Statistics

The current version of PhytAMP holds 271 antimicrobial plant peptides (AMPs), secreted by various families such as Amaranthaceae [9], Andropogoneae [10], Brassicaceae [36], Oryzeae [11], Santalaceae [11], Spermacoceae [17], Triticeae [34], Vicieae [12], Violaceae [51]. Classification has been proposed on the basis of primary structure (Garcia-Olmedo et al., 1998; Castro & Fontes, 2005). Viola (family Violaceae) and Arabidopsis (family Brassicaceae) appear to be the predominant genera among AMP producers, although this may be due to the extensive studies on these species. Plant AMPs in the database are classified as cyclotides[76], defensins [55], Hevein-like [14], Impatiens [4], knottins [4], lipid-transfer proteins [45], shepherins [2], snakins [20], thionins [43] or vicilin-like [6], plus MBP-1 and MiAMP1. An unrooted tree of the AMPs was generated, as shown in Figure 1.

 

figure

Figure 1: Unrooted phylogenetic tree of plant antimicrobial peptides (AMPs) compiled in the PhytAMP database.

A multiple sequence alignment of 271 plant AMPs was used to calculate a matrix with the genetic distances for each pair of the sequences. Based on this matrix, successive clustering of lineages was done to construct the unrooted tree with the neighbor-joining algorithm (Phylip package). Tree was generated using FigTree (http://tree.bio.ed.ac.uk/software/figtree/). Three-dimensional coordinates were obtained from the Protein Data Bank (http://www.rcsb.org/pdb/). PDB accession ID numbers: Viscotoxin A3: 1ED0; β-hordothionin: 1WUW; Nt-LTP1: 1T12; MiAMP1: 1CO1; Circulin A: 1BH4; Kalata B1: 1JJZ; Hevein: 1HEV; Pa-AMP1: 1DKC; VrD1: 1IT5; γ-1-purothionin: 1GPS. Pictures were generated using PyMOL software [10]. α-helices and β-sheets are shown in red and purple, respectively.

 

 

It is noteworthy that only 69% of the peptides have been sequenced directly, the remaining structures having been predicted from genome sequences. For 83.4%, the amino acid sequence length varies from 20 to 67 (Figure 2).

Figure 2: Histogram of peptide length distribution in the PhytAMP database.

 

Table 1 summarizes the amino acid percentages. It is generally presumed that AMPs are cysteine-rich proteins (CRPs) and this was apparent in our statistical results.

Table 1: Amino acid occurrence in the PhytAMP database

 

Amino acid

Number of residues

% of total residues

C (cysteine)

1975

14.59

G (glycine)

1384

10.23

S (serine)

1185

8.76

A (alanine)

979

7.23

K (lysine)

896

6.62

T (threonine)

853

6.30

R (arginine)

819

6.05

P (proline)

817

6.04

N (asparagine)

743

5.49

V (valine)

610

4.51

I (isoleucine)

562

4.15

L (leucine)

549

4.06

Y (tyrosine)

414

3.06

Q (glutamine)

410

3.03

D (aspartic acid)

335

2.48

E (glutamic acid)

322

2.38

F (phenylalanine)

291

2.15

H (histidine)

194

1.43

W (tryptophan)

123

0.91

M (methionine)

73

0.54

 

Glycine is also an abundant amino acid, 98.5% of these AMPs containing at least one glycine residue. The majority (84.9%) have net charges varying from 0 to +10, while less than 6% possess a positive charge superior to +10, the highest being +17 (PHYT00099). In addition, only 9.2% have a net negative charge, the most negative being -6 (PHYT00259). As a result, the average net charge of all AMPs in PhytAMP is +4.6. Figure 3 shows the distribution of basic and acidic residues. The majority of sequences display a basic pattern, 53.1% having from six to eleven basic residues. In comparison, acidic residue content is more limited, 79.7% containing three or fewer acidic amino acids.

Figure 3: Bar graph of acidic and basic amino acid distribution among peptides in the PhytAMP database.

 

Current analysis revealed that three quarters of the plant AMPs contain between four and 13 hydrophobic residues. Only 39 were found to have 3D structures filed in the PDB database and resolved by NMR spectroscopy, crystallography or molecular modeling. Some of them nevertheless possess more than one structure in the PDB database, bringing the total number of 3D entries to 102. Only 39.5% are tested for biological activity. The majority possesses antifungal (51%), antibacterial (33%) and antiviral (10%) activities, as shown in Figure 4. These findings may be useful in isolating and characterizing novel plant AMPs or designing novel peptides with higher potency against pathogens or with broad antimicrobial spectra.

 

Figure 4: Chart of reported activities for plant peptides compiled in the PhytAMP database.