Structural Elucidation of the Bispecificity of A Domains as a Basis for Activating Non‐natural Amino Acids

Abstract

Many biologically active peptide secondary metabolites of bacteria are produced by modular enzyme complexes, the non-ribosomal peptide synthetases. Substrate selection occurs through an adenylation (A) domain, which activates the cognate amino acid with high fidelity. The recently discovered A domain of an Anabaenopeptin synthetase from Planktothrix agardhii (ApnA A1) is capable of activating two chemically distinct amino acids (Arg and Tyr). Crystal structures of the A domain reveal how both substrates fit into to binding pocket of the enzyme. Analysis of the binding pocket led to the identification of three residues that are critical for substrate recognition. Systematic mutagenesis of these residues created A domains that were monospecific, or changed the substrate specificity to tryptophan. The non-natural amino acid 4-azidophenylalanine is also efficiently activated by a mutant A domain, thus enabling the production of diversified non-ribosomal peptides for bioorthogonal labeling.

Synthesis of d -Amino Acid-Containing Dipeptides Using the Adenylation Domains of Nonribosomal Peptide Synthetase

“…The adenylation domain chosen as the target should activate D-amino acids. Enough information about adenylation domains that activate L-amino acids has been reported, such as the amino acid residues around the catalytic site and crystallization (12)(13)(14)(15). However, there are few reports about the D-amino acid-activating adenylation domains, and we needed to screen adenylation domains.…”

“…However, the module accepts restricted substrates because of the strict substrate specificity of both the adenylation domain and the condensation domain. Recent research has tended to analyze the adenylation domain alone (10,13,17) but has not tested substrate specificity toward D-amino acids. Thus, there is still little information about the adenylation domain itself.…”

Adenylation Domains in Nonribosomal Peptide Engineering

“… A) Domain swapping, B) subdomain swapping, and C) binding pocket mutagenesis are applied for reengineering NRPS specificity. D) The bispecific binding pocket of ApnA‐A 1 (PDB ID: 4D56) in complex with the ligands Arg‐AMS (cyan) and Tyr‐AMS (green). Mutations E204G/S243E and S243H promote activation of 4‐azido‐Phe.…”

Nicht‐ribosomale Peptidsynthese – Prinzipien und Perspektiven

“…Großbuchstaben kennzeichnen die Reste, die effektiv an der Substratbindung beteiligt sind, während der Stern die deutlich größere Bindungstasche der fungalen A‐Domäne SidNA3 anzeigt . (c)–(h) stellen die Bindungstaschen der A‐Domänen schematisch dar, die experimentell bestimmt wurden . Substrate sind blau, Asp1 und Lys10 rot und nicht‐wechselwirkende Reste grau dargestellt.…”

“…Eine häufige Beobachtung in Studien über A‐Domänen ist die relaxierte Substratspezifität, die als eine Strategie von NRP‐produzierenden Organismen zur Erweiterung ihres Naturstoffspektrums angesehen werden kann . Diese Promiskuität geht hauptsächlich auf den degenerierten nicht‐ribosomalen Code selbst zurück, da diverse Kombinationen von Aminosäureseitenketten in der Substratbindungstasche eine gewisse Plastizität gegenüber chemisch ähnlichen (Arg/Lys) oder chemisch unterschiedlichen Substraten (Arg/Tyr) ermöglichen. Darüber hinaus zeigen aktuelle Untersuchungen, dass C‐Domänen das Substratspezifitätsprofil ihrer benachbarten A‐Domäne beeinflussen können .…”