Phytocystatin 6 is a context-dependent, tight-binding inhibitor of Arabidopsis thaliana legumain isoform ß.

Plant legumains are crucial for processing seed storage proteins and are critical regulators of plant programmed cell death. Although research on legumains boosted recently, little is known about their activity regulation. In our study, we used pull-down experiments to identify AtCYT6 as a natural inhibitor of legumain isoform ß (AtLEGß) in Arabidopsis thaliana. Biochemical analysis revealed that AtCYT6 inhibits both AtLEGß and papain-like cysteine proteases through two separate cystatin domains. The N-terminal domain inhibits papain-like proteases, while the C-terminal domain inhibits AtLEGß. Furthermore, we showed that AtCYT6 interacts with legumain in a substrate-like manner, facilitated by a conserved asparagine residue in its reactive center loop. Complex formation was additionally stabilized by charged exosite interactions, contributing to pH-dependent inhibition. Processing of AtCYT6 by AtLEGß suggests a context-specific regulatory mechanism with implications for plant physiology, development, and programmed cell death. These findings enhance our understanding of AtLEGß regulation and its broader physiological significance.

Insight into the biochemical characterization of phytocystatin from Glycine max and its interaction with Cd+2 and Ni+2.

Phytocystatins are cysteine proteinase inhibitors ubiquitously present in plants and animals. They are known to carry out various significant physiological functions and also maintain the balance of protease-antiprotease activity. In the present disquisition, a phytocystatin after preliminary treatment has been isolated and purified to homogeneity from soybean (Glycine max) by a simple two-step stratagem using ammonium sulfate fractionation and gel filtration chromatography performed on Sephacryl S-100-HR. Soybean phytocystatin (SBPC) was purified with a fold purification of 635 and percent yield of 77.6%. A single band was observed on native gel electrophoresis confirming the homogeneity of the purified SBPC. The molecular weight of SBPC was found to be 19.05 kDa as determined by SDS-PAGE. The SBPC was found to be devoid of carbohydrate moieties and sulfhydryl group content. The binding stoichiometry of SBPC-papain interaction was determined by isothermal calorimetry suggesting 1:1 complex, and the value of binding constant (K) was found to be 2.78 × 105  M-1 The affinity of binding (Kd ) value obtained through ITC was 3.59 × 10-6  M. The purified SBPC was found to be stable in the pH range of 3 to 7 and is thermostable up to 50°C. The UV-visible and fluorescence studies showed significant changes in the conformation upon the formation of the SBPC-papain complex. Furthermore, fluorescence spectroscopy, ANS binding, and caseinolytic activity assay were conducted out to explore the effect of metal ions on SBPC which showed that there was a loss in the inhibitory activity along with conformational changes of SBPC upon complex formation with Cd+2 and Ni+2 .

The wheat multidomain cystatin TaMDC1 displays antifungal, antibacterial, and insecticidal activities in planta.

KEY MESSAGE: Expression of the TaMDC1 in transgenic tomato plants confer resistance to bacterial and fungal pathogens, as well as an insect pest and thus prove in planta function of the wheat cystatin. Cystatins are the polypeptides with cysteine proteinase inhibitory activities. Plant cystatins or phytocystatins are known to contribute to plant resistance against insect pests. Recently, increasing data proved that some of the phytocystatins also have antifungal activities in vitro. Here, we functionally characterized a wheat multidomain cystatin, TaMDC1, using in planta assays. Expression of TaMDC1 in wheat seedlings is up-regulated in response to methyl jasmonate and salicylic acid, indicating that TaMDC1 is involved in biotic stress responses mediated by these plant hormones. The TaMDC1 cDNA was integrated in tomato genome and expressed under cauliflower mosaic virus 35S promoter. Four transgenic plants that show high level of the transgene expression were selected by RNA gel blot and immunoblot analysis and utilized to assess biotic stress resistance against the bacterial pathogen Pseudomonas syringae, the fungal pathogens Botrytis cinerea and Alternaria alternata, and the insect pest Colorado potato beetle (CPB, Leptinotarsa decemlineata). Detached leaf inoculation assays revealed that the tomato plants expressing TaMDC1 showed high levels of resistance against P. syringae and A. alternata, and elevated tolerance against B. cinerea. Sustenance of L. decemlineata larvae to the transgenic plants demonstrated inhibition of CPB larvae growth. Inhibitory activity of TaMDC1 against selected pathogens was also demonstrated by in vitro assays with total protein extracted from transgenic tomato plants. Taken together, the presented data suggest that TaMDC1 is involved in a broad spectrum biotic stress resistance in planta.

Exposure of carbendazim induces structural and functional alteration in garlic phytocystatin: An in vitro multi-spectroscopic approach.

Carbendazim is a broad spectrum benzimidazole fungicide which is used to ensure plants' protection from pest and pathogens' invasion. The present work describes the impact of carbendazim (CAR) on garlic phytocystatin (GPC) which is a crucial plant regulatory protein. Interaction of carbendazim with GPC has been investigated through various biophysical techniques viz. UV absorption, fluorescence spectroscopy, isothermal titration calorimetry, far-UV circular dichroism and FTIR spectroscopy which showed binding between them with consequent modulatory effects. Functional activity of GPC was monitored by the anti-papain inhibitory assay which suggests that incubation of GPC with the higher concentration of CAR disrupts the inhibitory function of GPC. UV spectroscopy confirmed the formation of GPC-CAR complex. Intrinsic fluorescence suggests binding of CAR to GPC which reflects the changes in microenvironment around tryptophan residues of GPC. Isothermal titration calorimetry suggests that interaction of CAR to GPC is an exothermic reaction. Secondary structure analysis was also performed which confirmed that binding of CAR decreases the alpha-helical content of GPC. Collectively, these results demonstrated that GPC exhibited significant structural and functional alteration upon interaction with carbendazim. Since GPC is involved in various regulatory processes, therefore, its structural or functional alteration may lead to disruption of physiological and biological balance within the plant. Hence, our study signifies that exposure of carbendazim to plant exerts physicochemical alteration within the plant.

Rice bifunctional phytocystatin is a dual modulator of legumain and papain-like proteases.

Phytocystatins are well-known inhibitors of C1A cysteine proteinases. However, previous research has revealed legumain (C13) protease inhibition via a carboxy-extended phytocystatin. Among the 12 phytocystatins genes in rice, OcXII is the only gene possessing this carboxy-terminal extension. The specific legumain inhibition activity was confirmed, in our work, using a recombinant OcXII harboring only the carboxy-terminal domain and this part did not exhibit any effect on papain-like activities. Meanwhile, rice plants silenced at the whole OcXII gene presented higher legumain and papain-like proteolytic activities, resulting in a faster initial seedling growth. However, when germinated under stressful alkaline conditions, OcXII-silenced plants exhibited impaired root formation and delayed shoot growth. Interestingly, the activity of OcXII promoter gene was detected in the rice seed scutellum region, and decreases with seedling growth. Seeds from these plants also exhibited slower growth at germination under ABA or alkaline conditions, while maintaining very high levels of OcXII transcriptional activation. This likely reinforces the proteolytic control necessary for seed germination and growth. In addition, increased legumain activity was detected in OcXII RNAi plants subjected to a fungal elicitor. Overall, the results of this study highlight the association of OcXII with not only plant development processes, but also with stress response pathways. The results of this study reinforce the bifunctional ability of carboxy-extended phytocystatins in regulating legumain proteases via its carboxy-extended domain and papain-like proteases by its amino-terminal domain.

Physico-chemical and in-silico analysis of a phytocystatin purified from Brassica juncea cultivar RoAgro 5444.

This study describes the isolation and purification of a phytocystatin from seeds of Brassica juncea (Indian mustard; cultivar RoAgro 5444), which is an important oilseed crop both agriculturally and economically. The protein was purified by gel filtration chromatography with 24.3% yield and 204-fold purification, and visualised by 2D gel electrophoresis. The 18.1 kDa mustard cystatin was highly specific for cysteine proteinases. The plant cystatin inhibited cathepsin B, confirming its role in conferring pest resistance. The inhibitor was highly stable over a pH range of 3-10 and retained significant inhibitory potential up to 70 °C. The stoichiometry of its interaction with papain, determined by isothermal calorimetry, suggests a 1:1 complex. Secondary structural elements calculated by far-UV circular dichroism (CD) spectroscopy show an 18.8% α-helical and 21% ß-sheet structure. The protein was a non-competitive inhibitor of thiol proteinases. The Stokes radius and frictional co-efficient were used to describe the shape and size of the protein. Homology modelling and docking studies proposed a prototype illustrating the Brassica phytocystatin mediated papain inhibition. Molecular dynamics (MD) study revealed the excellent stability of the papain-phytocystatin complex during a simulation for 100 ns. Detailed results identify the mustard cystatin as an important member of the phytocystatin family.

Purification and biochemical characterization of phytocystatin from Brassica alba.

Phytocystatins belong to the family of cysteine proteinases inhibitors. They are ubiquitously found in plants and carry out various significant physiological functions. These plant derived inhibitors are gaining wide consideration as potential candidate in engineering transgenic crops and in drug designing. Hence it is crucial to identify these inhibitors from various plant sources. In the present study a phytocystatin has been isolated and purified by a simple two-step procedure using ammonium sulfate saturation and gel filtration chromatography on Sephacryl S-100HR from Brassica alba seeds (yellow mustard seeds).The protein was purified to homogeneity with 60.3% yield and 180-fold of purification. The molecular mass of the mustard seed cystatin was estimated to be nearly 26,000 Da by sodium dodecyl sulfate polyacrylamide gel electrophoresis as well as by gel filtration chromatography. The stokes radius and diffusion coefficient of the mustard cystatin were found to be 23A° and 9.4 × 10(-7) cm(2) s(-1) respectively. The isolated phytocystatin was found to be stable in the pH range of 6-8 and is thermostable up to 60 °C. Kinetic analysis revealed that the phytocystatin exhibited non-competitive type of inhibition and inhibited papain more efficiently (K(i) = 3 × 10(-7) M) than ficin (K(i) = 6.6 × 10(-7) M) and bromelain (K(i) = 7.7 × 10(-7) M respectively). CD spectral analysis shows that it possesses 17.11% alpha helical content.

Phytocystatins: Defense Proteins against Phytophagous Insects and Acari.

This review deals with phytocystatins, focussing on their potential role as defence proteins against phytophagous arthropods. Information about the evolutionary, molecular and biochemical features and inhibitory properties of phytocystatins are presented. Cystatin ability to inhibit heterologous cysteine protease activities is commented on as well as some approaches of tailoring cystatin specificity to enhance their defence function towards pests. A general landscape on the digestive proteases of phytophagous insects and acari and the remarkable plasticity of their digestive physiology after feeding on cystatins are highlighted. Biotechnological approaches to produce recombinant cystatins to be added to artificial diets or to be sprayed as insecticide-acaricide compounds and the of use cystatins as transgenes are discussed. Multiple examples and applications are included to end with some conclusions and future perspectives.

Crystal structure and interconversion of monomers and domain-swapped dimers of the walnut tree phytocystatin.

Biotechnological applications of phytocystatins have garnered significant interest due to their potential applications in crop protection and improve crop resistance to abiotic stress factors. Cof1 and Wal1 are phytocystatins derived from Coffea arabica and Juglans regia, respectively. These plants hold significant economic value due to coffee's global demand and the walnut tree's production of valuable timber and widely consumed walnuts with culinary and nutritional benefits. The study involved the heterologous expression in E. coli Lemo 21(DE3), purification by immobilized metal ion affinity and size exclusion chromatography, and biophysical characterization of both phytocystatins, focusing on isolating and interconverting their monomers and dimers. The crystal structure of the domain-swapped dimer of Wal1 was determined revealing two domain-swapped dimers in the asymmetric unit, an arrangement reminiscent of the human cystatin C structure. Alphafold models of monomers and Alphafold-Multimer models of domain-swapped dimers of Cof1 and Wal1 were analyzed in the context of the crystal structure. The methodology and data presented here contribute to a deeper understanding of the oligomerization mechanisms of phytocystatins and their potential biotechnological applications in agriculture.

In Vitro Antibacterial Activity of Cysteine Protease Inhibitor from Kiwifruit (Actinidia deliciosa).

The need for replacing traditional pesticides with alternative agents for the management of agricultural pathogens is rising worldwide. In this study, a cysteine proteinase inhibitor (CPI), 11 kDa in size, was purified from green kiwifruit to homogeneity. We examined the growth inhibition of three plant pathogenic Gram-negative bacterial strains by kiwi CPI and attempted to elucidate the potential mechanism of the growth inhibition. CPI influenced the growth of phytopathogenic bacteria Agrobacterium tumefaciens (76.2 % growth inhibition using 15 µM CPI), Burkholderia cepacia (75.6 % growth inhibition) and, to a lesser extent, Erwinia carotovora (44.4 % growth inhibition) by inhibiting proteinases that are excreted by these bacteria. Identification and characterization of natural plant defense molecules is the first step toward creation of improved methods for pest control based on naturally occurring molecules.

Recombinant sugarcane cystatin CaneCPI-5 promotes osteogenic differentiation.

Cysteine proteases orchestrate bone remodeling, and are inhibited by cystatins. In reinforcing our hypothesis that exogenous and naturally obtained inhibitors of cysteine proteases (cystatins) act on bone remodeling, we decided to challenge osteoblasts with sugarcane-derived cystatin (CaneCPI-5) for up to 7 days. To this end, we investigated molecular issues related to the decisive, preliminary stages of osteoblast biology, such as adhesion, migration, proliferation, and differentiation. Our data showed that CaneCPI-5 negatively modulates both cofilin phosphorylation at Ser03, and the increase in cytoskeleton remodeling during the adhesion mechanism, possibly as a prerequisite to controlling cell proliferation and migration. This is mainly because CaneCPI-5 also caused the overexpression of the CDK2 gene, and greater migration of osteoblasts. Extracellular matrix remodeling was also evaluated in this study by investigating matrix metalloproteinase (MMP) activities. Our data showed that CaneCPI-5 overstimulates both MMP-2 and MMP-9 activities, and suggested that this cellular event could be related to osteoblast differentiation. Additionally, differentiation mechanisms were better evaluated by investigating Osterix and alkaline phosphatase (ALP) genes, and bone morphogenetic protein (BMP) signaling members. Altogether, our data showed that CaneCPI-5 can trigger biological mechanisms related to osteoblast differentiation, and broaden the perspectives for better exploring biotechnological approaches for bone disorders.

Maquiberry Cystatins: Recombinant Expression, Characterization, and Use to Protect Tooth Dentin and Enamel.

Phytocystatins are proteinaceous competitive inhibitors of cysteine peptidases involved in physiological and defensive roles in plants. Their application as potential therapeutics for human disorders has been suggested, and the hunt for novel cystatin variants in different plants, such as maqui (Aristotelia chilensis), is pertinent. Being an understudied species, the biotechnological potential of maqui proteins is little understood. In the present study, we constructed a transcriptome of maqui plantlets using next-generation sequencing, in which we found six cystatin sequences. Five of them were cloned and recombinantly expressed. Inhibition assays were performed against papain and human cathepsins B and L. Maquicystatins can inhibit the proteases in nanomolar order, except MaquiCPIs 4 and 5, which inhibit cathepsin B in micromolar order. This suggests maquicystatins' potential use for treating human diseases. In addition, since we previously demonstrated the efficacy of a sugarcane-derived cystatin to protect dental enamel, we tested the ability of MaquiCPI-3 to protect both dentin and enamel. Both were protected by this protein (by One-way ANOVA and Tukey's Multiple Comparisons Test, p < 0.05), suggesting its potential usage in dental products.

A Multifunctional Trypsin Protease Inhibitor from Yellow Bell Pepper Seeds: Uncovering Its Dual Antifungal and Hypoglycemic Properties.

Fungal infections are a growing public health concern worldwide and the emergence of antifungal resistance has limited the number of therapeutic options. Therefore, developing novel strategies for identifying and developing new antifungal compounds is an active area of research in the pharmaceutical industry. In this study, we purified and characterized a trypsin protease inhibitor obtained from Yellow Bell Pepper (Capsicum annuum L.) seeds. The inhibitor not only showed potent and specific activity against the pathogenic fungus Candida albicans, but was also found to be non-toxic against human cells. Furthermore, this inhibitor is unique in that it also inhibits α-1,4-glucosidase, positioning it as one of the first plant-derived protease inhibitors with dual biological activity. This exciting discovery opens new avenues for the development of this inhibitor as a promising antifungal agent and highlights the potential of plant-derived protease inhibitors as a rich source for the discovery of novel multifunctional bioactive molecules.

Molecular Cloning and Expression Analysis of Triticale Phytocystatins During Development and Germination of Seeds.

Three triticale cDNAs encoding inhibitors of cysteine endopeptidases, belonging to phytocystatins, have been identified and designated as TrcC-1, TrcC-4 and TrcC-5. Full-length cDNAs of TrcC-1 (617 bp) and TrcC-4 (940 bp), as well as a fragment of TrcC-5 cDNA (369 bp), were obtained. A high-level identity of the deduced amino acid sequence of TrcCs with other known phytocystatins, especially with wheat and barley, has been observed. Moreover, the presence of conserved domain, containing the G and W residues, the sequence of QxVxG and the sequence of LARFAV, characteristic for plant cysteine endopeptidase inhibitors, has been noted. The profiles of TrcC-1 and TrcC-5 mRNA levels in the developing seeds of two triticale cultivars that differ in their resistance to preharvest sprouting (Zorro and Disco) were similar. However, the expression of TrcC-4 was, higher in the developing seeds, and in the scutellum of germinating seeds of a cultivar more resistant to preharvest sprouting (Zorro) than in the less resistant (Disco). Additionally, the expression of TrcC-4 remained longer in developing seeds of Zorro as compared to Disco. The performed studies suggest that TrcC-4 might have an influence on the higher resistance of Zorro cultivar to preharvest sprouting.

Cyclic peptide engineered from phytocystatin inhibitory hairpin loop as an effective modulator of falcipains and potent antimalarial.

Cystatins are classical competitive inhibitors of C1 family cysteine proteases (papain family). Phytocystatin superfamily shares high sequence homology and typical tertiary structure with conserved glutamine-valine-glycine (Q-X-V-X-G) loop blocking the active site of C1 proteases. Here, we develop a cysteine-bounded cyclic peptide (CYS-cIHL) and linear peptide (CYS-IHL), using the conserved inhibitory hairpin loop amino acid sequence. Using an in silico approach based on modeling, protein-peptide docking, molecular dynamics simulations and calculation of free energy of binding, we designed and validated inhibitory peptides against falcipain-2 (FP-2) and -3 (FP-3), cysteine proteases from the malarial parasite Plasmodium falciparum. Falcipains are critical hemoglobinases of P. falciparum that are validated targets for the development of antimalarial therapies. CYS-cIHL was able to bind with micromolar affinity to FP-2 and modulate its binding with its substrate, hemoglobin in in vitro and in vivo assays. CYS-cIHL could effectively block parasite growth and displayed antimalarial activity in culture assays with no cytotoxicity towards human cells. These results indicated that cyclization can substantially increase the peptide affinity to the target. Furthermore, this can be applied as an effective strategy for engineering peptide inhibitory potency against proteases.

Recombinant sugarcane cystatin CaneCPI-5 down regulates inflammation and promotes angiogenesis and collagen deposition in a mouse subcutaneous sponge model.

Cystatins are natural inhibitors of cysteine peptidases that are found practically in all living organisms. CaneCPI-5 is a sugarcane cystatin with inhibitory activity against human cathepsins B, K and L, which are cysteine proteases highly expressed in a variety of pathological conditions, usually marked by persistent inflammation and processing of the extracellular matrix. This work evaluated the effects of daily administration of the recombinant cystatin CaneCPI-5 [0.01, 0.1 or 1.0 µg in 10 µL of Phosphate-Buffered Saline (PBS)] on the inflammatory, angiogenic and fibrogenic components during chronic inflammatory response induced by subcutaneous sponge implants. The anti-inflammatory effect of treatment with CaneCPI-5 was confirmed by reduction of the levels of the pro-inflammatory mediators TNF-α, CXCL1 and CCL2/JE/MCP-1, as well as the activity of the myeloperoxidase and n-acetyl-ß-D-glucosaminidase. Treatment with CaneCPI-5 promoted angiogenesis in the implants, increasing the production of cytokines VEGF and FGF and the formation of new blood vessels. Finally, the administration of the recombinant cystatin favored the production of the pro-fibrogenic cytokine TGF-ß1 and collagen deposition next to the implants. Together, these results show the potential therapeutic application of CaneCPI-5 as an anti-inflammatory agent, capable of favoring angiogenesis and fibrogenesis processes, necessary for tissue repair.

Purification and Characterization of a Novel Thermostable Papain Inhibitor from Moringa oleifera with Antimicrobial and Anticoagulant Properties.

Plant cystatins (or phytocystatins) comprise a large superfamily of natural bioactive small proteins that typically act as protein inhibitors of papain-like cysteine proteases. In this report, we present the purification and characterization of the first phytocystatin isolated from Moringa oleifera (MoPI). MoPI has a molecular mass of 19 kDa and showed an extraordinary physicochemical stability against acidic pHs and high temperatures. Our findings also revealed that MoPI is one of the most potent cysteine protease inhibitors reported to date, with Ki and IC50 values of 2.1 nM and 5.7 nM, respectively. More interestingly, MoPI presents a strong antimicrobial activity against human pathogens such as Enterococcus faecalis and Staphylococcus aureus. In addition, MoPI also showed important anticoagulant activity, which is an unprecedented property for this family of protease inhibitors. These results highlight the pharmaceutical potential of this plant and its derived bioactive molecules.

Proteolytic and Structural Changes in Rye and Triticale Roots under Aluminum Stress.

Proteolysis and structural adjustments are significant for defense against heavy metals. The purpose of this study was to evaluate whether the Al3+ stress alters protease activity and the anatomy of cereale roots. Azocaseinolytic and gelatinolytic measurements, transcript-level analysis of phytocystatins, and observations under microscopes were performed on the roots of Al3+-tolerant rye and tolerant and sensitive triticales exposed to Al3+. In rye and triticales, the azocaseinolytic activity was higher in treated roots. The gelatinolytic activity in the roots of rye was enhanced between 12 and 24 h in treated roots, and decreased at 48 h. The gelatinolytic activity in treated roots of tolerant triticale was the highest at 24 h and the lowest at 12 h, whereas in treated roots of sensitive triticale it was lowest at 12 h but was enhanced at 24 and 48 h. These changes were accompanied by increased transcript levels of phytocystatins in rye and triticale-treated roots. Light microscope analysis of rye roots revealed disintegration of rhizodermis in treated roots at 48 h and indicated the involvement of root border cells in rye defense against Al3+. The ultrastructural analysis showed vacuoles containing electron-dense precipitates. We postulate that proteolytic-antiproteolytic balance and structural acclimation reinforce the fine-tuning to Al3+.

Recombinant expression, characterization and phylogenetic studies of novels cystatins-like proteins of sweet orange (Citrus sinensis) and clementine (Citrus clementina).

Phytocystatins are plant cystatins that are related to several physiological processes regulating endogenous cysteine proteases involved in seed development and germination, programmed cell death and response to stress conditions. In addition, phytocystatins can act in plant defense against exogenous peptidases from herbivorous insects, pathogens and nematodes. Considering that Citrus fruits are important to human nutrition and represent a high value crop in worldwide agriculture, in the present work, we performed the identification of putative cystatins from Citrus sinensis and from Citrus clementine and submitted them to phylogenetic analysis. Six cystatins from each species were identified as orthologous and classified into three well supported phylogenetic groups. Five cystatins representative of the phylogenetic groups were recombinantly expressed and the in vitro studies revealed them to be potent inhibitors against the cysteine peptidases papain, legumain, human cathepsins (B, L, S, K) and a cathepsin B-like from Diaphorina citri (the Asian Citrus psyllid). Our findings provide the C. clementina and C. sinensis cystatins classification and an enzyme-inhibitor interactions profile, which may reflect an evolutionary process of Citrus cystatins related to gene functions as initial germination rates and seedlings development as well associated to plant defense against pathogens, as insects and nematodes.

Probing the binding effects of zinc and cadmium with garlic phytocystatin: Implication of the abiotic stress on garlic phytocystatin.

Abiotic stress induced by heavy metals retards the growth and development of plants. Therefore, it is essential to have an insight into the potential toxic effects of heavy metals. The present article investigates the effect of zinc and cadmium on the structure and function of garlic phytocystatin (GPhyCys). The cysteine proteinase inhibitory assay showed a reduction in the inhibitory activity upon binding with zinc and cadmium. UV-vis absorption spectroscopy revealed the complex formation of zinc and cadmium with garlic phytocystatin. Fluorescence quenching experiment confirmed the quenching of fluorophores upon binding of zinc and cadmium. Synchronous and 3-dimensional fluorescence spectroscopy suggest the alteration in the microenvironment around aromatic residues of garlic phytocystatin upon binding with the above metals. Circular dichroism showed a reduction in the alpha-helical content of native garlic phytocystatin. Scanning electron micrographs showed the morphological changes in the native garlic phytocystatin upon addition of zinc and cadmium. The observations confirmed the alteration in structure and conformation of garlic phytocystatin upon interaction with zinc and cadmium. It can be safely concluded that the high concentration of zinc and cadmium can alter the functioning of cysteine proteinase present in garlic and affects the growth and development of plants.