# Crystal Structure of Human Lysozyme Complexed with \(N\)-Acetyl-\(\alpha\)-D-glucosamine
## 1 Introduction  
In humans, lysozyme (muramidase or \(N\)-acetylmuramide glycanhydrolase) is found in several organs, tissues, and secretions. It is also found in the organs and secretions of other vertebrates, invertebrates, plants, and bacteria [1, 2]. This enzyme is functionally conserved and exhibits antimicrobial activity that is critical to host defense [3]. Lysozymes are classified into three main families based on their amino acid sequence and biochemical properties. These families include the chicken or conventional type (c-type), goose type (g-type), and invertebrate type (i-type) [2, 3]. In addition, phage type, bacterial type, and plant type lysozymes are also found in nature [4, 5, 6]. Bacterial cell walls are composed predominantly of peptidoglycan layers. Peptides dospicans consists of alternating units of \(N\)-acetylglucosamine (NAG or GlcNAc) and \(N\)-acetylmuramic acid (NAM or MurNAc). The peptide stems are covalently bound to a lactyl group in the NAM moiety [3]. Lysozymes enzymatically cleaves the peptidoglycan in bacterial cell walls by catalyzing the hydrolysis of the \(\beta\)-(1,4) linkages between the NAG and NAM saccharides [7]. These linkages are critical for the resistance of bacteria to osmotic stress, and their hydrolysis results in the loss of cellular membrane integrity and ultimately cell death by lysis [3, 8, 9]. Due to its broad antibacterial activity against pathogenic bacteria and viruses, lysozyme has been widely used in food, pharmaceutical, and clinical treatments, including various oral health care and antibiotic treatments [10, 11]. In humans, lysozyme is highly expressed in breast milk and acts as an important natural non-specific immune protein that participates in the immune response of infantsto bacterial [12]. For example, the concentration of lysozyme in the airway surface liquid of humans is estimated to be 20-100 ug/mL, which is sufficient to kill important lung pathogens, such as Gram-negative _Pseudomonas aeruginosa_ and Gram-positive _Staphylococcus aureus_[13]. Lysozyme enhances the immune system [14; 15], and in addition to antibacterial activity, it may also exhibit antitumor [16] and antiviral [17] activities. However, the molecular mechanism of these activities remains unclear. Many crystal structures of human lysozyme (hLZM) have been reported, but most crystal structures of hLZM have been used as model samples in X-ray crystallographic studies. For instance, only one crystal structure of saccharide-bound hLZM (PDB ID: 5 LSH, tetrasaccharide fragment from the O-chain of lipopolysaccharide from _Klebsiella pneumoniae_) [15] has been deposited in the Protein Data Bank (PDB). Accordingly, how hLZM recognizes various types of saccharide molecules has not been fully elucidated. On the other hand, the crystal structure of hLZM complexed with hexa-N-acetyl-chitohexose has previously been reported [18], but the coordinates and structural factors for this crystal structure are not deposited in PDB. Although the substrate recognition site of hLZM has been studied, it is not known how it recognizes single NAG or NAM sugar molecules constituting the substrate. The observation of single sugar molecule recognition of hLZM is expected to contribute to the initial first sugar molecule recognition when hLZM recognizes the actual substrate. In addition, it is unclear how hLZMs recognize sugars in bacterial cells and to what degree they are evolutionarily conserved with other lysozymes. To better understand saccharide recognition of hLZM, the crystal structures of native hLZM and hLZM complexed with \(N\)-acetyl-\(\alpha\)-D-glucosamine (\(\alpha\)-D-NAG) were determined at 1.30 A and 1.55/1.60 A resolution, respectively. The \(\alpha\)-D-NAG molecule was bound to the middle of subsites C and D in the active-site cleft of hLZM. The \(\alpha\)-D-NAG molecule was stabilized by conserved hydrophobic interactions, hydrogen bonds, and water bridges. The \(\alpha\)-D-NAG binding configuration of hLZM is unique when compared with crystal structure of sugar-bound lysozyme.