Directed Evolution Reveals the Functional Sequence Space of an Adenylation Domain Specificity Code.

Area of Science:

• Biochemistry
• Molecular Biology
• Natural Product Biosynthesis

Background:

• Nonribosomal peptides are crucial natural products synthesized by nonribosomal peptide synthetases (NRPSs).
• Adenylation (A) domains within NRPSs exhibit high substrate specificity, dictated by a 10-residue 'specificity code' in their binding pocket.
• Attempts to modify nonribosomal peptides via specificity code swapping have largely failed due to broadened specificity or loss of function.

Purpose of the Study:

• To deepen the understanding of A domain substrate selectivity.
• To analyze the specificity code of the EntF A domain, involved in enterobactin biosynthesis in Escherichia coli.
• To identify residues critical for strict specificity versus those tolerant of variation.

Main Methods:

• Utilized directed evolution techniques.
• Employed a genetic selection strategy.
• Performed detailed analysis of the EntF A domain specificity code.

Main Results:

• Determined which sites within the specificity code are strictly conserved and which allow for variation.
• Demonstrated that l-Ser-specific A domains, including EntF, possess a functional sequence space for l-Ser recognition, not a single fixed code.
• Discovered 152 novel l-Ser specificity codes, significantly expanding the known functional space.

Conclusions:

• Established that a functional sequence space, rather than a singular code, governs l-Ser recognition in A domains.
• Provided crucial insights into overcoming barriers to rational modification of A domain specificity.
• Opened new avenues for engineering NRPSs to produce novel nonribosomal peptides.