Multiple enzyme families can assemble oligopeptides without relying on ribosomal protein synthesis. This overview covers non-ribosomal peptide synthetases, ATP-grasp enzymes, α-amino acid ester acyltransferases, β-lactam acylases, and cyanophycinases, and it explains how these biocatalysts differ…
Non-ribosomal peptide synthesis is a core biochemical process that does not use the ribosome to assemble peptides. Instead, it is driven by enzymes called non-ribosomal peptide synthetases, or NRPSs for short. These enzymes are found in bacteria and fungi and are responsible for producing many therapeutically important peptides. The structures and biological activities of these products vary widely. Examples include penicillin, bleomycin, and cyclosporine.
Biocatalysts involved in non-ribosomal peptide synthesis can be sorted into two categories according to how they activate their substrates. The first category uses ATP and includes enzymes such as tRNA-dependent ligase, which activates amino acids through an aminoacyl-adenosine monophosphate intermediate. The second category does not use ATP. In this group, enzymes such as transacylase activate substrates through aminoacyl phosphate. Both routes ultimately allow an amide, or peptide, bond to form between amino acids.
A typical NRPS enzyme has a multidomain structure. The Protein Data Bank entry 2VSQ provides a representative view of this family. Such enzymes generally contain an adenylation domain A , a condensation domain C , a thioesterase domain Te , and a peptidyl carrier protein domain PCP . Together these domains catalyze amide bond formation.
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ATP-grasp enzymes are also known as ATP-dependent carboxylate-amine ligases. They activate acids by forming acylphosphate intermediates. These enzymes occur in many biological systems, including the de novo purine biosynthesis pathway. Notable members include biotin carboxylase, Ddl, and glutathione synthetase.
The name ATP-grasp comes from a distinctive structural feature. These enzymes generally have three conserved domains and a nonclassical ATP binding fold that encloses an ATP molecule. In the active site, most of these enzymes require a magnesium ion Mg2+ that is coordinated by ATP. This ion helps position the nucleotide for catalysis. An example of this enzyme class is glycinamide ribonucleotide synthetase. A structural model for this enzyme can be found in the Protein Data Bank under identifier 2IP4. This system illustrates how ATP-grasp enzymes form peptide bonds.
Kenzo and colleagues reported an efficient enzymatic method for producing oligopeptides from unprotected amino acids with high yield. The method used the bacterial strain Empedobacter brevis ATCC 14234. From this strain, the researchers found an enzyme catalyst called carboxypeptidase Y that helped rapidly produce certain oligopeptides. At the time, the report did not include the amino acid sequence, coding gene sequence, or three-dimensional crystal structure of this catalyst.
Later work by Isao ABE and team described the first cloning and expression of α-amino ester acyltransferases, abbreviated AETs, from the same strain and from another strain. The amino acid sequences of these AETs were 35% and 36% identical to the α-amino acid ester hydrolase from Acetobacter pasteurianus. AETs have dual activity. They act as dipeptidyl peptidases and as transferases. They also show strong specificity for both acyl donors and nucleophiles. To date, there have been no studies on the three-dimensional structure of AET or its reaction mechanisms.
β-lactam antibiotics contain a β-lactam ring in their chemical structure. This class includes penicillin, cephalosporins, and thiamycins. These drugs are widely used as anti-infective agents and carry considerable importance in the pharmaceutical industry. Enzymatic routes to β-lactam antibiotics are increasingly adopted by modern pharmaceutical companies because they offer an environmentally friendly and cost-effective way to synthesize these compounds.
β-lactam acylases are enzymes that historically process β-lactam antibiotics. They can also be used to make semi-synthetic β-lactam antibiotics. Several types of these enzymes have been studied for biosynthesis. These include penicillin acylase PA , glutaryl acylase GA , and β-amino acid ester hydrolase AEH .
Penicillin acylases are generally produced by a wide range of microorganisms. They are grouped into two categories depending on their substrate specificity. In industry, PAs are widely applied to produce 6-aminopenicillanic acid 6-APA , an active pharmaceutical intermediate. They are also used to synthesize semi-synthetic antibiotics and may be useful for developing new drugs. In addition, penicillin acylases can catalyze peptide synthesis, resolve racemic mixtures, and generate both achiral and chiral compounds used as pharmaceutical intermediates.
Cyanophycin granule polypeptide CGP , also called multi-L-arginyl-poly, is an intracellular storage polymer found in most cyanobacteria. The polymer has an aspartic acid backbone with equimolar concentrations of arginine and aspartic acid. Arginine moieties are linked to the β-carboxyl group of each aspartic acid through the arginine α-amino group.
In most genera of cyanobacteria, the cyanophycin synthetase gene cphA has been identified and verified to synthesize CGP. The intracellular and extracellular degradation of CGP is catalyzed by cyanophycinases named CphB and CphE. These enzymes release dipeptides such as β-Asp-Arg.
β-Asp-Arg can be efficiently synthesized by producing CGP and CGPase at the same time. This dipeptide has possible applications in feed or food that requires arginine Arg content. Production and isolation of CGP have already been successfully established in several recombinant hosts, including Escherichia coli, Nicotiana tabacum, Pseudomonas putida, and Pseudomonas alcaligenes DIP1.
These successes make it feasible to produce dipeptides such as β-Asp-Arg through metabolic engineering of suitable hosts and chemo-enzymatic strategies. In one recent example, CGP and CGPase were co-expressed in Nicotiana tabacum. Further analysis showed that this synthetic model can achieve sufficient storage and efficient transport of arginine and β-Asp-Arg dipeptides.
Reference: Wang, T., et al. Strategy for the biosynthesis of short oligopeptides: Green and sustainable chemistry. Biomolecules. 2019, 9 11 : 733.
Peptides referenced: Glutathione.
Related reading: Six biocatalytic strategies for enzymatic oligopeptide synthesis, Solid-Phase Peptide Synthesis: Resins and Working Protocols, Peptide-Receptor Systems for Tumor Imaging: A Field Guide, How cyclic peptides cross lipid membranes: a four-step mechanism.