Peptides 33 (2012) 220–229 Contents lists available at SciVerse ScienceDirect Peptides j our na l ho me p age : www.elsevier.com/locate/peptides Enhanced antifungal and insect -amylase inhibitory activities of Alpha-TvD1, a peptide variant of Tephrosia villosa defensin (TvD1) generated through in vitro mutagenesis S. Vijayan a,1 , J. Imani b,1 , K. Tanneeru c , L. Guruprasad c,∗ , K.H. Kogel b,∗ , P.B. Kirti a,∗ a Department of Plant Sciences, University of Hyderabad, Hyderabad 500046, India b Research Center for BioSystems, Land Use and Nutrition (IFZ Giessen), Justus Liebig University, Giessen, Germany c School of Chemistry, University of Hyderabad, Hyderabad 500046, India a r t i c l e i n f o Article history: Received 11 November 2011 Received in revised form 27 December 2011 Accepted 29 December 2011 Available online 8 January 2012 Keywords: Tephrosia villosa Wild type TvD1 Alpha-TvD1 Tenebrio molitor -amylase inhibition Protein–protein docking a b s t r a c t TvD1 is a small, cationic, and highly stable defensin from the weedy legume, Tephrosia villosa with demon- strated in vitro antifungal activity. We show here peptide modifications in TvD1 that lead to enhanced antifungal activities. Three peptide variants, S32R, D37R, and Alpha-TvD1 (-G-M-T-R-T-) with variations in and around the 2–3 loop region that imposes the two -strands, 2 and 3 were generated through in vitro mutagenesis. Alpha-TvD1 exhibited enhanced antifungal activity against the fungal pathogens, Fusarium culmorum and Fusarium oxysporum with respective IC 50 values of 2.5 M and 3.0 M, when compared to S32R (<5.0 M and >5.0 M), D37R (5.5 M and 4.5 M), and the wild type TvD1 (6.5 M). Because of the enhanced antifungal activity, this variant peptide was characterized further. Growth of F. culmorum in the presence of Alpha-TvD1 showed deformities in hyphal walls and nuclear damage. With respect to the plant pathogenic bacterium, Pseudomonas syringae pv. tomato strain DC3000, both Alpha-TvD1 and the wild type TvD1 showed comparable antibacterial activity. Both wild type TvD1 and Alpha-TvD1 displayed inhibitory activity against the -amylase of the mealworm beetle, Tenebrio molitor (TMA) with the latter showing enhanced activity. The human salivary as well as barley -amylase activ- ities were not inhibited even at concentrations of up to 50 M, which has been predicted to be due to differences in the pocket size and the size of the interacting loops. Present study shows that the variant Alpha-TvD1 exhibits enhanced antifungal as well as insect -amylase inhibitory activity. © 2012 Elsevier Inc. All rights reserved. 1. Introduction Plant defensins are small (5–10 kDa), highly stable, cysteine-rich peptides, which can act as broad-spectrum antimicrobial peptides with potent antifungal activity against certain fungal species [44]. The characteristic feature of plant defensin is the presence of four disulfide bridges between cysteine residues in the mature peptide forming the cysteine stabilized motif, but some plant defensins like PhD1 from Petunia hybrida exhibit five disulfide brides [18]. They carry one -helix and three anti-parallel -strands stabilized by four disulfide bridges folding into a CS configuration with three extra loops and net positive charge [5]. They were implicated in host defense and detrimental to fungal pathogens and bacteria [29,30,39,42]. ∗ Corresponding authors. E-mail addresses: lgpsc@uohyd.ernet.in (L. Guruprasad), Karl-Heinz.Kogel@agrar.uni-giessen.de (K.H. Kogel), pbksl@uohyd.ernet.in (P.B. Kirti). 1 Contributed equally to the investigation. The mode of action of plant defensins is still not precisely understood. They have been found to interact with components in the plasma membrane of higher fungi resulting in membrane permeabilization [41]. Their antifungal activities could be due to the presence of glycosyl ceramide residues in the fungal cell wall, whose interaction with the defensin peptides results in membrane damage followed by pore formation, ion leakage, and cell death eventually due to ionic imbalance [40,43]. In addition to antimicrobial activities, plant defensins are also reported to exhibit plant developmental [1,44], antiprolifer- ative [45], HIV-1 reverse transcriptase inhibitor [28], proteinase inhibitory [46] and -amylase inhibitory [3,22,31] activities and also inhibit plant parasites [47]. Plant defensins, such as VrD1 (Vigna radiata defensin) and VuD1 (V. unguiculata defensin), exhibit insect -amylase inhibitory activity, which was detrimental to insect pests like Tenebrio molitor and Z. subfasciatus, respectively [21,22,31]. The defensins with -amylase inhibitory activity inter- fere in insect digestion leading to deprivation of energy from starch [6]. In VrD1, the amino acids such as -G-M-T-R-T- present in the loop-3 region have been shown to be associated with the gut -amylase inhibitory activity in T. molitor, whereas VrD2 has 0196-9781/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2011.12.020