A key enzyme in the biosynthetic pathway of Nisin

Nisin is a class of lantibiotics synthesized by Lactococcus lactis. Its biosynthesis is a complex process involving multiple enzymatic reactions, which essentially relies on the synergistic action of a series of key enzymes. The functions of these enzymes determine the efficiency of modification, processing, and maturation of the nisin precursor peptide. Below is a review of the research progress on core enzymes, starting from the key stages of the synthesis pathway:

I. Enzymes Related to Precursor Peptide Synthesis and Transport

The biosynthesis of nisin begins with the synthesis of the precursor peptide (NisA). The key enzymes in this stage are mainly responsible for the transcriptional coding and transmembrane transport of the precursor peptide, laying the foundation for subsequent modifications.

Enzymes Related to NisA Precursor Peptide Synthesis

The structural gene nisA of Nisin is transcribed into mRNA on the ribosomes of Lactococcus lactis, and then translated into a 57-amino-acid nisin precursor peptide (comprising a 23-amino-acid signal peptide and a 34-amino-acid mature peptide domain) with the participation of aminoacyl-tRNA synthetase and other enzymes. Among these, aminoacyl-tRNA synthetase ensures the accuracy of the amino acid sequence of the precursor peptide by accurately recognizing the corresponding amino acids and tRNAs, which is a prerequisite for the correct synthesis of "raw materials" for subsequent modification reactions.

ABC Transporter NisT

After the synthesis of the precursor peptide, it needs to be transported across the membrane to the inner side of the cell membrane (the space between the inner cell membrane and the peptidoglycan layer) via NisT, a member of the ABC transporter protein family, creating "spatial conditions" for subsequent post-translational modifications. Studies have shown that NisT has ATPase activity and can provide energy by hydrolyzing ATP, driving the specific recognition between the signal peptide of the precursor peptide and its own binding domain to achieve the directional transport of the precursor peptide. If the nisT gene mutates, the precursor peptide accumulates in the cytoplasm, preventing the initiation of subsequent modifications and directly blocking Nisin synthesis.

II. Key Enzymes for Post-Translational Modification

Post-translational modification is the core step for Nisin to form its active structure. With the action of enzymes such as lanthionine synthetase and dehydratase, special modified groups like lanthionine (Lan) and β-methyllanthionine (MeLan) are introduced into the mature peptide domain. These modifications determine the antimicrobial active conformation of Nisin.

Dehydratase NisB

NisB is a dehydratase dependent on S-adenosylmethionine (SAM), whose core function is to catalyze the dehydration of specific serine (Ser) and threonine (Thr) residues in the mature domain of the precursor peptide, generating dehydroalanine (Dha) and dehydrobutyrine (Dhb). Studies have shown that NisB first binds to the mature domain of the precursor peptide through its N-terminal domain, then uses SAM as a cofactor to activate the hydroxyl groups of Ser/Thr by transferring methyl groups, and subsequently catalyzes the elimination reaction of hydroxyl groups and adjacent hydrogen atoms to generate unsaturated Dha/Dhb. Moreover, NisB exhibits strict specificity for modification sites, acting only on 8 specific Ser/Thr residues (e.g., Ser3, Thr7) in the mature domain. These dehydration products are key intermediates for the subsequent formation of lanthionine.

Lanthionine Synthetase NisC

NisC is a redox enzyme containing a [4Fe-4S] cluster, responsible for catalyzing the addition reaction between the dehydration products (Dha/Dhb) and the sulfhydryl groups of cysteine (Cys) residues to form thioether bonds, thereby generating lanthionine (Lan) or β-methyllanthionine (MeLan). Specifically, the [4Fe-4S] cluster of NisC can stabilize reaction intermediates through electron transfer, promoting the nucleophilic attack of the double bond of Dha/Dhb by the sulfhydryl group of Cys. Studies have confirmed that there is an interprotein interaction between NisC and NisB, and the two can form a complex to act synergistically: after NisB completes dehydration, NisC can quickly bind to the modified precursor peptide, avoiding the degradation of intermediates and improving modification efficiency. If the [4Fe-4S] cluster structure of NisC is destroyed (e.g., by adding an iron chelating agent), the formation of thioether bonds is completely blocked, and the generated Nisin precursor peptide has no antimicrobial activity.

III. Key Enzymes for Precursor Peptide Processing and Maturation

The precursor peptide that has undergone post-translational modification still needs to have its signal peptide removed and undergo further processing to form mature Nisin with complete antimicrobial activity. The key enzymes in this stage are mainly responsible for signal peptide cleavage and product release.

Signal Peptidase NisP

NisP is a serine protease localized on the outer side of the cell membrane, whose function is to specifically cleave the signal peptide (23 amino acids) of the modified precursor peptide, releasing a 34-amino-acid mature Nisin precursor (containing modified groups). Studies have found that the catalytic domain of NisP can recognize the specific amino acid sequence (Ala-Val-Ala) at the junction of the signal peptide and the mature domain, and attack the peptide bond through the hydroxyl group of the serine residue to achieve precise cleavage of the signal peptide. If the active site of NisP (e.g., Ser231) mutates, the signal peptide cannot be removed, and the modified precursor peptide is anchored on the cell membrane surface, unable to be released outside the cell to exert its antimicrobial effect.

Transport and Maturation Auxiliary Enzyme NisI

Although NisI is not an enzyme directly involved in peptide bond cleavage, as a membrane-bound protein, it can bind to mature Nisin. On one hand, it protects Nisin from degradation by the host’s own proteases; on the other hand, it assists NisP in transporting the mature Nisin (after signal peptide cleavage) to the outside of the cell. Studies have shown that the binding site of NisI does not overlap with the antimicrobial active site of Nisin, which neither affects the subsequent antimicrobial function of Nisin nor improves the stability of mature Nisin. Thus, it is an important "protection and transport auxiliary factor" in the Nisin maturation process.

IV. Regulatory Enzymes

The biosynthesis of Nisin is also strictly regulated. Some enzymes indirectly affect the synthesis efficiency of key enzymes by participating in signal transduction or gene expression regulation, thereby controlling Nisin production.

Histidine Kinase NisK and Response Regulator NisR

These two constitute a typical two-component signal transduction system (TCS) and are the core regulatory modules for Nisin synthesis. When a certain concentration of Nisin accumulates in the environment, NisK (a histidine kinase on the cell membrane) senses this signal and undergoes autophosphorylation, then transfers the phosphate group to NisR (a response regulator protein) in the cell. Phosphorylated NisR binds to the promoter region of the nis gene cluster (including nisA, nisB, nisC, etc.), activates the transcription of related genes, and promotes the synthesis of key enzymes such as NisA, NisB, and NisC, forming a positive feedback regulation of "Nisin-induced self-synthesis". Studies have shown that the signal recognition domain of NisK has high specificity for Nisin and only responds to the induction of homologous Nisin, while the phosphorylation efficiency of NisR directly determines the transcription intensity of the nis gene cluster, making it a key node for regulating Nisin synthesis rate.

The biosynthesis of Nisin is the result of the synergistic action of key enzymes such as NisB, NisC, NisT, NisP, and NisK/NisR. These enzymes play irreplaceable roles in links such as "raw material synthesis-transport-modification-processing-regulation". Current research on these key enzymes not only reveals the synthesis mechanism of lantibiotics but also provides important targets for improving Nisin yield and optimizing its antimicrobial performance through genetic engineering to modify enzyme activity (e.g., enhancing the dehydration efficiency of NisB and the signal sensitivity of NisK).