencing of several different P. pastoris strains has provided the foundation for understanding these cellular functions in recent genomics, transcriptomics and proteomics experiments. This experimentation has identified mistakes, gaps and incorrectly annotated open reading frames in the previously published draft genome sequences. Here, a refined reference genome is presented, generated with genome and transcriptome sequencing data from multiple P. pastoris strains. Twelve major sequence gaps from 20 to 6000 base pairs were closed and 5111 out of 5256 putative open reading frames were

ACS Chemical Biology, 2016

Flavin-containing mono-oxygenases are known as potent drug-metabolizing enzymes, providing comple... more Flavin-containing mono-oxygenases are known as potent drug-metabolizing enzymes, providing complementary functions to the well-investigated cytochrome P450 mono-oxygenases. While human FMO isoforms are typically involved in the oxidation of soft nucleophiles, the biocatalytic activity of human FMO5 (along its physiological role) has long remained unexplored. In this study, we demonstrate the atypical in vitro activity of human FMO5 as a Baeyer-Villiger mono-oxygenase on a broad range of substrates, revealing the first example to date of a human protein catalyzing such reactions. The isolated and purified protein was active on diverse carbonyl compounds, whereas soft nucleophiles were mostly non- or poorly reactive. The absence of the typical characteristic sequence motifs sets human FMO5 apart from all characterized Baeyer-Villiger mono-oxygenases so far. These findings open new perspectives in human oxidative metabolism.

Beilstein Journal of Organic Chemistry, 2015

Many synthetically useful reactions are catalyzed by cofactor-dependent enzymes. As cofactors rep... more Many synthetically useful reactions are catalyzed by cofactor-dependent enzymes. As cofactors represent a major cost factor, methods for efficient cofactor regeneration are required especially for large-scale synthetic applications. In order to generate a novel and efficient host chassis for bioreductions, we engineered the methanol utilization pathway of Pichia pastoris for improved NADH regeneration. By deleting the genes coding for dihydroxyacetone synthase isoform 1 and 2 (DAS1 and DAS2), NADH regeneration via methanol oxidation (dissimilation) was increased significantly. The resulting Δdas1 Δdas2 strain performed better in butanediol dehydrogenase (BDH1) based whole-cell conversions. While the BDH1 catalyzed acetoin reduction stopped after 2 h reaching ~50% substrate conversion when performed in the wild type strain, full conversion after 6 h was obtained by employing the knock-out strain. These results suggest that the P. pastoris Δdas1 Δdas2 strain is capable of supplying th...

Microbial Cell Factories, 2015

Background: Getting access to authentic human drug metabolites is an important issue during the d... more Background: Getting access to authentic human drug metabolites is an important issue during the drug discovery and development process. Employing recombinant microorganisms as whole-cell biocatalysts constitutes an elegant alternative to organic synthesis to produce these compounds. The present work aimed for the generation of an efficient whole-cell catalyst based on the flavin monooxygenase isoform 2 (FMO2), which is part of the human phase I metabolism. Results: We show for the first time the functional expression of human FMO2 in E. coli. Truncations of the C-terminal membrane anchor region did not result in soluble FMO2 protein, but had a significant effect on levels of recombinant protein. The FMO2 biocatalysts were employed for substrate screening purposes, revealing trifluoperazine and propranolol as FMO2 substrates. Biomass cultivation on the 100 L scale afforded active catalyst for biotransformations on preparative scale. The whole-cell conversion of trifluoperazine resulted in perfectly selective oxidation to 48 mg (46% yield) of the corresponding N 1-oxide with a purity >98%. Conclusions: The generated FMO2 whole-cell catalysts are not only useful as screening tool for human metabolites of drug molecules but more importantly also for their chemo-and regioselective preparation on the multi-milligram scale.

Chemical Communications, 2015

2A peptides enabling the coordinate expression of nine genes in P. pastoris represent a valuable ... more 2A peptides enabling the coordinate expression of nine genes in P. pastoris represent a valuable tool for pathway construction and engineering.

Microbial Cell Factories, 2012

Background Yarrowia lipolytica efficiently metabolizes and assimilates hydrophobic compounds such... more Background Yarrowia lipolytica efficiently metabolizes and assimilates hydrophobic compounds such as n-alkanes and fatty acids. Efficient substrate uptake is enabled by naturally secreted emulsifiers and a modified cell surface hydrophobicity and protrusions formed by this yeast. We were examining the potential of recombinant Y. lipolytica as a biocatalyst for the oxidation of hardly soluble hydrophobic steroids. Furthermore, two-liquid biphasic culture systems were evaluated to increase substrate availability. While cells, together with water soluble nutrients, are maintained in the aqueous phase, substrates and most of the products are contained in a second water-immiscible organic solvent phase. Results For the first time we have co-expressed the human cytochromes P450 2D6 and 3A4 genes in Y. lipolytica together with human cytochrome P450 reductase (hCPR) or Y. lipolytica cytochrome P450 reductase (YlCPR). These whole-cell biocatalysts were used for the conversion of poorly solub...

Database, 2012

Mutational events as well as the selection of the optimal variant are essential steps in the evol... more Mutational events as well as the selection of the optimal variant are essential steps in the evolution of living organisms. The same principle is used in laboratory to extend the natural biodiversity to obtain better catalysts for applications in biomanufacturing or for improved biopharmaceuticals. Furthermore, single mutation in genes of drug-metabolizing enzymes can also result in dramatic changes in pharmacokinetics. These changes are a major cause of patient-specific drug responses and are, therefore, the molecular basis for personalized medicine. MuteinDB systematically links laboratory-generated enzyme variants (muteins) and natural isoforms with their biochemical properties including kinetic data of catalyzed reactions. Detailed information about kinetic characteristics of muteins is available in a systematic way and searchable for known mutations and catalyzed reactions as well as their substrates and known products. MuteinDB is broadly applicable to any known protein and their variants and makes mutagenesis and biochemical data searchable and comparable in a simple and easy-to-use manner. For the import of new mutein data, a simple, standardized, spreadsheetbased data format has been defined. To demonstrate the broad applicability of the MuteinDB, first data sets have been incorporated for selected cytochrome P450 enzymes as well as for nitrilases and peroxidases.

Chemistry & Biodiversity, 2008

The reaction of aged carboplatin (reaction of carboplatin in 24 mM NaHCO 3 for 45 h, 37 o C, pH 8... more The reaction of aged carboplatin (reaction of carboplatin in 24 mM NaHCO 3 for 45 h, 37 o C, pH 8.6) with pBR322 DNA for 0 ≤ r ≤ 2.8, where r = [drug]/[DNAbp], in 24 mM HEPES buffer, pH 7.4, for 24 h followed by agarose gel electrophoresis showed DNA mobility changes consistent with unwinding closed circular DNA. However, identical experiments conducted in a two buffer system, 24mM HEPES plus 24mM carbonate, showed no DNA mobility changes, indicating that carbonate blocks formation of the 1,2-intrastrand crosslink on DNA. Studies with aged carboplatin and with cisplatin carried out with 2.0 ≤ r ≤ 10.0 in the two buffer system show that some DNA binding and unwinding occurs for both drugs. Since carbonate inhibits the binding of aged carboplatin and cisplatin to DNA, carbonate present in the body likely modulates the reactivity of these drugs with a variety of biological targets including DNA.

Figure S1. 1H NMR of trifluoperazine metabolite; Figure S2. 2D HSQC NMR of trifluoperazine metabo... more Figure S1. 1H NMR of trifluoperazine metabolite; Figure S2. 2D HSQC NMR of trifluoperazine metabolite; Figure S3. 2D HMBC NMR of trifluoperazine metabolite; Figure S4. 1H NMR of benzydamine metabolite and benzydamine; Figure S5. Comparison of DEPT NMR of benzydamine metabolite with 13C NMR of parent benzydamine.

Chimica Oggi-chemistry Today, 2013

for a correctly incorporated heme-cofactor and the need for the presence of their electron transf... more for a correctly incorporated heme-cofactor and the need for the presence of their electron transfer partners. If employed as whole-cell biocatalysts, the host systems should also exhibit features advantageous for process relevant parameters such as scalability and down-stream processing. During the recent years the methylotrophic yeast Pichia pastoris has emerged from a tool for single-cell protein production to a highly successful expression host for many proteins which are diffi cult to express in more simple hosts such as E. coli (4). Its success can be attributed to several factors including the ease of genetic manipulation, the presence of the strong and controllable AOX1 promoter allowing high intraand extracellular recombinant protein levels as well as the presence of a typical eukaryotic post-translational protein modifi cation machinery. Furthermore, P. pastoris can be grown on cheap media to cell densities yielding up to 130 gCDW/liter (4), thus allowing the simple product...

Biotechnology Advances, 2020

Competitive sustainable production in industry demands new and better biocatalysts, optimized bio... more Competitive sustainable production in industry demands new and better biocatalysts, optimized bioprocesses and cost-effective product recovery. Our review sheds light on the progress made for the individual steps towards these goals, starting with the discovery of new enzymes and their corresponding genes. The enzymes are subsequently engineered to improve their performance, combined in reaction cascades to expand the reaction scope and integrated in whole cells to provide an optimal environment for the bioconversion. Strain engineering using synthetic biology methods tunes the host for production, reaction design optimizes the reaction conditions and downstream processing ensures the efficient recovery of commercially viable products. Selected examples illustrate how modified enzymes can revolutionize future-oriented applications ranging from the bioproduction of bulk-, specialty-and fine chemicals, active pharmaceutical ingredients and carbohydrates, over the conversion of the greenhouse-gas CO 2 into valuable products and biocontrol in agriculture, to recycling of synthetic polymers and recovery of precious metals.

Nature Communications, 2018

The original HTML version of this Article included an incomplete 'Description of Additional Suppl... more The original HTML version of this Article included an incomplete 'Description of Additional Supplementary Files' which lacked legends for each file. The HTML version of the Article has been updated to include a corrected version of the 'Description of Additional Supplementary Files'.

Nature Communications, 2018

Numerous synthetic biology endeavors require well-tuned co-expression of functional components fo... more Numerous synthetic biology endeavors require well-tuned co-expression of functional components for success. Classically, monodirectional promoters (MDPs) have been used for such applications, but MDPs are limited in terms of multi-gene co-expression capabilities. Consequently, there is a pressing need for new tools with improved flexibility in terms of genetic circuit design, metabolic pathway assembly, and optimization. Here, motivated by nature’s use of bidirectional promoters (BDPs) as a solution for efficient gene co-expression, we generate a library of 168 synthetic BDPs in the yeast Komagataella phaffii (syn. Pichia pastoris), leveraging naturally occurring BDPs as a parts repository. This library of synthetic BDPs allows for rapid screening of diverse expression profiles and ratios to optimize gene co-expression, including for metabolic pathways (taxadiene, β-carotene). The modular design strategies applied for creating the BDP library could be relevant in other eukaryotic ho...

Angewandte Chemie, 2018

Schema 1. CYP2C9 katalysiert die Hydroxylierungvon Diclofenac (1). Schema 2. AOX-katalysierte Oxi... more Schema 1. CYP2C9 katalysiert die Hydroxylierungvon Diclofenac (1). Schema 2. AOX-katalysierte Oxidation von Phenanthridin (3). Schema 3. AOX-katalysierte Oxidation von Famciclovir (5).

Biotechnology and bioengineering, Jan 27, 2017

Carbon source regulated promoters are well-studied standard tools for controlling gene expression... more Carbon source regulated promoters are well-studied standard tools for controlling gene expression. Acquiring control over the natural regulation of promoters is important for metabolic engineering and synthetic biology applications. In the commonly used protein production host Komagataella phaffii (Pichia pastoris), methanol-inducible promoters are used because of their tight regulation and exceptional strength. Yet, induction with toxic and flammable methanol can be a considerable safety risk and cannot be applied in many existing fermentation plants. Here we studied new regulatory circuits based on the most frequently used alcohol oxidase 1 promoter (PAOX1 ), which is tightly repressed in presence of repressing carbon sources and strongly induced by methanol. We compared different overexpression strategies for putative carbon source dependent regulators identified by a homology search in related yeasts and previously published literature in order to convert existing methanol depen...

AIMS Bioengineering, 2015

With the emergence of synthetic biology and the vast knowledge about individual biocatalytic reac... more With the emergence of synthetic biology and the vast knowledge about individual biocatalytic reactions, the challenge nowadays is to implement whole natural or synthetic pathways into microorganisms. For this purpose balanced enzyme activities throughout the pathway need to be achieved in addition to simple functional gene expression to avoid bottlenecks and to obtain high titers of the desired product. As the optimization of pathways in a specific biological context is often hard to achieve by rational design, combinatorial approaches have been developed to address this issue. Here, current strategies and proof of concepts for combinatorial pathway assembly in yeasts are reviewed. By exploiting its ability to join multiple DNA fragments in a very efficient and easy manner, the yeast Saccharomyces cerevisiae does not only constitute an attractive host for heterologous pathway expression, but also for assembling pathways by recombination in vivo.

Journal of Biotechnology, 2016

Strains of the species Komagataella phaffii are the most frequently used "Pichia pastoris" strain... more Strains of the species Komagataella phaffii are the most frequently used "Pichia pastoris" strains employed for recombinant protein production as well as studies on peroxisome biogenesis, autophagy and secretory pathway analyses. Genome sequencing of several different P. pastoris strains has provided the foundation for understanding these cellular functions in recent genomics, transcriptomics and proteomics experiments. This experimentation has identified mistakes, gaps and incorrectly annotated open reading frames in the previously published draft genome sequences. Here, a refined reference genome is presented, generated with genome and transcriptome sequencing data from multiple P. pastoris strains. Twelve major sequence gaps from 20 to 6000 base pairs were closed and 5111 out of 5256 putative open reading frames were

ACS Chemical Biology, 2016

Flavin-containing mono-oxygenases are known as potent drug-metabolizing enzymes, providing comple... more Flavin-containing mono-oxygenases are known as potent drug-metabolizing enzymes, providing complementary functions to the well-investigated cytochrome P450 mono-oxygenases. While human FMO isoforms are typically involved in the oxidation of soft nucleophiles, the biocatalytic activity of human FMO5 (along its physiological role) has long remained unexplored. In this study, we demonstrate the atypical in vitro activity of human FMO5 as a Baeyer-Villiger mono-oxygenase on a broad range of substrates, revealing the first example to date of a human protein catalyzing such reactions. The isolated and purified protein was active on diverse carbonyl compounds, whereas soft nucleophiles were mostly non- or poorly reactive. The absence of the typical characteristic sequence motifs sets human FMO5 apart from all characterized Baeyer-Villiger mono-oxygenases so far. These findings open new perspectives in human oxidative metabolism.

Beilstein Journal of Organic Chemistry, 2015

Many synthetically useful reactions are catalyzed by cofactor-dependent enzymes. As cofactors rep... more Many synthetically useful reactions are catalyzed by cofactor-dependent enzymes. As cofactors represent a major cost factor, methods for efficient cofactor regeneration are required especially for large-scale synthetic applications. In order to generate a novel and efficient host chassis for bioreductions, we engineered the methanol utilization pathway of Pichia pastoris for improved NADH regeneration. By deleting the genes coding for dihydroxyacetone synthase isoform 1 and 2 (DAS1 and DAS2), NADH regeneration via methanol oxidation (dissimilation) was increased significantly. The resulting Δdas1 Δdas2 strain performed better in butanediol dehydrogenase (BDH1) based whole-cell conversions. While the BDH1 catalyzed acetoin reduction stopped after 2 h reaching ~50% substrate conversion when performed in the wild type strain, full conversion after 6 h was obtained by employing the knock-out strain. These results suggest that the P. pastoris Δdas1 Δdas2 strain is capable of supplying th...

Microbial Cell Factories, 2015

Background: Getting access to authentic human drug metabolites is an important issue during the d... more Background: Getting access to authentic human drug metabolites is an important issue during the drug discovery and development process. Employing recombinant microorganisms as whole-cell biocatalysts constitutes an elegant alternative to organic synthesis to produce these compounds. The present work aimed for the generation of an efficient whole-cell catalyst based on the flavin monooxygenase isoform 2 (FMO2), which is part of the human phase I metabolism. Results: We show for the first time the functional expression of human FMO2 in E. coli. Truncations of the C-terminal membrane anchor region did not result in soluble FMO2 protein, but had a significant effect on levels of recombinant protein. The FMO2 biocatalysts were employed for substrate screening purposes, revealing trifluoperazine and propranolol as FMO2 substrates. Biomass cultivation on the 100 L scale afforded active catalyst for biotransformations on preparative scale. The whole-cell conversion of trifluoperazine resulted in perfectly selective oxidation to 48 mg (46% yield) of the corresponding N 1-oxide with a purity >98%. Conclusions: The generated FMO2 whole-cell catalysts are not only useful as screening tool for human metabolites of drug molecules but more importantly also for their chemo-and regioselective preparation on the multi-milligram scale.

Chemical Communications, 2015

2A peptides enabling the coordinate expression of nine genes in P. pastoris represent a valuable ... more 2A peptides enabling the coordinate expression of nine genes in P. pastoris represent a valuable tool for pathway construction and engineering.

Microbial Cell Factories, 2012

Background Yarrowia lipolytica efficiently metabolizes and assimilates hydrophobic compounds such... more Background Yarrowia lipolytica efficiently metabolizes and assimilates hydrophobic compounds such as n-alkanes and fatty acids. Efficient substrate uptake is enabled by naturally secreted emulsifiers and a modified cell surface hydrophobicity and protrusions formed by this yeast. We were examining the potential of recombinant Y. lipolytica as a biocatalyst for the oxidation of hardly soluble hydrophobic steroids. Furthermore, two-liquid biphasic culture systems were evaluated to increase substrate availability. While cells, together with water soluble nutrients, are maintained in the aqueous phase, substrates and most of the products are contained in a second water-immiscible organic solvent phase. Results For the first time we have co-expressed the human cytochromes P450 2D6 and 3A4 genes in Y. lipolytica together with human cytochrome P450 reductase (hCPR) or Y. lipolytica cytochrome P450 reductase (YlCPR). These whole-cell biocatalysts were used for the conversion of poorly solub...

Database, 2012

Mutational events as well as the selection of the optimal variant are essential steps in the evol... more Mutational events as well as the selection of the optimal variant are essential steps in the evolution of living organisms. The same principle is used in laboratory to extend the natural biodiversity to obtain better catalysts for applications in biomanufacturing or for improved biopharmaceuticals. Furthermore, single mutation in genes of drug-metabolizing enzymes can also result in dramatic changes in pharmacokinetics. These changes are a major cause of patient-specific drug responses and are, therefore, the molecular basis for personalized medicine. MuteinDB systematically links laboratory-generated enzyme variants (muteins) and natural isoforms with their biochemical properties including kinetic data of catalyzed reactions. Detailed information about kinetic characteristics of muteins is available in a systematic way and searchable for known mutations and catalyzed reactions as well as their substrates and known products. MuteinDB is broadly applicable to any known protein and their variants and makes mutagenesis and biochemical data searchable and comparable in a simple and easy-to-use manner. For the import of new mutein data, a simple, standardized, spreadsheetbased data format has been defined. To demonstrate the broad applicability of the MuteinDB, first data sets have been incorporated for selected cytochrome P450 enzymes as well as for nitrilases and peroxidases.

Chemistry & Biodiversity, 2008

The reaction of aged carboplatin (reaction of carboplatin in 24 mM NaHCO 3 for 45 h, 37 o C, pH 8... more The reaction of aged carboplatin (reaction of carboplatin in 24 mM NaHCO 3 for 45 h, 37 o C, pH 8.6) with pBR322 DNA for 0 ≤ r ≤ 2.8, where r = [drug]/[DNAbp], in 24 mM HEPES buffer, pH 7.4, for 24 h followed by agarose gel electrophoresis showed DNA mobility changes consistent with unwinding closed circular DNA. However, identical experiments conducted in a two buffer system, 24mM HEPES plus 24mM carbonate, showed no DNA mobility changes, indicating that carbonate blocks formation of the 1,2-intrastrand crosslink on DNA. Studies with aged carboplatin and with cisplatin carried out with 2.0 ≤ r ≤ 10.0 in the two buffer system show that some DNA binding and unwinding occurs for both drugs. Since carbonate inhibits the binding of aged carboplatin and cisplatin to DNA, carbonate present in the body likely modulates the reactivity of these drugs with a variety of biological targets including DNA.