Crystal structure of a newly identified M61 family aminopeptidase with broad substrate specificity that is solely responsible for recycling acidic amino acids.

Aminopeptidases with varied substrate specificities are involved in different crucial physiological processes of cellular homeostasis. They also have wide applications in food and pharma industries. Within the bacterial cell, broad specificity aminopeptidases primarily participate in the recycling of amino acids by degrading oligopeptides generated via primary proteolysis mediated by cellular ATP-dependent proteases. However, in bacteria, a truly broad specificity enzyme, which can cleave off acidic, basic, Gly and hydrophobic amino acid residues, is extremely rare. Here, we report structure-function of a putative glycyl aminopeptidase (M61xc) from Xanthomonas campestris pv campestris (Xcc) belonging to the M61 peptidase family. The enzyme exhibits broad specificity and cleaves Ala, Leu, Asp, Glu, Met, Ser, Phe, Tyr, Gly, Arg, and Lys at the N terminus, optimally of peptides with a length of 3-7 amino acids. Further, we report the high-resolution crystal structure of M61xc in the apo form (2.1 Å) and bestatin-bound form (1.95 Å), detailing its catalytic and substrate preference mechanisms. Comparative analysis of enzyme activity in crude cell extracts from both wild-type and m61xc-knockout mutant strains of Xcc has elucidated the unique intracellular role of M61xc. This study suggests that M61xc is the exclusive enzyme in these bacteria that is responsible for liberating Asp/Glu residues from the N-termini of peptides. Also, in view of its broad specificity and peptide degradation ability, it could be considered equivalent to M1 or other oligomeric peptidases from families like M17, M18, M42 or S9, who have an important auxiliary role in post-proteasomal protein degradation in prokaryotes.

Characterization of a secreted aminopeptidase of M28 family from B. fragilis and its possible role in protein metabolism in the gut.

Products of microbial protein metabolism in the gut can influence the health of the host in many ways. Members of the Bacteriodales, major commensals of the human colon have been associated with long-term intake of high-protein diets. Undigested proteins or peptides that reach the colon can be hydrolyzed by extra-cellular proteases found in some Bacteroides species into amino acids and peptides which can be further catabolized. In this communication, we have characterized one such secreted aminopeptidase (BfAP) from Bacteroides fragilis belonging to the M28 family which is capable of degrading peptides released from soybean protein after predigestion in the small intestine. The BfAP enzyme was cloned, expressed in E. coli, and purified to homogeneity. It is a metallopeptidase requiring Co2+ ion for optimum activity at 55 °C and pH 8 and preferentially cleaves neutral aliphatic (Met/Leu) and positively charged (Arg/Lys) amino acids from the N-terminus of peptides. It showed high specificity for long peptides as well as proteins like ß-casein. Structural analysis of BfAP and its orthologues using AlphaFold2 reveal a shared highly conserved M28 domain, but vary with respect to their N-terminal region with some of them possessing an additional cap domain which may be important for regulation of substrate binding. Although BfAP lacks the typical cap domain, it shows small extensions that can form a loop adjacent to the proposed active site and may affect substrate binding. We suggest that this secreted enzyme may play an important role in protein metabolism in the colon where Bacteroides species are abundant.

Modifying rheological properties of psyllium by gamma irradiation enables development of low glycaemic index food with a predicted gastrointestinal tolerance.

Gel forming dietary fibre like psyllium (PS) is effective in slowing down rate of digestion as well as absorption of glucose thereby reducing the postprandial glucose level and hence is used to develop functional foods for diabetic patients. The fortification level is however limited which otherwise elicit unwanted rheological response and poor sensorial quality in final product. In the present study this limitation was overcome by improving the functionality of the fibre by gamma radiation processing of the polysaccharides. We assessed the changes in rheological properties of radiation processed PS (RPPS) at different doses which enabled us to optimise the irradiation dose and levels of fortification of the RPPS in wheat flour for preparation of Indian unleavened bread (chapati). We observed that PS processed at a dose of 25 kGy could be incorporated to a level as high as 14 % in wheat flour yielding a sensorially better product compared to unfortified wheat flour. Further, the most striking effect observed for RPPS fortified chapati was reduction in the release of glucose upon subjecting to simulated gastrointestinal digestion. Finally, clinical and in vitro fermentation studies also confirmed a low GI value and high gastrointestinal tolerance of RPPS fortified chapati.

Gamma-irradiation of inulin improves its biological functionality and feasibility as a functional ingredient in synbiotic food.

Inulin, a dietary fibre, is widely used as a prebiotic, sugar replacer, and texture modifier in the food industry. In this study, we have shown that irradiation affects the physicochemical properties of inulin, which in turn improves its biological functionality and feasibility as a functional ingredient in synbiotic foods. The biological functionality of 25 kGy-irradiated inulin (IRI) was assessed in terms of antioxidant capacity, protective action against intracellular ROS, and prebiotic activity. Antioxidant assays revealed that irradiated inulin had improved antioxidant activity, which was even greater than that of fructooligosaccharides. Furthermore, IRI was found to be comparatively more effective in maintaining low intracellular ROS levels. The in vitro fermentation studies showed that IRI had higher bifidogenic efficacy than fructooligosaccharides and unirradiated inulin. A synbiotic low-fat yogurt containing IRI (8.5 %) was prepared. In terms of sensory attributes, the developed product was comparable to a commercially available non-synbiotic and high-fat containing product.

Autoproteolysis of Procerain and Procerain B mediated by structural changes.

Procerain (Pc) and Procerain B (PcB) are two latex proteases from Calotropis procera having potential applications in food and other industries. However, autolytic degradation of these proteases limits their potential use in industry. Nevertheless, basic mechanism underlying the autoproteolysis has not been detailed. In order to understand the same, we subjected the enzymes to various denaturing and activating conditions. The results showed that structural changes induced by different denaturing conditions trigger their autoproteolysis. We also observed differential response of Pc, PcB and other papain-like proteases towards autocatalysis in presence of reducing agent in-spite of sharing the same structural fold, including the number of disulfide bonds. The possible reason underlying this intriguing observation is also discussed. Further, present work establishes that structural changes in the proteases lead to autoproteolysis and the enzymes are stable unless they experience structural perturbation. These findings could thus be useful for their practical applications in industries.

Structural basis for the unusual substrate specificity of unique two-domain M1 metallopeptidase.

M1 metallopeptidases regulate many important biological processes such as angiogenesis, tumour growth, hormone regulation, and immune cell development. Knowledge of substrate specificity mechanism in this family is valuable. An M1 peptidase from Deinococcus radiodurans (M1dr) with preference for bulky hydrophobic residues at N-terminus of peptide substrates was recently reported. In contrast to Escherichia coli aminopeptidase N, a previously characterized M1 peptidase, M1dr exhibits reduced activity towards peptides with N-terminal Arg or Ala residue. In order to illuminate structural basis of substrate specificity, we report several crystal structures of M1dr with different amino acids bound to the active site. Structural analysis indicated that the enzyme makes subtle adjustments to multiple residues leading to significant volume change of the active site cavity to accommodate residues of varying sizes (Leu to Trp). This study further reveals that the low preference for Arg at N-terminus of peptide substrate arises from a non-productive conformation in which many of the Arg molecules bind where they block the proton donor essential for the peptidase reaction. Hence, this study illuminates the substrate-binding mechanism and also reveals the structural basis for the substrate specificity of M1dr enzyme.

Two-domain aminopeptidase of M1 family: Structural features for substrate binding and gating in absence of C-terminal domain.

Zinc metallopeptidases of the M1 family (M1 peptidases) with unique metal binding motif HEXXH(X)18E regulate many important biological processes such as tumor growth, angiogenesis, hormone regulation, and immune cell development. Typically, these enzymes exist in three-domain [N-terminal domain (N-domain), catalytic domain, and C-terminal domain (C-domain)] or four-domain (N-domain, catalytic domain, middle domain, and C-domain) format in which N-domain and catalytic domain are more conserved. The C-domain plays important roles in substrate binding and gating. In this study we report the first structure of a two-domain (N-domain and catalytic domain) M1 peptidase at 2.05 Šresolution. Despite the lack of C-domain, the enzyme is active and prefers peptide substrates with large hydrophobic N-terminal residues. Its substrate-bound structure was determined at 1.9 Šresolution. Structural analyses supported by site directed mutagenesis and molecular dynamics simulations reveal structural features that could compensate for the lack of C-domain. A unique loop insertion (loop A) in the N-domain has important roles in gating and desolvation of active site. Three Arg residues of the catalytic domain are involved in substrate-binding roles typically played by positively charged residues of C-domain in other M1 peptidases. Further, its unique exopeptidase sequence motif, LALET, creates a more hydrophobic environment at the S1 subsite (which binds N-terminal residue of the substrate in aminopeptidases) than the more common GXMEN motif in the family. This leads to high affinity for large hydrophobic residues in the S1 subsite, which contributes towards efficient substrate binding in absence of C-domain.

Catalytic triad heterogeneity in S51 peptidase family: Structural basis for functional variability.

Peptidase E (PepE) is a nonclassical serine peptidase with a Ser-His-Glu catalytic triad. It is specific for dipeptides with an N-terminal aspartate residue (Asp-X dipeptidase activity). Its homolog from Listeria monocytogenes (PepElm) has a Ser-His-Asn "catalytic triad." Based on sequence alignment we predicted that the PepE homolog from Deinococcus radiodurans (PepEdr) would have a Ser-His-Asp "catalytic triad." We confirmed this by solving the crystal structure of PepEdr to 2.7 Å resolution. We show that PepElm and PepEdr lack the Asp-X dipeptidase activity. Our analyses suggest that absence of P1 pocket in the active site could be the main reason for this lack of typical activity. Sequence and structural data reveal that the PepE homologs can be divided into long and short PepEs based on presence or absence of a C-terminal tail which adopts a ß-hairpin conformation in the canonical PepE from Salmonella enterica. A long PepE from Bacillus subtilis with Ser-His-Asp catalytic triad exhibits Asp-X dipeptidase activity. Whereas the three long PepEs enzymatically characterized till date have been found to possess the Asp-X dipeptidase activity, the three enzymatically characterized short PepEs lack this activity irrespective of the nature of their catalytic triads. This study illuminates the structural and functional heterogeneity in the S51 family and also provides structural basis for the functional variability among PepE homologs.

Effect of enzymatic hydrolysis on the functional, antioxidant, and angiotensin I-converting enzyme (ACE) inhibitory properties of whole horse gram flour.

Horse gram hydrolysate (HGH) with different degree of hydrolysis (DH) (20, 25, 35, 40, and 45%) was prepared from whole horse gram flour using alcalase. The amino acid composition of HGH showed the presence of essential amino acids. The alcalase hydrolysis (DH ≥ 20%) increased protein solubility with a notable difference in the pH range of 3-5 (p < 0.05). The emulsifying activity and stability of HGH improved with increase in pH, especially at DH ≥ 25% (p < 0.05). With increase in DH, the foaming properties reduced while the antioxidant and angiotensin I-converting enzyme inhibitory activities increased. Sensory evaluation showed no significant difference (p > 0.05) in preference between control soup and soup mixed with HGH. Thus, these results suggest the possibility of HGH to be used as an appropriate functional ingredient with different food applications including in management of oxidative stress as well as in controlling hypertension .

Structures and activities of widely conserved small prokaryotic aminopeptidases-P clarify classification of M24B peptidases.

M24B peptidases cleaving Xaa-Pro bond in dipeptides are prolidases whereas those cleaving this bond in longer peptides are aminopeptidases-P. Bacteria have small aminopeptidases-P (36-39 kDa), which are diverged from canonical aminopeptidase-P of Escherichia coli (50 kDa). Structure-function studies of small aminopeptidases-P are lacking. We report crystal structures of small aminopeptidases-P from E. coli and Deinococcus radiodurans, and report substrate-specificities of these proteins and their ortholog from Mycobacterium tuberculosis. These are aminopeptidases-P, structurally close to small prolidases except for absence of dipeptide-selectivity loop. We noticed absence of this loop and conserved arginine in canonical archaeal prolidase (Maher et al., Biochemistry. 43, 2004, 2771-2783) and questioned its classification. Our enzymatic assays show that this enzyme is an aminopeptidase-P. Further, our mutagenesis studies illuminate importance of DXRY sequence motif in bacterial small aminopeptidases-P and suggest common evolutionary origin with human XPNPEP1/XPNPEP2. Our analyses reveal sequence/structural features distinguishing small aminopeptidases-P from other M24B peptidases.

Carboxypeptidase in prolyl oligopeptidase family: Unique enzyme activation and substrate-screening mechanisms.

Serine peptidases of the prolyl oligopeptidase (POP) family are of substantial therapeutic importance because of their involvement in diseases such as diabetes, cancer, neurological diseases, and autoimmune disorders. Proper annotation and knowledge of substrate specificity mechanisms in this family are highly valuable. Although endopeptidase, dipeptidyl peptidase, tripeptidyl peptidase, and acylaminoacyl peptidase activities have been reported previously, here we report the first instance of carboxypeptidase activity in a POP family member. We determined the crystal structures of this carboxypeptidase, an S9C subfamily member from Deinococcus radiodurans, in its active and inactive states at 2.3-Å resolution, providing an unprecedented view of assembly and disassembly of the active site mediated by an arginine residue. We observed that this residue is poised to bind substrate in the active structure and disrupts the catalytic triad in the inactive structure. The assembly of the active site is accompanied by the ordering of gating loops, which reduces the effective size of the oligomeric pore. This prevents the entry of larger peptides and constitutes a novel mechanism for substrate screening. Furthermore, we observed structural adaptations that enable its carboxypeptidase activity, with a unique loop and two arginine residues in the active site cavity orienting the peptide substrate for catalysis. Using these structural features, we identified homologs of this enzyme in the POP family and confirmed the presence of carboxypeptidase activity in one of them. In conclusion, we have identified a new type within POP enzymes that exhibits not only unique activity but also a novel substrate-screening mechanism.

Crystal structure of a novel prolidase from Deinococcus radiodurans identifies new subfamily of bacterial prolidases.

Xaa-Pro peptidases (XPP) are dinuclear peptidases of MEROPS M24B family that hydrolyze Xaa-Pro iminopeptide bond with a trans-proline at the second position of the peptide substrate. XPPs specific towards dipeptides are called prolidases while those that prefer longer oligopeptides are called aminopeptidases P. Though XPPs are strictly conserved in bacterial and archaeal species, the structural and sequence features that distinguish between prolidases and aminopeptidases P are not always clear. Here, we report 1.4 Å resolution crystal structure of a novel XPP from Deinococcus radiodurans (XPPdr). XPPdr forms a novel dimeric structure via unique dimer stabilization loops of N-terminal domains such that their C-terminal domains are placed far apart from each other. This novel dimerization is also the consequence of a different orientation of N-terminal domain in XPPdr monomer than those in other known prolidases. The enzymatic assays show that it is a prolidase with broad substrate specificity. Our structural, mutational, and molecular dynamics simulation analyses show that the conserved Arg46 of N-terminal domain is important for the dipeptide selectivity. Our BLAST search found XPPdr orthologs with conserved sequence motifs which correspond to unique structural features of XPPdr, thus identify a new subfamily of bacterial prolidases.

Active site gate of M32 carboxypeptidases illuminated by crystal structure and molecular dynamics simulations.

Enzyme gates are important dynamic features that regulate function. Study of these features is critical for understanding of enzyme mechanism. In this study, the active-site gate of M32 carboxypeptidases (M32CP) is illuminated. Only a handful of members of this family have been structurally and functionally characterized and various aspects of their activity and mechanism are yet not clarified. Here, crystal structure of putative M32CP from Deinococcus radiodurans (M32dr) was solved to 2.4Å resolution. Enzymatic assays confirmed its identity as a carboxypeptidase. Open and relatively closed conformations observed in the structure provided supporting evidence for previously hypothesized hinge motion in this family of enzymes. Molecular dynamics simulations of 1.5µs displayed distinct open and closed conformations revealing amplitude of the motion to be beyond what was observed in the crystal structure. Hinge region and anchoring region of this shell-type gate were identified. A small displacement of 3Å and a helical tilt of 9° propagated by the hinge region translates into a 10Å motion at the top of the gate. The dynamics of the gate was supported by our mutagenesis experiment involving formation of disulphide bond across helices of the gate. The nearly inactive mutant enzyme showed 65-fold increase in the enzymatic activity in presence of reducing agent. Further, while a previously proposed structural basis would have led to its classification in subfamily II, experimentally observed substrate length restriction places M32dr in subfamily I of M32CPs.

Structure of the human aminopeptidase XPNPEP3 and comparison of its in vitro activity with Icp55 orthologs: Insights into diverse cellular processes.

The human aminopeptidase XPNPEP3 is associated with cystic kidney disease and TNF-TNFR2 cellular signaling. Its yeast and plant homolog Icp55 processes several imported mitochondrial matrix proteins leading to their stabilization. However, the molecular basis for the diverse roles of these enzymes in the cell is unknown. Here, we report the crystal structure of human XPNPEP3 with bound apstatin product at 1.65 Å resolution, and we compare its in vitro substrate specificity with those of fungal Icp55 enzymes. In contrast to the suggestions by earlier in vivo studies of mitochondrial processing, we found that these enzymes are genuine Xaa-Pro aminopeptidases, which hydrolyze peptides with proline at the second position (P1'). The mitochondrial processing activity involving cleavage of peptides lacking P1' proline was also detected in the purified enzymes. A wide proline pocket as well as molecular complementarity and capping at the S1 substrate site of XPNPEP3 provide the necessary structural features for processing the mitochondrial substrates. However, this activity was found to be significantly lower as compared with Xaa-Pro aminopeptidase activity. Because of similar activity profiles of Icp55 and XPNPEP3, we propose that XPNPEP3 plays the same mitochondrial role in humans as Icp55 does in yeast. Both Xaa-Pro aminopeptidase and mitochondrial processing activities of XPNPEP3 have implications toward mitochondrial fitness and cystic kidney disease. Furthermore, the presence of both these activities in Icp55 elucidates the unexplained processing of the mitochondrial cysteine desulfurase Nfs1 in yeast. The enzymatic and structural analyses reported here provide a valuable molecular framework for understanding the diverse cellular roles of XPNPEP3.

Crystal structure and biochemical investigations reveal novel mode of substrate selectivity and illuminate substrate inhibition and allostericity in a subfamily of Xaa-Pro dipeptidases.

Xaa-Pro dipeptidase (XPD) catalyzes hydrolysis of iminopeptide bond in dipeptides containing trans-proline as a second residue. XPDs are found in all living organisms and are believed to play an essential role in proline metabolism. Here, we report crystal structures and extensive enzymatic studies of XPD from Xanthomonas campestris (XPDxc), the first such comprehensive study of a bacterial XPD. We also report enzymatic activities of its ortholog from Mycobacterium tuberculosis (XPDmt). These enzymes are strictly dipeptidases with broad substrate specificities. They exhibit substrate inhibition and allostericity, as described earlier for XPD from Lactococcus lactis (XPDll). The structural, mutational and comparative data have revealed a novel mechanism of dipeptide selectivity and substrate binding in these enzymes. Moreover, we have identified conserved sequence motifs that distinguish these enzymes from other prolidases, thus defining a new subfamily. This study provides a suitable structural template for explaining unique properties of this XPDxc subfamily. In addition, we report unique structural features of XPDxc protein like an extended N-terminal tail region and absence of a conserved Tyr residue near the active site.

The members of M20D peptidase subfamily from Burkholderia cepacia, Deinococcus radiodurans and Staphylococcus aureus (HmrA) are carboxydipeptidases, primarily specific for Met-X dipeptides.

Three members of peptidase family M20D from Burkholderia cepacia (BcepM20D; Uniprot accession no. A0A0F7GQ23), Deinococcus radiodurans R1 (DradM20D; Uniprot accession no. Q9RTP6) and Staphylococcus aureus (HmrA; Uniprot accession no. Q99Q45) were characterized in terms of their preference for various substrates. The results thus reveal that all the enzymes including HmrA lack endopeptidase as well as aminopeptidase activities and possess strong carboxypeptidase activity. Further, the amidohydrolase activity exerted on other substrates like N-Acetyl-Amino acids, N-Carbobenzoxyl-Amino acids and Indole acetic acid (IAA)-Amino acids is due to the ability of these enzymes to accommodate different types of chemical groups other than the amino acid at the S1 pocket. Further, data on peptide hydrolysis strongly suggests that all the three enzymes are primarily carboxydipeptidases exhibiting highest catalytic efficiency (kcat/Km 5-36 × 10(5) M(-1) s(-1)) for Met-X substrates, where -X could be Ala/Gly/Ser/Tyr/Phe/Leu depending on the source organism. The supportive evidence for the substrate specificities was also provided with the molecular docking studies carried out using structure of SACOL0085 and homology modelled structure of BcepM20D. The preference for different substrates, their binding at active site of the enzyme and possible role of these enzymes in recycling of methionine are discussed in this study.

Crystallization and preliminary X-ray diffraction analysis of Xaa-Pro dipeptidase from Xanthomonas campestris.

Xaa-Pro dipeptidase (XPD; prolidase; EC 3.4.13.9) specifically hydrolyzes dipeptides with a prolyl residue at the carboxy-terminus. Xanthomonas spp. possess two different isoforms of XPD (48 and 43 kDa) which share ∼24% sequence identity. The XPD of 43 kDa in size (XPD43) from Xanthomonas spp. is unusual as it lacks the strictly conserved tyrosine residue (equivalent to Tyr387 in Escherichia coli aminopeptidase P) that is suggested to be important in the proton-shuttle transfer required for catalysis in the M24B (MEROPS) family. Here, the crystallization and preliminary X-ray analysis of XPD43 from X. campestris (GenBank accession No. NP_637763) are reported. Recombinant XPD43 was crystallized using the microbatch-under-oil technique. Diffraction data were collected on the recently commissioned protein crystallography beamline (PX-BL21) at the Indian synchrotron (Indus-2, 2.5 GeV) to 1.83 Šresolution with 100% completeness. The crystal belonged to space group P212121, with unit-cell parameters a = 84.32, b = 105.51, c = 111.35 Å. Two monomers are expected to be present in the asymmetric unit of the crystal, corresponding to a solvent content of 58%. Structural analysis of XPD43 will provide new insights into the role of the conserved residues in catalysis in the M24B family.

Expression, purification, crystallization and preliminary X-ray diffraction analysis of acylpeptide hydrolase from Deinococcus radiodurans.

Acylpeptide hydrolase (APH; EC 3.4.19.1), which belongs to the S9 family of serine peptidases (MEROPS), catalyzes the removal of an N-acylated amino acid from a blocked peptide. The role of this enzyme in mammalian cells has been suggested to be in the clearance of oxidatively damaged proteins as well as in the degradation of the ß-amyloid peptides implicated in Alzheimer's disease. Detailed structural information for the enzyme has been reported from two thermophilic archaea; both of the archaeal APHs share a similar monomeric structure. However, the mechanisms of substrate selectivity and active-site accessibility are totally different and are determined by inter-domain flexibility or the oligomeric structure. An APH homologue from a bacterium, Deinococcus radiodurans (APHdr), has been crystallized using microbatch-under-oil employing the random microseed matrix screening method. The protein crystallized in space group P21, with unit-cell parameters a = 77.6, b = 189.6, c = 120.4 Å, ß = 108.4°. A Matthews coefficient of 2.89 Å(3) Da(-1) corresponds to four monomers, each with a molecular mass of ∼73 kDa, in the asymmetric unit. The APHdr structure will reveal the mechanisms of substrate selectivity and active-site accessibility in the bacterial enzyme. It will also be helpful in elucidating the functional role of this enzyme in D. radiodurans.

Comparative study on antioxidant activity of different varieties of commonly consumed legumes in India.

Legumes are rich source of proteins, dietary fiber, micronutrients and bioactive phytochemicals. Thirty different varieties of commonly consumed legumes in India, were screened for phenolic content and antioxidant activity using, radical scavenging [(1,1-diphenyl-2-picryl-hydrazyl (DPPH·) and 2,2'-azino-bis (3-ethylbenz-thiazoline-6-sulfonic acid, (ABTS·âº], Ferric Reducing Antioxidant Power (FRAP) and metal ion (Fe²âº) chelation assays. Legumes varied largely in their antioxidant activity. Horse gram, common beans, cowpea (brown and red) and fenugreek showed high DPPH· radical scavenging activity (>400 units/g), while lablab bean (cream and white), chickpea (cream and green), butter bean and pea (white and green) showed low antioxidant activity (<125 units/g). Green gram, black gram, pigeon pea, lentils, cowpea (white) and common bean (maroon) showed intermediate activity. Similar trend was observed when the activity was assessed with ABTS·âº and FRAP assays. Thus most of the varieties having light color seed coat, except soybean exhibited low antioxidant activity. While legumes having dark color seed coat did not always possessed high antioxidant activity (e.g. moth bean, black pea, black gram, lentils). Antioxidant activity showed positive correlation (r²>0.95) with phenolic contents, in DPPH·, ABTS·âº and FRAP assays, whereas poor correlation (r²=0.297) was observed between Fe²âº chelating activity of the legumes and phenolic contents.