Nediljko Budisa

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Organization: Technische Universit?t Berlin , Germany

Department: Institut für Chemie

Title: (PhD)

Chemicals
References

Amide rotation trajectories probed by symmetry

Co-reporter:Vladimir Kubyshkin

Organic & Biomolecular Chemistry 2017 vol. 15(Issue 32) pp:6764-6772

Publication Date(Web):2017/08/16

DOI:10.1039/C7OB01421J

Amide rotation of peptidyl–prolyl fragments is an important factor in backbone structure organization of proteins. Computational studies have indicated that this rotation preferentially proceeds through a defined transition-state structure (syn/exo). Here, we complement the computational findings by determining the amide bond rotation barriers for derivatives of the two symmetric proline analogues, meso and racemic pyrrolidine-2,5-dicarboxylic acids. The rotations around these residues represent syn/exo–syn/exo and anti/endo–syn/exo hybrid transition states for the meso and racemic diastereomer, respectively. The rotation barriers are lower for the former rotation by about 9 kJ mol−1 (aqueous medium), suggesting a strong preference for the syn/exo (clockwise) rotation over the anti/endo (anticlockwise) rotation. The results show that both hybrid rotation processes are enthalpically driven but respond differently to solvent polarity changes due to the different transition state dipole–dipole interactions.

Biocatalysis with Unnatural Amino Acids: Enzymology Meets Xenobiology

Co-reporter:M. Sc. Federica Agostini;Dr. Jan-Stefan Völler; Dr. Beate Koksch;Dr. Carlos G. Acevedo-Rocha;Dr. Vladimir Kubyshkin; Dr. Nediljko Budisa

Angewandte Chemie International Edition 2017 Volume 56(Issue 33) pp:9680-9703

Publication Date(Web):2017/08/07

DOI:10.1002/anie.201610129

AbstractThe goal of xenobiology is to design biological systems endowed with unusual biochemical functions, whereas enzymology concerns the study of enzymes, the workhorses of biocatalysis. Biocatalysis employs enzymes and organisms to perform useful biotransformations in synthetic chemistry and biotechnology. During the past few years, the effects of incorporating noncanonical amino acids (ncAAs) into enzymes with potential applications in biocatalysis have been increasingly investigated. In this Review, we provide an overview of the effects of new chemical functionalities that have been introduced into proteins to improve various facets of enzymatic catalysis. We also discuss future research avenues that will complement unnatural mutagenesis with standard protein engineering to produce novel and versatile biocatalysts with applications in synthetic organic chemistry and biotechnology.

Biokatalyse mit nicht-natürlichen Aminosäuren: Enzymologie trifft Xenobiologie

Co-reporter:M. Sc. Federica Agostini;Dr. Jan-Stefan Völler; Dr. Beate Koksch;Dr. Carlos G. Acevedo-Rocha;Dr. Vladimir Kubyshkin; Dr. Nediljko Budisa

Angewandte Chemie 2017 Volume 129(Issue 33) pp:9810-9835

Publication Date(Web):2017/08/07

DOI:10.1002/ange.201610129

AbstractDas Ziel der Xenobiologie ist das Design von biologischen Systemen, die mit nicht-natürlichen biochemischen Funktionen ausgestattet sind, während die Enzymologie sich mit dem Verständnis von Enzymen auseinandersetzt, den Routinewerkzeugen der Biokatalyse. In der Biokatalyse werden Enzyme und Organismen dazu verwendet, um nützliche und leistungsfähige Biotransformationen in der synthetischen Chemie und Biotechnologie durchzuführen. In den vergangenen Jahren sind die Auswirkungen des Einbaus von nicht-kanonischen Aminosäuren (nkAAs) in Enzyme, welche potentiell interessante Anwendung für die Biokatalyse aufweisen, mit gesteigerter Aufmerksamkeit untersucht worden. Mit diesem Aufsatz wollen wir einen Überblick über die Effekte und Auswirkungen neuer chemischer Funktionalitäten geben, mit denen Proteine ausgestattet wurden, um eine Vielzahl von Aspekten der enzymatischen Biokatalyse zu verbessern. Ein weiteres Hauptaugenmerk liegt auf zukünftigen Forschungsperspektiven, welche die nicht-natürliche Mutagenese durch die Möglichkeiten des herkömmlichen Protein-Engineerings ergänzen werden, um neue und vielseitige Biokatalysatoren mit Anwendungen in der synthetischen organischen Chemie und Biotechnologie zu generieren.

Coupling genetic code expansion and metabolic engineering for synthetic cells

Co-reporter:Jan-Stefan Völler, Nediljko Budisa

Current Opinion in Biotechnology 2017 Volume 48(Volume 48) pp:

Publication Date(Web):1 December 2017

DOI:10.1016/j.copbio.2017.02.002

•Orthogonal translation (OT) of proteins with novel amino acids is well-established.•Only few attempts made to couple OT and metabolic engineering (ME).•ME is the basic prerequisite to transform OT to industrial biotechnology.•OT with synthetic metabolism is a basis for synthetic cells.•Synthetic cells are tools to study the core chemistry of life.Orthogonal protein translation with noncanonical amino acids (ncAAs) has become a standard method in biosciences. Whereas much effort is made to broaden the chemical space of ncAAs, only few attempts on their systematic low-cost in situ production are reported until now. The main aim is to engineer cells with newly designed biosynthetic pathways coupled with orthogonal aminoacyl-tRNA synthetase/tRNA pairs (o-pairs). These should provide cost-effective solutions to industrially relevant bio-production problems, such as peptide/protein production beyond the canonical set of natural molecules and to expand the arsenal of chemistries available for living cells. Therefore, coupling genetic code expansion (GCE) with metabolic engineering is the basic prerequisite to transform orthogonal translation from a standard technique in academic research to industrial biotechnology.Download high-res image (248KB)Download full-size image

Construction of a polyproline structure with hydrophobic exterior using octahydroindole-2-carboxylic acid

Co-reporter:Vladimir Kubyshkin

Organic & Biomolecular Chemistry 2017 vol. 15(Issue 3) pp:619-627

Publication Date(Web):2017/01/18

DOI:10.1039/C6OB02306A

The proline analogue (2S,3aS,7aS)-octahydroindole-2-carboxylic acid (Oic) has been previously applied as a proline substitute in pharmocologically active peptides and as a structural component of the antihypertensive drug Perindopril. Herein, we describe the formation of an oligoproline structure by an Oic oligomer. A series of Oic oligomers were investigated to show the structural and energetic contribution of appended residues to the structure. NMR investigation of these oligomers revealed an all-trans amide bond structure, and we provide evidence that a cascade-like mechanism is responsible for the all-trans folding cooperativity. X-ray crystallography of the Oic-hexapeptide clearly demonstrates that the all-trans structure of the Oic oligomer is a polyproline II helix. Thus, as a hydrophobic proline analog with a highly stable trans-amide bond, Oic represents an ideal building block for hydrophobic sites of polyproline II structures in biologically relevant contexts.

Global substitution of hemeproteins with noncanonical amino acids in Escherichia coli with intact cofactor maturation machinery

Co-reporter:Jan-Stefan Völler, Tuyet Mai Thi To, Hernan Biava, Beate Koksch, Nediljko Budisa

Enzyme and Microbial Technology 2017 Volume 106(Volume 106) pp:

Publication Date(Web):1 November 2017

DOI:10.1016/j.enzmictec.2017.06.014

•We developed an effective SPI method for incorporation of ncAAs into hemeproteins.•The designed experimental setup allows temporal control of heme production.•This guarantees intact cofactor maturation while the protein is engineered.•Our approach is probably applicable for other cofactor-containing proteins.•Presented strategy is valuable for biotechnological production of novel biocatalysts.Global substitution of canonical amino acids (cAAs) with noncanonical (ncAAs) counterparts in proteins whose function is dependent on post-translational events such as cofactor binding is still a methodically challenging and difficult task as ncAA insertion generally interferes with the cofactor biosynthesis machinery. Here, we report a technology for the expression of fully substituted and functionally active cofactor-containing hemeproteins. The maturation process which yields an intact cofactor is timely separated from cAA → ncAA substitutions. This is achieved by an optimised expression and fermentation procedure which includes pre-induction of the heme cofactor biosynthesis followed by an incorporation experiment at multiple positions in the protein sequence. This simple strategy can be potentially applied for engineering of other cofactor-containing enzymes.Download high-res image (167KB)Download full-size image

Energetic contribution to both acidity and conformational stability in peptide models

Co-reporter:Vladimir Kubyshkin, Patrick Durkin and Nediljko Budisa

New Journal of Chemistry 2016 vol. 40(Issue 6) pp:5209-5220

Publication Date(Web):31 Mar 2016

DOI:10.1039/C5NJ03611A

The acidity of N-acyl amino acids is dependent upon the rotameric state of the amide bond. In this work we systematically investigated the acidity difference of the rotamers (ΔpKa) in the frames of various acetylated amino acids. Our results indicated a mutual interaction of two carbonyl groups of an attractive type. We observed conservative ΔpKas for acyclic amino acids (2.2–3.0 kJ mol−1), whereas in the case of alicyclic amino acids, the experimental values revealed a strong dependency on the structural context (1.5–4.4 kJ mol−1). In homologous amino acids (α-, β-, γ-, etc.), the strength of the attraction decays in an exponential fashion. Furthermore, the interaction can accumulate through a chain of amide bonds in a cascade fashion, as demonstrated by an Ac-Pro-Pro dipeptide. As a result, we demonstrate that ΔpKa is an experimental parameter to estimate increments in the carbonyl–carbonyl alignment, as determined by the amino acid or peptidyl context. This parameter is also important in understanding the roles of amino acids in both protein folding and translation in biological systems as well as their evolutionary appearance in the genetic code.

Entropic Contribution of Elongation Factor P to Proline Positioning at the Catalytic Center of the Ribosome

Co-reporter:Lili K. Doerfel; Ingo Wohlgemuth; Vladimir Kubyshkin; Agata L. Starosta; Daniel N. Wilson; Nediljko Budisa;Marina V. Rodnina

Journal of the American Chemical Society 2015 Volume 137(Issue 40) pp:12997-13006

Publication Date(Web):September 18, 2015

DOI:10.1021/jacs.5b07427

The peptide bond formation with the amino acid proline (Pro) on the ribosome is slow, resulting in translational stalling when several Pro have to be incorporated into the peptide. Stalling at poly-Pro motifs is alleviated by the elongation factor P (EF-P). Here we investigate why Pro is a poor substrate and how EF-P catalyzes the reaction. Linear free energy relationships of the reaction on the ribosome and in solution using 12 different Pro analogues suggest that the positioning of Pro-tRNA in the peptidyl transferase center is the major determinant for the slow reaction. With any Pro analogue tested, EF-P decreases the activation energy of the reaction by an almost uniform value of 2.5 kcal/mol. The main source of catalysis is the favorable entropy change brought about by EF-P. Thus, EF-P acts by entropic steering of Pro-tRNA toward a catalytically productive orientation in the peptidyl transferase center of the ribosome.

Chemical Evolution of a Bacterial Proteome

Co-reporter:Dr. Michael Georg Hoesl;M.Sc. Stefan Oehm;Dr. Patrick Durkin;Dr. Elise Darmon;Dr. Lauri Peil;Dr. Hans-Rudolf Aerni;Dr. Juri Rappsilber;Dr. Jesse Rinehart;Dr. David Leach;Dr. Dieter Söll;Dr. Nediljko Budisa

Angewandte Chemie International Edition 2015 Volume 54( Issue 34) pp:10030-10034

Publication Date(Web):

DOI:10.1002/anie.201502868

Abstract

We have changed the amino acid set of the genetic code of Escherichia coli by evolving cultures capable of growing on the synthetic noncanonical amino acid L-β-(thieno[3,2-b]pyrrolyl)alanine ([3,2]Tpa) as a sole surrogate for the canonical amino acid L-tryptophan (Trp). A long-term cultivation experiment in defined synthetic media resulted in the evolution of cells capable of surviving Trp[3,2]Tpa substitutions in their proteomes in response to the 20 899 TGG codons of the E. coli W3110 genome. These evolved bacteria with new-to-nature amino acid composition showed robust growth in the complete absence of Trp. Our experimental results illustrate an approach for the evolution of synthetic cells with alternative biochemical building blocks.

Chemische Evolution eines bakteriellen Proteoms

Angewandte Chemie 2015 Volume 127( Issue 34) pp:10168-10172

Publication Date(Web):

DOI:10.1002/ange.201502868

Abstract

Der natürliche Satz an Aminosäuren innerhalb des genetischen Codes wurde durch kontinuierliche Evolution von Escherichia coli so modifiziert, dass diese in Gegenwart der synthetischen Aminosäure L-β-(Thieno[3,2-b]pyrrolyl)alanin ([3,2]Tpa) als alleiniges Surrogat zur kanonischen Aminosäure L-Tryptophan (Trp) wachsen. Mittels Langzeit-Kultivierung in synthetischem Nährmedium konnten Bakterien evolviert werden, die diese Substitution im Proteom an allen 20899 TGG-Codons im Genom von E. coli W3110 aufweisen. Die erzeugten Bakterien mit naturfremder Aminosäurezusammensetzung zeigen robustes Wachstum selbst in Abwesenheit von Tryptophan. Unsere Experimente stellen einen richtungweisenden Ansatz zur Evolution synthetischer Zellen mithilfe alternativer biochemischer Basiskomponenten dar.

Innentitelbild: Chemische Evolution eines bakteriellen Proteoms (Angew. Chem. 34/2015)

Angewandte Chemie 2015 Volume 127( Issue 34) pp:

Publication Date(Web):

DOI:10.1002/ange.201506522

Inside Cover: Chemical Evolution of a Bacterial Proteome (Angew. Chem. Int. Ed. 34/2015)

Angewandte Chemie International Edition 2015 Volume 54( Issue 34) pp:

Publication Date(Web):

DOI:10.1002/anie.201506522

Applying -Substituted Prolines in the Foldon Peptide: Polarity Contradicts Preorganization

Co-reporter:Dennis Dietz;Dr. Vladimir Kubyshkin ; Dr. Nediljko Budisa

ChemBioChem 2015 Volume 16( Issue 3) pp:403-406

Publication Date(Web):

DOI:10.1002/cbic.201402654

Abstract

Rational choice of chemical modifications to proline residues allows the preorganization principle to be exploited for more stable assembly of the foldon domain as a tag for trimerization. With systematic knowledge of how chemical and steric variations of the ring substituents affect the relative stabilities of exo and endo puckers, the preorganization principle should then be usable in biotechnologically synthesized foldon mutants and applicable for protein tagging elsewhere.

Expanded genetic code for the engineering of ribosomally synthetized and post-translationally modified peptide natural products (RiPPs)

Co-reporter:Nediljko Budisa

Current Opinion in Biotechnology (August 2013) Volume 24(Issue 4) pp:591-598

Publication Date(Web):1 August 2013

DOI:10.1016/j.copbio.2013.02.026

The number of constituent amino acids in ribosomally synthetized and post-translationally modified peptide natural products (RiPPs) is restricted to the 20 canonical amino acids. Microorganisms with an engineered genetic code are capable of delivering the biological, chemical, or physical properties of many unnatural or synthetic noncanonical amino acids, ncAAs (in different combinations of their numbers and chemistry) precisely defined by the chemist at the bench. In this way, post-translational modifications (PTMs) which make RiPPs chemically extremely rich can be augmented by the co-translational insertion of ncAAs. This will dramatically expand the chemical and functional space of these molecules and enable the design of novel and unique sequence combinations with improved specificity, stability, membrane permeability and even better oral availability.Graphical abstractDownload high-res image (272KB)Download full-size imageHighlights► RiPPs have high capabilities of target interaction far beyond the field of anti-infectives. ► RiPPs are especially suitable for design and engineering with the genetic code expansion. ► First published studies yielded RiPPs with unnatural/synthetic amino acids. ► Achievement: expanded scope of the in vivo (mainly bacterial) RiPPs synthesis. ► Perspective: design of RiPPS with novel chemistries and unique sequence combinations.

Construction of a polyproline structure with hydrophobic exterior using octahydroindole-2-carboxylic acid

Co-reporter:Vladimir Kubyshkin and Nediljko Budisa

Organic & Biomolecular Chemistry 2017 - vol. 15(Issue 3) pp:NaN627-627

Publication Date(Web):2016/12/08

DOI:10.1039/C6OB02306A