Amino Acid Dehydrogenase Research Articles

Synthesis of C-N bonds by nicotinamide-dependent oxidoreductase: an overview.

Compounds containing chiral C-N bonds play a vital role in the composition of biologically active natural products and small pharmaceutical molecules. Therefore, the development of efficient and convenient methods for synthesizing compounds containing chiral C-N bonds is a crucial area of research. Nicotinamide-dependent oxidoreductases (NDOs) emerge as promising biocatalysts for asymmetric synthesis of chiral C-N bonds due to their mild reaction conditions, exceptional stereoselectivity, high atom economy, and environmentally friendly nature. This review aims to present the structural characteristics and catalytic mechanisms of various NDOs, including imine reductases/ketimine reductases, reductive aminases, EneIRED, and amino acid dehydrogenases. Additionally, the review highlights protein engineering strategies employed to modify the stereoselectivity, substrate specificity, and cofactor preference of NDOs. Furthermore, the applications of NDOs in synthesizing essential medicinal chemicals, such as noncanonical amino acids and chiral amine compounds, are extensively examined. Finally, the review outlines future perspectives by addressing challenges and discussing the potential of utilizing NDOs to establish efficient biosynthesis platforms for C-N bond synthesis. In conclusion, NDOs provide an economical, efficient, and environmentally friendly toolbox for asymmetric synthesis of C-N bonds, thus contributing significantly to the field of pharmaceutical chemical development.

Kinetic and stability studies of amino acid metal-organic frameworks for encapsulating of amino acid dehydrogenase

Zr-MOFs was applied for the immobilization of hyperthermophilic and halophilic amino acid dehydrogenase (Zr-MOFs-NTAaDH) by physical adsorption for the biosynthesis of L-homophenylalanine. Activity of Zr-MOFs-NTAaDH was enhanced by 3.3-fold of the free enzyme at 70°C. And the enzyme activity of Zr-MOFs-NTAaDH was maintained at 4.16 U/mg at pH 11, which was 7.8 folds of that of NTAaDH. Kinetic parameters indicated catalytic efficiency of Zr-MOFs-NTAaDH was increased compared to the free enzyme as kcat of Zr-MOFs-NTAaDH was 12.3-fold of that of free enzyme. After 7 recycles, the activity of Zr-MOFs-NTAaDH remained 68 %. And Zr-MOFs-NTAaDH exhibited high ionic liquid tolerance which indicated the great potential for industrial application.

Design of motifs interfacial interactions by co‐evolved analysis of D‐amino acid dehydrogenase for stability enhancement

AbstractTo design D‐amino acid dehydrogenase (DAADH) for enhanced stability, the interactions of the subunit interfaces of DAADH were analyzed. Interaction network analysis of DAADH indicated that there are only weak interactions between the A and B subunits. Several co‐evolved residue pairs were selected for mutation to enhance interfacial interactions of subunits, and 11 designed DAADHs were obtained. DA06 and DA11 were selected for experimental verification for their salt bridges are 1.4 and 1.2‐fold of that of DAwild, respectively. DA11 can maintain 93% activity in 80°C, while it was only 40% for DAwild. Thermostability study indicated the half‐life of DA11 was 2‐fold of DAwild. Molecular dynamics simulations revealed that the extraordinary stability of the DA11 was due to the formation of extra interfacial salt bridges. The article provides a strategy of mutations outside the active site of enzyme by co‐evolutionary analysis which can reduce the effect of the activity‐thermostability trade‐off.

Asymmetric Reductive Amination in Organocatalysis and Biocatalysis

AbstractThis review summarizes the recent progress of organocatalytic and biocatalytic asymmetric reductive amination (ARA), a challenging but important topic for drug discovery and the pharmaceutical industry. At present, ARA can be divided into three categories: metal catalysis, organic catalysis, and biocatalysis. In the past decade, transition metal‐catalysed ARA has been well established. Organocatalytic ARA has emerged as a powerful alternative to metal‐catalysed ARA, the hydrogen sources used in organocatalytic ARA are usually Hantzsch esters, benzothiazolines, boranes, and hydrosilanes, which require Lewis base or phosphoric acid catalysts to activate them to give secondary chiral amines. It is worth mentioning that biocatalytic ARA has made remarkable progress in the last decade, amino acid dehydrogenases, amine dehydrogenases, opine dehydrogenases and imine reductases have been successfully used in ARA.

The Symmetric Active Site of Enantiospecific Enzymes

Biomolecules are frequently chiral compounds, existing in enantiomeric forms. Amino acids represent a meaningful example of chiral biological molecules. Both L- and D-amino acids play key roles in the biochemical structure and metabolic processes of living organisms, from bacteria to mammals. In this review, we explore the enantiospecific interaction between proteins and chiral amino acids, introducing theoretical models and describing the molecular basis of the ability of some of the most important enzymes involved in the metabolism of amino acids (i.e., amino acid oxidases, dehydrogenases, and aminotransferases) to discriminate the opposite enantiomers. Our analysis showcases the power of natural evolution in shaping biological processes. Accordingly, the importance of amino acids spurred nature to evolve strictly enantioselective enzymes both through divergent evolution, starting from a common ancestral protein, or through convergent evolution, starting from different scaffolds: intriguingly, the active sites of these enzymes are frequently related by a mirror symmetry.

NADP+-dependent l-arginine dehydrogenase from Pseudomonas velonii: Purification, characterization and application to an l-arginine assay

l-Arginine dehydrogenase (L-ArgDH) is an amino acid dehydrogenase which catalyzes the reversible oxidative deamination of l-arginine to the oxo analog in the presence of NAD(P)+. We here found the gene homolog of L-ArgDH in genome data of Pseudomonas veronii and succeeded in expression of P. veronii JCM11942 gene in E. coli. The gene product exhibited strong NADP+-dependent L-ArgDH activity. The enzyme was unstable, but markedly stabilized by the addition of 10% glycerol. The enzyme first purified to homogeneity consisted of a homodimeric protein with a molecular mass of about 65 kDa. The enzyme selectively catalyzed NADP+-dependent l-arginine oxidation with maximal activity at pH 9.5. The apparent Km values for l-arginine and NADP+ were 2.5 and 0.21 mM, respectively. The nucleotide sequence coding the enzyme gene was determined and the amino acid sequence was deduced from the nucleotide sequence. The simple colorimetric microassay for l-arginine using the enzyme was achieved.

Substrate-Specific Engineering of Amino Acid Dehydrogenase Superfamily for Synthesis of a Variety of Chiral Amines and Amino Acids

Amino acid dehydrogenases (AADHs) are a group of enzymes that catalyze the reversible reductive amination of keto acids with ammonia to produce chiral amino acids using either nicotinamide adenine dinucleotide (NAD+) or nicotinamide adenine dinucleotide phosphate (NADP+) as cofactors. Among them, glutamate dehydrogenase, valine dehydrogenase, leucine dehydrogenase, phenylalanine dehydrogenase, and tryptophan dehydrogenase have been classified as a superfamily of amino acid dehydrogenases (s-AADHs) by previous researchers because of their conserved structures and catalytic mechanisms. Owing to their excellent stereoselectivity, high atom economy, and low environmental impact of the reaction pathway, these enzymes have been extensively engineered to break strict substrate specificities for the synthesis of high value-added chiral compounds (chiral amino acids, chiral amines, and chiral amino alcohols). Substrate specificity engineering of s-AADHs mainly focuses on recognition engineering of the substrate side chain R group and substrate backbone carboxyl group. This review summarizes the reported studies on substrate specificity engineering of s-AADHs and reports that this superfamily of enzymes shares substrate specificity engineering hotspots (the inside of the pocket, substrate backbone carboxyl anchor sites, substrate entrance tunnel, and hinge region), which sheds light on the substrate-specific tailoring of these enzymes.

Characterization and kinetic study of immobilized of phenylalanine dehydrogenase on metal ions coordinated polydopamine-coated MWNTs

Biosynthesis of L-homophenylalanine by immobilized hyperthermophilic and halophilic amino acid dehydrogenase (AaDH) was studied. Polydopamine (PDA) modified MWNTs (MWNTs-PDA) was applied for AaDH immobilization. Coordinated with metal ions, MWNTs-PDA-Ni exhibit high loading capacity and biocompatibility. MWNTs-PDA-AaDH and MWNTs-PDA-Ni-AaDH enhanced activity by 3.2 and 7.4 -fold than free enzyme, respectively. Compared with the MWNTs-PDA-AaDH, nickel ions coordinated MWNTs-PDA improve enzyme recovery of immobilization by twice, and the enzyme activity remained as 58.0% after 7 recycles. Compared with free enzyme, stability of MWNTs-PDA-Ni-AaDH and MWNTs-PDA-AaDH in 30% cyclohexane were enhanced by 2.0 and 3.2 -fold, respectively. Kinetic parameters of MWNTs-PDA-AaDH, MWNTs-PDA-Ni-AaDH were caculated and compared. Mechanism of immobilization was investigated from the perspectives of multi-level interactions. This study offers an easy application of conductive hybride materials for enzyme immobilization, which can further expand the application for bioelectrodes.

Combined active pocket and hinge region engineering to develop an NADPH-dependent phenylglycine dehydrogenase

NADPH-dependent amino acid dehydrogenases (AADHs) are favorable enzymes to construct artificial biosynthetic pathways in whole-cell for high-value noncanonical amino acids (NcAAs) production. Glutamate dehydrogenases (GluDHs) represent attractive candidates for the development of novel NADPH-dependent AADHs. Here, we report the development of a novel NADPH-dependent phenylglycine dehydrogenase by combining active pocket engineering and hinge region engineering of a GluDH from Pseudomonas putida (PpGluDH). The active pocket of PpGluDH was firstly tailored to optimize its binding mode with bulky substrate α-oxobenzeneacetic acid (α-OA), and then, the hinge region was further engineered to tune the protein conformational dynamics, which finally resulted in a mutant M3 (T196A/T121I/L123D) with a 103-fold increase of catalytic efficiency (kcat/Km) toward α-OA. The M3 mutant exhibited high catalytic performance in both in vitro biocatalysis preparation and in vivo biosynthesis of l-phenylglycine, indicating its promising practical applications. Our results demonstrated that co-engineering of the active pocket and hinge region is an effective strategy for developing novel NADPH-dependent AADHs from GluDHs for NcAAs production.

Biosynthesis of Chiral Amino Alcohols via an Engineered Amine Dehydrogenase in E. coli.

Chiral amino alcohols are prevalent synthons in pharmaceuticals and synthetic bioactive compounds. The efficient synthesis of chiral amino alcohols using ammonia as the sole amino donor under mild conditions is highly desired and challenging in organic chemistry and biotechnology. Our previous work explored a panel of engineered amine dehydrogenases (AmDHs) derived from amino acid dehydrogenase (AADH), enabling the one-step synthesis of chiral amino alcohols via the asymmetric reductive amination of α-hydroxy ketones. Although the AmDH-directed asymmetric reduction is in a high stereoselective manner, the activity is yet fully excavated. Herein, an engineered AmDH derived from a leucine dehydrogenase from Sporosarcina psychrophila (SpAmDH) was recruited as the starting enzyme, and the combinatorial active-site saturation test/iterative saturation mutagenesis (CAST/ISM) strategy was applied to improve the activity. After three rounds of mutagenesis in an iterative fashion, the best variant wh84 was obtained and proved to be effective in the asymmetric reductive amination of 1-hydroxy-2-butanone with 4-fold improvements in k cat /K m and total turnover number (TTN) values compared to those of the starting enzyme, while maintaining high enantioselectivity (ee >99%) and thermostability (T 50 15 >53°C). In preparative-scale reaction, the conversion of 100 and 200 mM 1-hydroxy-2-butanone catalyzed by wh84 was up to 91–99%. Insights into the source of an enhanced activity were gained by the computational analysis. Our work expands the catalytic repertoire and toolbox of AmDHs.

Procalcitonin metabolomics in the critically ill reveal relationships between inflammation intensity and energy utilization pathways

Procalcitonin is a biomarker of systemic inflammation and may have importance in the immune response. The metabolic response to elevated procalcitonin in critical illness is not known. The response to inflammation is vitally important to understanding metabolism alterations during extreme stress. Our aim was to determine if patients with elevated procalcitonin have differences in the metabolomic response to early critical illness. We performed a metabolomics study of the VITdAL-ICU trial where subjects received high dose vitamin D3 or placebo. Mixed-effects modeling was used to study changes in metabolites over time relative to procalcitonin levels adjusted for age, Simplified Acute Physiology Score II, admission diagnosis, day 0 25-hydroxyvitamin D level, and the 25-hydroxyvitamin D response to intervention. With elevated procalcitonin, multiple members of the short and medium chain acylcarnitine, dicarboxylate fatty acid, branched-chain amino acid, and pentose phosphate pathway metabolite classes had significantly positive false discovery rate corrected associations. Further, multiple long chain acylcarnitines and lysophosphatidylcholines had significantly negative false discovery rate corrected associations with elevated procalcitonin. Gaussian graphical model analysis revealed functional modules specific to elevated procalcitonin. Our findings show that metabolite differences exist with increased procalcitonin indicating activation of branched chain amino acid dehydrogenase and a metabolic shift.

Transamination-Like Reaction Catalyzed by Leucine Dehydrogenase for Efficient Co-Synthesis of α-Amino Acids and α-Keto Acids.

α-Amino acids and α-keto acids are versatile building blocks for the synthesis of several commercially valuable products in the food, agricultural, and pharmaceutical industries. In this study, a novel transamination-like reaction catalyzed by leucine dehydrogenase was successfully constructed for the efficient enzymatic co-synthesis of α-amino acids and α-keto acids. In this reaction mode, the α-keto acid substrate was reduced and the α-amino acid substrate was oxidized simultaneously by the enzyme, without the need for an additional coenzyme regeneration system. The thermodynamically unfavorable oxidation reaction was driven by the reduction reaction. The efficiency of the biocatalytic reaction was evaluated using 12 different substrate combinations, and a significant variation was observed in substrate conversion, which was subsequently explained by the differences in enzyme kinetics parameters. The reaction with the selected model substrates 2-oxobutanoic acid and L-leucine reached 90.3% conversion with a high total turnover number of 9.0 × 106 under the optimal reaction conditions. Furthermore, complete conversion was achieved by adjusting the ratio of addition of the two substrates. The constructed reaction mode can be applied to other amino acid dehydrogenases in future studies to synthesize a wider range of valuable products.

Abstract 10770: Trans-Ancestry Multivariate Genome-Wide Analysis Highlights the Role of Common Genetic Variation in Cardiac Structure, Function, and Heart Failure-Related Traits

Introduction: The contribution of common genetic variation to heart failure (HF) risk has not been fully elucidated. Here, we applied multi-ancestry and multivariate methods to summary genetic data from >1 million individuals to improve power to identify common genetic variants, genes, cells, tissues, and circulating proteins/metabolites associated with HF and related cardiac imaging traits. Methods: Trans-ancestry meta-analysis of HF (56,722 cases and 1,133,054 controls) was performed using METAL. Multivariate analysis including GWAS of HF and imaging traits (MRI and echocardiogram) was performed using N-GWAMA. Downstream transcriptome-wide association studies (TWAS; S-PrediXcan), tissue/cell enrichment (LDSC-SEG using RNAseq and snRNAseq of human left ventricle samples from the MAGnet consortium), and Mendelian randomization (MR) analyses were performed. Results: The multi-ancestry HF GWAS identified 15 loci associated with all-cause HF (p < 5 x 10 -8 ). Multivariate analysis identified 48 (16 novel) loci (p < 5 x 10 -8 ), with enrichment for loci near Mendelian cardiomyopathy genes (p < 1 x 10 -4 ). Genetic associations were enriched (FDR < 0.05) for cardiac and musculoskeletal gene expression and chromatin marks. Gene expression (p = 0.007) and splicing events (p = 0.01) were enriched for established cardiomyopathy genes. Branch chain amino acid dehydrogenase ( BCKDHA ) expression was prioritized in TWAS, and MR identified causal associations between circulating branch chain amino acids and cardiac imaging traits: LVEDV MRI (leucine β = -0.137, 95% CI -0.25 to -0.022, p = 0.02; isoleucine β = -0.276, 95% CI -0.38 to -0.17, p = 3 x 10 -7 ) and LVSEV MRI (leucine β = -.131, 95% CI -0.24 to -0.026, p = 0.01; isoleucine β = -0.217, 95% CI -0.33 to -0.11, p = 1 x 10 -4 ). Unbiased proteome-wide MR of 725 circulating proteins identified 18 significant (FDR < 0.05) causal protein-trait associations, including between lipoprotein(a) and HF (OR 1.09 per 1-SD increase in lipoprotein(a), 95% CI 1.06 to 1.11, p = 1.6 x 10 -11 ). Conclusion: These analyses implicate novel common genetic variation in the pathogenesis of HF, highlight Mendelian cardiomyopathy genes in common HF, and identify circulating metabolites and proteins that may represent new treatment targets.

Assembly of peptide linker to amino acid dehydrogenase and immobilized with metal–organic framework

AbstractBACKGROUNDThe metal–organic framework (MOF) ZIF‐8 has the advantages of a large specific surface area and high stability. A new strategy for the assembly of amino acid dehydrogenase with peptide linker and for improving enzyme immobilization is proposed.RESULTSPeptide linker enhanced enzyme activity after immobilization by 138.7%, and maintained 81.2% of its initial activity after seven recycles. The stability of PheDH_1D02/ZIF‐8 was greatly enhanced at 70–80 °C and pH 10–11. The catalytic efficiency of PheDH_1D02/ZIF‐8 was also enhanced compared with PheDH_1C1D/ZIF‐8 and two free enzymes.CONCLUSIONSMetal ions coordinated with the peptide linker and reoriented immobilization of enzyme onto the MOF with high enzyme loading capacity were achieved. The mesoporous structure of PheDH_1D02/ZIF‐8 facilitates substrate diffusion. Combining the assembly of peptide linker and MOF immobilization is a simple, economical and efficient method for improving dehydrogenase stability. © 2021 Society of Chemical Industry (SCI).

Effect of ionic liquids on catalytic characteristics of hyperthermophilic and halophilic phenylalanine dehydrogenase and mechanism study

Biosynthesis of an unnatural amino acid, L-Homophenylalanine, by hyperthermophilic and halophilic phenylalanine dehydrogenase were studied. The catalytic characteristics of phenylalanine dehydrogenase from Natranaerobius thermophiles (NTAaDH) in the reaction systems involving ionic liquids (ILs) were examined. [EMIM]BF4 activated enzyme activity by 132%. Effect of several ionic liquids with different anions on enzyme activity was studied with associated conformational changes. Immobilization capacity of 0.550 mg/mg for NTAaDH was achieved. Kinetic parameters of reductive amination were calculated and compared. GO-PEI-ILs integrates beneficial characteristics of ionic liquids and polymers and enhanced the activity of NTAaDH. The mechanism of the effect of ionic liquids on GO-PEI for enzyme immobilization was investigated.

Superior production of heavy pamamycin derivatives using a bkdR deletion mutant of Streptomyces albus J1074/R2

BackgroundPamamycins are macrodiolides of polyketide origin which form a family of differently large homologues with molecular weights between 579 and 663. They offer promising biological activity against pathogenic fungi and gram-positive bacteria. Admittedly, production titers are very low, and pamamycins are typically formed as crude mixture of mainly smaller derivatives, leaving larger derivatives rather unexplored so far. Therefore, strategies that enable a more efficient production of pamamycins and provide increased fractions of the rare large derivatives are highly desired. Here we took a systems biology approach, integrating transcription profiling by RNA sequencing and intracellular metabolite analysis, to enhance pamamycin production in the heterologous host S. albus J1074/R2.ResultsSupplemented with l-valine, the recombinant producer S. albus J1074/R2 achieved a threefold increased pamamycin titer of 3.5 mg L−1 and elevated fractions of larger derivatives: Pam 649 was strongly increased, and Pam 663 was newly formed. These beneficial effects were driven by increased availability of intracellular CoA thioesters, the building blocks for the polyketide, resulting from l-valine catabolism. Unfavorably, l-valine impaired growth of the strain, repressed genes of mannitol uptake and glycolysis, and suppressed pamamycin formation, despite the biosynthetic gene cluster was transcriptionally activated, restricting production to the post l-valine phase. A deletion mutant of the transcriptional regulator bkdR, controlling a branched-chain amino acid dehydrogenase complex, revealed decoupled pamamycin biosynthesis. The regulator mutant accumulated the polyketide independent of the nutrient status. Supplemented with l-valine, the novel strain enabled the biosynthesis of pamamycin mixtures with up to 55% of the heavy derivatives Pam 635, Pam 649, and Pam 663: almost 20-fold more than the wild type.ConclusionsOur findings open the door to provide rare heavy pamamycins at markedly increased efficiency and facilitate studies to assess their specific biological activities and explore this important polyketide further.

Efficient Amino Donor Recycling in Amination Reactions: Development of a New Alanine Dehydrogenase in Continuous Flow and Dialysis Membrane Reactors

Transaminases have arisen as one of the main biocatalysts for amine production but despite their many advantages, their stability is still a concern for widespread application. One of the reasons for their instability is the need to use an excess of the amino donor when trying to synthesise amines with unfavourable equilibria. To circumvent this, recycling systems for the amino donor, such as amino acid dehydrogenases or aldolases, have proved useful to push the equilibria while avoiding high amino donor concentrations. In this work, we report the use of a new alanine dehydrogenase from the halotolerant bacteria Halomonas elongata which exhibits excellent stability to different cosolvents, combined with the well characterised CbFDH as a recycling system of L-alanine for the amination of three model substrates with unfavourable equilibria. In a step forward, the amino donor recycling system has been co-immobilised and used in flow with success as well as re-used as a dialysis enclosed system for the amination of an aromatic aldehyde.

Crystal Structures and Catalytic Mechanism of l-erythro-3,5-Diaminohexanoate Dehydrogenase and Rational Engineering for Asymmetric Synthesis of β-Amino Acids.

Amino acid dehydrogenases (AADHs) have shown considerable potential as biocatalysts in the asymmetric synthesis of chiral amino acids. However, compared to the widely studied α-AADHs, limited knowledge is available about β-AADHs that enable the synthesis of β-amino acids. Herein, we report the crystal structures of a l-erythro-3,5-diaminohexanoate dehydrogenase and its variants, the only known member of β-AADH family. Crystal structure analysis, site-directed mutagenesis studies and quantum chemical calculations revealed the differences in the substrate binding and catalytic mechanism from α-AADHs. A number of rationally engineered variants were then obtained with improved activity (by 110-800 times) toward various aliphatic β-amino acids without an enantioselectivity trade-off. Two β-amino acids were prepared by using the outstanding variants with excellent enantioselectivity (>99 % ee) and high isolated yields (86-87 %). These results provide important insights into the molecular mechanism of 3,5-DAHDH, and establish a solid foundation for further design of β-AADHs for the asymmetric synthesis of β-amino acids.

Production of bio-xylitol from d-xylose by an engineered Pichia pastoris expressing a recombinant xylose reductase did not require any auxiliary substrate as electron donor

BackgroundXylitol is a five-carbon sugar alcohol that has numerous beneficial health properties. It has almost the same sweetness as sucrose but has lower energy value compared to the sucrose. Metabolism of xylitol is insulin independent and thus it is an ideal sweetener for diabetics. It is widely used in food products, oral and personal care, and animal nutrition as well. Here we present a two-stage strategy to produce bio-xylitol from d-xylose using a recombinant Pichia pastoris expressing a heterologous xylose reductase gene. The recombinant P. pastoris cells were first generated by a low-cost, standard procedure. The cells were then used as a catalyst to make the bio-xylitol from d-xylose.ResultsPichia pastoris expressing XYL1 from P. stipitis and gdh from B. subtilis demonstrated that the biotransformation was very efficient with as high as 80% (w/w) conversion within two hours. The whole cells could be re-used for multiple rounds of catalysis without loss of activity. Also, the cells could directly transform d-xylose in a non-detoxified hemicelluloses hydrolysate to xylitol at 70% (w/w) yield.ConclusionsWe demonstrated here that the recombinant P. pastoris expressing xylose reductase could transform d-xylose, either in pure form or in crude hemicelluloses hydrolysate, to bio-xylitol very efficiently. This biocatalytic reaction happened without the external addition of any NAD(P)H, NAD(P)+, and auxiliary substrate as an electron donor. Our experimental design & findings reported here are not limited to the conversion of d-xylose to xylitol only but can be used with other many oxidoreductase reactions also, such as ketone reductases/alcohol dehydrogenases and amino acid dehydrogenases, which are widely used for the synthesis of high-value chemicals and pharmaceutical intermediates.

Biochemical changes during Seed development and maturation in little millet cv.CO (Samai) 4

Study on seed development and maturation studies in little millet revealed that optimum stage of physiological maturity was attained at 40 days after anthesis (DAA). The individual panicles of bulk crop raised in the field and were tagged at the time of flower opening. The panicles were collected at 5 days intervals up to 45 DAA and subjected to the biochemical changes viz., electrical conductivity, leachate amino acid content, leachate sugars, protein content and dehydrogenase activity in seeds. Biochemical parameters were superior in the seeds collected at 40 DAA.