Bioinformatics of cyanophycin metabolism genes and characterization of promiscuous isoaspartyl dipeptidases that catalyze the final step of cyanophycin degradation

Cyanophycin is a bacterial biopolymer used for storage of fixed nitrogen. It is composed of a backbone of L-aspartate residues with L-arginines attached to each of their side chains. Cyanophycin is produced by cyanophycin synthetase 1 (CphA1) using Arg, Asp and ATP, and is degraded in two steps. First, cyanophycinase breaks down the backbone peptide bonds, releasing β-Asp-Arg dipeptides. Then, these dipeptides are broken down into free Asp and Arg by enzymes with isoaspartyl dipeptidase activity. Two bacterial enzymes are known to possess promiscuous isoaspartyl dipeptidase activity: isoaspartyl dipeptidase (IadA) and isoaspartyl aminopeptidase (IaaA). We performed a bioinformatic analysis to investigate whether genes for cyanophycin metabolism enzymes cluster together or are spread around the microbial genomes. Many genomes showed incomplete contingents of known cyanophycin metabolizing genes, with different patterns in various bacterial clades. Cyanophycin synthetase and cyanophycinase are usually clustered together when recognizable genes for each are found within a genome. Cyanophycinase and isoaspartyl dipeptidase genes typically cluster within genomes lacking cphA1. About one-third of genomes with genes for CphA1, cyanophycinase and IaaA show these genes clustered together, while the proportion is around one-sixth for CphA1, cyanophycinase and IadA. We used X-ray crystallography and biochemical studies to characterize an IadA and an IaaA from two such clusters, in Leucothrix mucor and Roseivivax halodurans, respectively. The enzymes retained their promiscuous nature, showing that being associated with cyanophycin-related genes did not make them specific for β-Asp-Arg dipeptides derived from cyanophycin degradation.


F o r M a n u s c r i p t R e v i e w
Page 2 Full wwPDB X-ray Structure Validation Report (*For Manuscript Review*) 8DQM 1 Overall quality at a glance i ○ The following experimental techniques were used to determine the structure:

X-RAY DIFFRACTION
The reported resolution of this entry is 2.70 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. The table below summarises the geometric issues observed across the polymeric chains and their fit to the electron density. The red, orange, yellow and green segments of 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 fit to the electron density. The numeric value is given above the bar.

Metric
Mol Chain Length Quality of chain 2 Entry composition i ○ There are 4 unique types of molecules in this entry. The entry contains 8662 atoms, of which 0 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 Isoaspartyl aminopeptidase.     Full wwPDB X-ray Structure Validation Report (*For Manuscript Review*) 8DQM

Mol Chain Residues
3 Residue-property plots i ○ These plots are drawn for all protein, RNA, DNA and oligosaccharide chains in the entry. The first 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 fit 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.
• Molecule 1: Isoaspartyl aminopeptidase Chain A: Xtriage's analysis on translational NCS is as follows: The analyses of the Patterson function reveals a significant off-origin peak that is 20.60 % of the origin peak, indicating pseudo-translational symmetry. The chance of finding a peak of this or larger height randomly in a structure without pseudo-translational symmetry is equal to 8.3866e-03. The detected translational NCS is most likely also responsible for the elevated intensity ratio. The Z score for a bond length (or angle) is the number of standard deviations the observed value is removed from the expected value. A bond length (or angle) with |Z| > 5 is considered an outlier worth inspection. RMSZ is the root-mean-square of all Z scores of the bond lengths (or angles). There are no bond angle outliers.

Mol Chain
There are no chirality outliers.
There are no planarity outliers.

Too-close contacts i ○
In the following  Full wwPDB X-ray Structure Validation Report (*For Manuscript Review*) 8DQM Continued from previous page... 2  D  950  0  926  12  0  2  F  941  0  920  5  0  2  H  998  0  969  6  0  3  A  1  The all-atom clashscore is defined as the number of clashes found per 1000 atoms (including hydrogen atoms). The all-atom clashscore for this structure is 3.

Mol Chain Non-H H(model) H(added) Clashes Symm-Clashes
All (51) close contacts within the same asymmetric unit are listed below, sorted by their clash magnitude.    Full wwPDB X-ray Structure Validation Report (*For Manuscript Review*) 8DQM

Protein backbone i ○
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.
Mol Chain Analysed Favoured Allowed Outliers Percentiles There are no Ramachandran outliers to report.

Protein sidechains i ○
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.   5.4 Non-standard residues in protein, DNA, RNA chains i ○ There are no non-standard protein/DNA/RNA residues in this entry.

Carbohydrates i ○
There are no monosaccharides in this entry.

Ligand geometry i ○
Of 4 ligands modelled in this entry, 4 are monoatomic -leaving 0 for Mogul analysis.
There are no bond length outliers.
There are no bond angle outliers.
There are no chirality outliers.
There are no torsion outliers.
There are no ring outliers. Full wwPDB X-ray Structure Validation Report (*For Manuscript Review*) 8DQM No monomer is involved in short contacts.

Other polymers i ○
There are no such residues in this entry.

Polymer linkage issues i ○
There are no chain breaks in this entry.
F o r M a n u s c r i p t

R e v i e w
Page 16 Full wwPDB X-ray Structure Validation Report (*For Manuscript Review*) 8DQM 6 Fit of model and data i ○ 6.1 Protein, DNA and RNA chains i ○ In the following table, the column labelled '#RSRZ> 2' contains the number (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.  Full wwPDB X-ray Structure Validation Report (*For Manuscript Review*) 8DQM

Mol Chain
6.2 Non-standard residues in protein, DNA, RNA chains i ○ There are no non-standard protein/DNA/RNA residues in this entry.

Carbohydrates i ○
There are no monosaccharides in this entry.

Ligands i ○
In the following

Other polymers i ○
There are no such residues in this entry.