Lysozyme—also known as N-acetyl-muramidase or muramidase—is an archetypal example of an antimicrobial enzyme with profound structural and mechanistic significance. Alexander Fleming discovered lysozyme in 1921, and it was first characterized by X-ray crystallography in the mid-1960s (Phillips, 1965). Lysozyme was the first enzyme whose three-dimensional structure was resolved via X-ray diffraction. Due to its structure, catalytic mechanism, and stability, lysozyme has become a canonical model in enzymology and structural biology.

At Creative Enzymes, we offer a diverse range of high-quality lysozyme products tailored for various applications. Discover more about lysozyme's molecular architecture and its significance in this article.

Primary and Tertiary Structure

Primary Sequence and Disulfide Architecture

The most widely studied form, hen egg-white lysozyme (HEWL), comprises 129 amino acids and has a molecular weight of approximately 14.3 kDa. It contains six tryptophan (Trp), three tyrosine (Tyr), and three phenylalanine (Phe) residues. The four disulfide bonds—between Cys6-Cys127, Cys30-Cys115, Cys64-Cys80, and Cys76-Cys94—enforce a compact and rigid tertiary fold.

Fold Architecture

Lysozyme adopts a compact, globular fold composed of two domains:

• An α-helical domain (~residues 1-35 and 89-129)
• A β-sheet domain (~residues 36-88).

These domains form a deep catalytic cleft that accommodates peptidoglycan chains, lined with six carbohydrate-binding subsites (A-F). The enzyme is highly cationic (isoelectric point ~11.3 for HEWL), facilitating strong electrostatic attraction to bacterial surfaces under physiological pH.

Figure 1. Unfolded lysozyme and folded lysozyme. (Dilip et al., 2022)

Catalytic Mechanism

At the core of lysozyme's antimicrobial function lies its ability to catalyze the hydrolysis of glycosidic bonds within bacterial cell wall peptidoglycan. Specifically, lysozyme targets the β-1,4 glycosidic linkage between N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG), which are the repeating sugar units critical to bacterial cell wall integrity. By cleaving this bond, lysozyme compromises the structural stability of the bacterial cell wall, ultimately leading to cell lysis. This activity is essential for its defensive role in innate immunity across many organisms.

Key Active-Site Residues

The enzymatic activity of lysozyme is mediated by two essential catalytic residues within its active site: Glutamic acid 35 (Glu35) and Aspartic acid 52 (Asp52). Glu35 acts as a general acid, donating a proton to facilitate the departure of the leaving group during bond cleavage. Asp52 functions as a nucleophile, stabilizing the oxocarbenium ion-like transition state or forming a transient covalent intermediate. The substrate binds across six well-defined subsites, labeled A through F, where the NAM moiety typically occupies subsite B and the NAG moiety binds at subsite A—this orientation aligns the scissile bond precisely for catalytic attack.

Reaction Pathway

Lysozyme has two widely discussed proposed mechanisms: the Koshland mechanism and the Phillips mechanism.

The Koshland mechanism suggests a retaining double-displacement pathway. In this model, Glu35 acts as a proton donor to facilitate glycosidic bond cleavage, while Asp52 performs a nucleophilic attack on the anomeric carbon, forming a covalent glycosyl-enzyme intermediate. Subsequently, a water molecule hydrolyzes this intermediate, restoring the enzyme and retaining the configuration of the sugar.

In contrast, the Phillips mechanism proposes a single-step SN1-like process. Here, the glycosidic bond is cleaved to generate an oxocarbenium ion-like transition state. Asp52 stabilizes this positive charge, while Glu35 donates a proton to assist bond cleavage. A water molecule then acts as a nucleophile to complete the reaction.

While both mechanisms offer insight into lysozyme's catalytic function, experimental evidence such as detection of covalent intermediates has lent greater support to the Koshland mechanism.

Figure 2. Two possible mechanisms of lysozyme.

Substrate Recognition

Effective catalysis by lysozyme depends not only on the chemistry of the active site but also on precise substrate recognition and positioning. This is achieved through multiple non-covalent interactions, including hydrogen bonds and hydrophobic stacking, involving residues such as Trp62, Trp63, Asp101, and others. These residues help anchor the peptidoglycan chain within the active cleft. However, chemical modifications to the peptidoglycan—such as O-acetylation of NAM or N-deacetylation of NAG—can hinder these interactions. Such modifications are used by bacteria like Staphylococcus aureus to evade lysozyme attack, representing a significant form of resistance.

Structural Diversity and Evolution

Lysozyme Families

Lysozymes are a structurally and functionally diverse group of enzymes found across a wide range of organisms. Based on their sequence homology, structural folds, and catalytic mechanisms, lysozymes are categorized into several families:

• C-Type (Chicken-Type) Lysozymes: These are the most widely studied, with a molecular weight of approximately 14 kDa, and are prevalent in vertebrates. They are characterized by a conserved α/β fold and are typically active against Gram-positive bacteria.
• G-Type (Goose-Type) Lysozymes: These enzymes are generally larger (20–22 kDa) and are predominantly found in birds and reptiles. Despite a different fold and catalytic mechanism, they serve a similar antimicrobial function.
• I-Type (Invertebrate-Type) Lysozymes: These exhibit greater structural variability and are found in various invertebrates. Their function can extend beyond antimicrobial defense to roles in digestion and immune modulation, reflecting their diverse evolutionary pressures.

Figure 3. Phylogenetic analysis of lysozymes. The names of lysozyme genes used in the analysis were shown as scientific name of species followed by GenBank accession number of this specific gene. The Ostrinia lysozymes are marked in red. The branches specific for invertebrate c-type, vertebrate c-type, i-type, and g-type lysozymes are shaded in yellow, blue, green, and orange, respectively. (Liu et al., 2014)

Evolutionary Conservation and Divergence

Despite divergence across taxa, core catalytic architecture—particularly the α/β fold and active-site residues—remains conserved, highlighting lysozyme's ancient role in innate immunity. Yet sequence variations in surface loops modulate substrate preferences, stability, and noncanonical functions.

Stability, Folding, and Biophysical Properties

• Thermal and pH Stability: HEWL exhibits high thermal stability (melting temperature up to -72 °C at pH 5), making it ideal for industrial applications. By contrast, human lysozyme is less thermostable and deactivates at lower temperatures. Lysozyme retains activity across a wide pH range (pH 6-9).
• Intrinsic Fluorescence and Structural Probing: The six tryptophan residues (notably Trp62 and Trp108) serve as intrinsic fluorophores, enabling studies on fold dynamics, ligand binding, and protein aggregation via fluorescence spectroscopy and molecular dynamics simulations.
• Amyloidosis and Aggregation Tendencies: Under destabilizing conditions (e.g., high SDS concentration), lysozyme can form amyloid-like fibrils, which serve as models for protein aggregation in systemic amyloidosis research.

Functional Roles in Host Defense

Antimicrobial Activity

Lysozyme plays a central role in the innate immune system by exerting bacteriolytic activity, primarily against Gram-positive bacteria. These organisms possess an exposed and thick peptidoglycan layer, which is readily accessible to lysozyme's enzymatic activity. In contrast, Gram-negative bacteria have an additional outer membrane that shields the peptidoglycan layer, rendering them generally more resistant to lysozyme unless the outer barrier is compromised by detergents, antimicrobial peptides, or host-derived factors. Importantly, lysozyme's antimicrobial effect is not solely dependent on its enzymatic cleavage of the β-1,4 glycosidic bond in peptidoglycan. Even when catalytic activity is diminished or inhibited, lysozyme retains bactericidal properties through alternative mechanisms, such as electrostatic interactions with negatively charged bacterial surfaces and induction of bacterial autolysins, enzymes that cause self-digestion of the bacterial cell wall.

Immunomodulatory Interactions

In addition to its direct antimicrobial role, lysozyme contributes significantly to immune modulation. The enzymatic degradation of bacterial peptidoglycan by lysozyme results in the release of muropeptides, small fragments that are recognized by intracellular pattern recognition receptors (PRRs), particularly NOD1 and NOD2 (nucleotide-binding oligomerization domain-containing proteins). These receptors initiate signaling cascades that promote pro-inflammatory responses, cytokine production, and immune cell recruitment, thereby enhancing the host's defense against infection. Furthermore, lysozyme has been shown to facilitate the processing and presentation of bacterial antigens, particularly in mucosal tissues, where it helps orchestrate a balanced immune response that resolves inflammation without excessive tissue damage. This immunomodulatory function is crucial in maintaining mucosal homeostasis, especially in environments such as the gastrointestinal and respiratory tracts.

Lectin-like Binding

Beyond its enzymatic and immune signaling functions, lysozyme exhibits lectin-like behavior, allowing it to bind directly to bacterial surface carbohydrates, such as lipopolysaccharides (LPS) and capsular polysaccharides, even in the absence of enzymatic activity. This non-catalytic binding facilitates bacterial agglutination, promotes immune recognition, and enhances phagocytosis by host immune cells. Such activity is particularly important when pathogens evolve mechanisms to inhibit lysozyme's catalytic function, as the lectin-like binding provides a secondary line of defense.

Structure-Function Relationships

• Relationship between Structure and Substrate Specificity: The structural configuration of lysozyme's active-site cleft, comprising subsites A through F, governs its substrate specificity by determining how peptidoglycan chains are accommodated during catalysis. Modifications to the bacterial cell wall, such as O-acetylation of NAM residues, introduce steric hindrance and prevent effective substrate binding. Such modifications are employed by several pathogenic bacteria, including Staphylococcus aureus, to evade lysozyme-mediated degradation, representing a major mechanism of resistance.
• Electrostatic Surface and Non-Catalytic Activity: Lysozyme features a strongly cationic (positively charged) surface, which promotes interaction with the anionic (negatively charged) surfaces of bacterial cells, particularly teichoic acids and phospholipid membranes. This electrostatic affinity not only enhances binding but also contributes to non-enzymatic bactericidal effects, including membrane permeabilization and activation of endogenous bacterial autolysins. These features underscore lysozyme's multifunctional role in host defense, extending beyond simple enzymatic hydrolysis.
• Structural Determinants of Stability: Lysozyme's remarkable structural robustness stems from its compact α/β fold, stabilized by four disulfide bridges, tight helix-sheet packing, and well-constrained loop regions. These features confer exceptional stability under conditions of heat, pH variation, and proteolytic challenge. As a result, lysozyme remains functional across diverse environmental conditions, making it suitable for therapeutic, diagnostic, and industrial applications where long-term activity and stability are critical.

In summary, lysozyme remains a molecular marvel: a modestly sized enzyme whose structural elegance underpins diverse biological functions—from cleaving bacterial walls to tuning immune responses. It is a keystone in enzymology, structural biology, immunology, and biotechnology.

At Creative Enzymes, we provide premium-grade lysozyme products designed to meet the highest standards of research and industrial application. Contact us today to find the optimal lysozyme for your needs.