Die Bedeutung von additiven und nicht‐additiven Mutationseffekten beim Protein‐Engineering

Reetz, Manfred T.

doi:10.1002/ange.201207842

Cited by 30 publications

(8 citation statements)

References 128 publications

(105 reference statements)

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“…If the effect of combining the mutations was additive, a twofold increase in reverse transcriptase activity would be expected. However, our results suggest an interaction between the individual mutations in a non‐additive manner that results in a synergistic effect of the mutations 10…”

Section: Specific Activities Of Ktq Variants On Dna or Rna Templatementioning

confidence: 62%

Structure and Function of an RNA‐Reading Thermostable DNA Polymerase

Blatter

Bergen

Nolte³

et al. 2013

Angew Chem Int Ed

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The high substrate specificity of DNA-dependent DNA polymerases is essential for the stability of the genome as well as many biotechnological applications. [1] The discrimination between ribo-and deoxyribonucleotides and between RNA and DNA, particularly in cells, is fundamental since the concentration of ribo moieties by far exceeds that of deoxyribo analogues. Although the selection mechanisms for the incorporation of nucleotides have been investigated intensively for DNA and RNA polymerases, [2] much less is known on how DNA-dependent DNA polymerases discriminate between the different nucleic acid templates (DNA versus RNA). Some viral DNA polymerases (e.g. reverse transcriptases) can utilize both DNA and RNA as a template for nucleic acid synthesis. Crystal-structure analysis of these enzymes, complexed either to an RNA or DNA template, have contributed significantly to our understanding of this process. [3] However, similar structural data of DNA-dependent DNA polymerases that have a poor propensity to process RNA templates are lacking, presumably because the crystallization is hampered by the formation of unstable complexes that leads to structural heterogeneity.Structure analysis of KlenTaq DNA polymerase, a shortened form of Thermus aquaticus DNA polymerase, has added significant contributions to the understanding of how DNA polymerases recognize the cognate substrate, [4] process abasic sites, [5] and non-natural nucleotides. [4d-g] Since our attempts to obtain suitable crystals of KlenTaq complexed to RNA failed, we set out to engineer the enzyme in such a way that it is capable of processing an RNA template more efficiently, which might result in improved crystallization properties. Indeed, we were able to obtain a significantly improved KlenTaq variant from which we obtained structural insights into a DNA-dependent DNA polymerase while processing RNA as a template for the first time. Furthermore, the generated KlenTaq variant turned out to be a thermostable DNA polymerase with significant reverse transcriptase activity, thus resulting in it being a valuable tool for crucial applications in clinical diagnostics and molecular biology, such as transcriptome analysis as well as the detection of pathogens and disease-specific markers.For the generation of an improved enzyme variant the mutation sites of two KlenTaq (KTq) variants M1 (L322M, L459M, S515R, I638F, S739G, E773G) [6] and M747K [7] were recombined by DNA shuffling. Both variants, M1 and M747K, were reported to possess either some reverse transcriptase activity or an expanded substrate spectrum. DNA shuffling was employed since the M1 variant, previously evolved in error-prone PCR, comprises six mutations distributed over the enzyme scaffold, but the individual contributions of the mutations are unknown. We generated a library of 1570 clones to obtain high coverage of all mutation combinations (as described in the Supporting Information). Proteins were expressed in 96-well plates and, after lysis and heat denaturation of the host proteins, use...

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Section: Specific Activities Of Ktq Variants On Dna or Rna Templatementioning

confidence: 62%

Structure and Function of an RNA‐Reading Thermostable DNA Polymerase

Blatter

Bergen

Nolte³

et al. 2013

Angew Chem Int Ed

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“…They interact with one another cooperatively (more than mathematical additivity). This is one of the most important lessons that we have learned from research in directed evolution, which has significant ramifications for enzymology in general 35…”

Section: Restructuring the Max‐planck‐institut Für Kohlenforschungmentioning

confidence: 94%

One Hundred Years of the Max‐Planck‐Institut für Kohlenforschung

Reetz

2014

Angew Chem Int Ed

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This Essay is an account of the institutional and scientific development of the Max‐Planck‐Institut für Kohlenforschung in Mülheim an der Ruhr (Germany), which is the successor to the Kaiser‐Wilhelm‐Institut für Kohlenforschung founded in 1914. The Essay is divided into four main parts, corresponding to the four major periods which are closely associated with the respective Directors of the Institute from 1914 to 2014: 1) Franz Fischer; 2) Karl Ziegler; 3) Günther Wilke; and 4) the period beginning with Manfred T. Reetz, who established a directorate comprising five Directors of equal status, each heading a different research department under the banner of catalysis. Along with key historical events associated with the Institute, research highlights of the four periods are featured.

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“…Vielmehr interagieren sie kooperativ miteinander (mehr als mathematische Additivität). Dies gehört zu den wichtigsten Lehren, die wir aus der Forschung in gerichteter Evolution gezogen haben, was erhebliche Auswirkungen auf die Enzymologie im Allgemeinen haben dürfte 35…”

Section: Umstrukturierung Des Max‐planck‐instituts Für Kohlenforscunclassified

100 Jahre Max‐Planck‐Institut für Kohlenforschung

Reetz

2014

Angewandte Chemie

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Dieser Essay beschreibt die institutionelle und wissenschaftliche Entwicklung des Max‐Planck‐Instituts für Kohlenforschung in Mülheim an der Ruhr, des Nachfolge‐Instituts des 1914 gegründeten Kaiser‐Wilhelm‐Instituts für Kohlenforschung. Die vier Hauptteile des Essays entsprechen den vier historischen Perioden, die mit den jeweiligen Direktoren zusammenhängen: 1) Franz Fischer; 2) Karl Ziegler; 3) Günther Wilke; 4) die von Manfred T. Reetz initiierte Umstrukturierung, in deren Folge ein Direktorium bestehend aus fünf gleichberechtigten Direktoren mit jeweils einer Abteilung etabliert wurde. Zusätzlich zu den rein historischen und politischen Entwicklungen werden auch Höhepunkte der Forschung aus den vier Perioden beschrieben.

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Die Bedeutung von additiven und nicht‐additiven Mutationseffekten beim Protein‐Engineering

Cited by 30 publications

References 128 publications

Structure and Function of an RNA‐Reading Thermostable DNA Polymerase

Structure and Function of an RNA‐Reading Thermostable DNA Polymerase

One Hundred Years of the Max‐Planck‐Institut für Kohlenforschung

100 Jahre Max‐Planck‐Institut für Kohlenforschung

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