Fungal GH25 muramidases: New family members with applications in animal nutrition and a crystal structure at 0.78Å resolution

Muramidases/lysozymes hydrolyse the peptidoglycan component of the bacterial cell wall. They are found in many of the glycoside hydrolase (GH) families. Family GH25 contains muramidases/lysozymes, known as CH type lysozymes, as they were initially discovered in the Chalaropsis species of fungus. The characterized enzymes from GH25 exhibit both β-1,4-N-acetyl- and β-1,4-N,6-O-diacetylmuramidase activities, cleaving the β-1,4-glycosidic bond between N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) moieties in the carbohydrate backbone of bacterial peptidoglycan. Here, a set of fungal GH25 muramidases were identified from a sequence search, cloned and expressed and screened for their ability to digest bacterial peptidoglycan, to be used in a commercial application in chicken feed. The screen identified the enzyme from Acremonium alcalophilum JCM 736 as a suitable candidate for this purpose and its relevant biochemical and biophysical and properties are described. We report the crystal structure of the A. alcalophilum enzyme at atomic, 0.78 Å resolution, together with that of its homologue from Trichobolus zukalii at 1.4 Å, and compare these with the structures of homologues. GH25 enzymes offer a new solution in animal feed applications such as for processing bacterial debris in the animal gut.

1 Overall quality at a glance i O The following experimental techniques were used to determine the structure:

X-RAY DIFFRACTION
The reported resolution of this entry is 0.78 Å.
Percentile scores (ranging between 0-100) for global validation metrics of the entry are shown in the following graphic. The table shows the number of entries on which the scores are based.

Metric
Whole archive (#Entries) Similar resolution (#Entries, resolution range(Å)) R The table below summarises the geometric issues observed across the polymeric chains and their t to the electron density. The red, orange, yellow and green segments on the lower bar indicate the fraction of residues that contain outliers for >=3, 2, 1 and 0 types of geometric quality criteria respectively. A grey segment represents the fraction of residues that are not modelled. The numeric value for each fraction is indicated below the corresponding segment, with a dot representing fractions <=5% The upper red bar (where present) indicates the fraction of residues that have poor t to the electron density. The numeric value is given above the bar.
Mol Chain Length Quality of chain 1 A 208 Full wwPDB X-ray Structure Validation Report 6ZM8 2 Entry composition i O There are 2 unique types of molecules in this entry. The entry contains 3666 atoms, of which 1585 are hydrogens and 0 are deuteriums.
In the tables below, the ZeroOcc column contains the number of atoms modelled with zero occupancy, the AltConf column contains the number of residues with at least one atom in alternate conformation and the Trace column contains the number of residues modelled with at most 2 atoms.
Molecule 1 is a protein called muramidase. 3 Residue-property plots i O These plots are drawn for all protein, RNA, DNA and oligosaccharide chains in the entry. The rst graphic for a chain summarises the proportions of the various outlier classes displayed in the second graphic. The second graphic shows the sequence view annotated by issues in geometry and electron density. Residues are color-coded according to the number of geometric quality criteria for which they contain at least one outlier: green = 0, yellow = 1, orange = 2 and red = 3 or more. A red dot above a residue indicates a poor t to the electron density (RSRZ > 2). Stretches of 2 or more consecutive residues without any outlier are shown as a green connector. Residues present in the sample, but not in the model, are shown in grey.

Mol Chain Residues
• Molecule 1: muramidase The all-atom clashscore is dened as the number of clashes found per 1000 atoms (including hydrogen atoms). The all-atom clashscore for this structure is 2.
All (8) O   In the following table, the Percentiles column shows the percent Ramachandran outliers of the chain as a percentile score with respect to all X-ray entries followed by that with respect to entries of similar resolution.
The Analysed column shows the number of residues for which the backbone conformation was analysed, and the total number of residues.

Protein sidechains i O
In the following table, the Percentiles column shows the percent sidechain outliers of the chain as a percentile score with respect to all X-ray entries followed by that with respect to entries of similar resolution.
The Analysed column shows the number of residues for which the sidechain conformation was analysed, and the total number of residues.
Mol Chain Analysed Rotameric Outliers Percentiles  (and percentage) of RSRZ outliers, followed by percent RSRZ outliers for the chain as percentile scores relative to all X-ray entries and entries of similar resolution. The OWAB column contains the minimum, median, 95 th percentile and maximum values of the occupancy-weighted average B-factor per residue. The column labelled`Q< 0.9' lists the number of (and percentage) of residues with an average occupancy less than 0.9.