Structural Biochemistry/Nonribosomal Peptide Synthestases (NRPSs)

What are nonribosomal peptide synthetases (NRPSs) and what do they make?
-NRPSs are large, multimodular catalysts that assemble peptides.

-NRPSs are NOT ribosomes, but they synthesize peptides.

-The peptides that NRPSs make are called non-ribosomal peptides (NRPs).

-The reactions that NRPSs catalyze are stereospecific and regiospecific.

-NRPs have many clinical applications, including antibiotics, anitumors, and antifungals.

-NRPs are secondary metabolites produced by bacteria and fungi.

What is the structure of a NRPS?
-NRPSs are composed of modules.

-Each module incorporates one monomer into the NRP product.

-Each module can be subdivided into catalytic domains.

-The four catalytic domains that are found on most NRPSs are:
 * 1) Adenylation (A) domain
 * 2) Thiolation (T) or peptidyl carrier protein (PCP) domain
 * 3) Condensation (C) domain
 * 4) Thioesterase (TE) domain
 * -The function and reaction of each domain is discussed below.


 * -Knowledge on each of the domains was gained through NMR and x-ray crystal structure studies.
 * -Gramicidin S synthetase (GrsA) has been used to study the A and T domains.


 * A domain


 * -Activates the amino acid.


 * -There are about 550 amino acids that make up an A domain.


 * -Very selective “gatekeeper”: only allows certain monomers to be incorporated into the NRP product. The selectivity of the A domain is conferred by 10 amino acids, which are responsible for substrate binding in the A domain active site. There is a specific A domain for each amino acid that is incorporated into the NRP product.


 * -The A domain starts in an open conformation, which is proposed to able to bind to the amino acid and ATP. An adenylating intermediate is formed when the substrate (amino acid, Mg2+, and ATP) docks. The A domain closed formation is generated when the phosphoester bond of ATP is cleaved concurrently with aminoacyl adenylate formation and pyrophosphate release. The activated amino acid is then transferred onto the PCP domain and the A domain returns to its open conformation.


 * -The A domain can catalyze amino acid activation independently, without being connected to other domains.


 * GrsA studies


 * -The A domain of GrsA has been expressed in E. Coli and its 3D structure solved, it is described as being composed of two folded sub-domains.


 * -The A domain of GrsA is similar in structure to firefly luciferase, though the proteins only have 16% identical primary structure.


 * T/PCP domain


 * -Propagates the growing peptide chain.


 * -There are about 100 amino acids that make up a PCP domain.


 * -The PCP domain must be able to reach the A and C domains, which are involved in peptide elongation.


 * -The PCP domain can exist in three different conformations: apo (A), apo/holo (A/H), or holo (H). The PCP domain is converted from the apo to the holo form by post-translation priming with the 4’-phophopantetheine (ppant) cofactor. . This modification occurs at a conserved serine residue on the PCP domain and is carried out by ppant-transferases, like Bacillus subtilis ppant-transferase Sfp.


 * C domain


 * -Links amino acids via a condensation reaction.


 * -There are about 450 amino acids that make up a C domain.


 * -Possesses a V-shaped structure: two equal-sized subdomains are connected by a small hinge region at the base of the V and by a loop in the center of the V. The catalytically active amino acid is located in the middle of the V as well. The V shape allows the PCP domains that are sandwiching the C domain to position condensation substrates.


 * -Database analysis of C domains does not suggest homologous enzymes with similar activity. However, within the super-family of peptide synthetases, C domains are homologous to one another.


 * TE domain


 * -Releases the peptide product through hydrolysis or macrocyclization.


 * -There are about 250 amino acids that make up a TE domain.


 * -The genes that code for bacterial TE domains are homologous to genes of mammalian cells that code for the TE domains of fatty acid biosynthesis.


 * -A conserved aspartic residue is found in the TE domain.

Constructing new products from the domains
-Since domains are responsible for discrete functions in NRP product formation, it is possible that domains can be mixed and matched together to generate a template that can make new NRP products. Knowledge of the domains’ structure and biochemical function advance the possibility of constructing such a template.

-Advancements have been made in modifying existing NRPS systems, but a completely engineered template has not yet been achieved.

-The designed template must contain the correct order and number of activation modules.

-An approach to modifying existing NRPS systems could be accomplished by changing the amino acid activating module. The recombination method for changing the module involves two steps. The gene that codes for the activating module is targeted and is replaced with a hybrid gene. The hybrid gene codes for a new activating module that recognizes different substrates. The new substrate specificity results in different amino acids that are substituted into the NRP product. This approach was successfully used to re-engineer a surfactin synthetase found in B. subtilis


 * -The two step recombination requires genetic information about the NRPS system that is being targeted. Few NRPS systems are characterized thoroughly at the genetic level. Advancement in identifying peptide synthetase genes is facilitated by conservation of motif structures in the peptide synthetase domains.

-As random changes to NRPS products are unlikely to enhance their desired properties, modeling tools could be helpful in directing NRPS engineering efforts.

Questions that remain about NRPSs
-What is the mechanism of activated amino acid transfer from the A domain to the PCP domain?