Article

Computational Structural Analysis and Kinetic Studies of a Cytosolic Glutamine Synthetase from Camellia sinensis (L.) O. Kuntze

Biotechnology Division, Institute of Himalayan Bioresource Technology, CSIR, Palampur 176061, India.
The Protein Journal (Impact Factor: 0.91). 11/2009; 28(9-10):428-34. DOI: 10.1007/s10930-009-9210-3
Source: PubMed

ABSTRACT

Structural analysis of a cytosolic glutamine synthetase from Camellia sinensis (CsGS) has been conducted employing computational techniques. This was conducted to compare its structural aspects with other known structures of GS. The disordered residues and their distribution in CsGS are in close comparison to earlier reported GS. The 3-D structure of CsGS also showed high degree of similarity with the only known crystal structure of GS from Zea mays. The K
m
values observed with recombinant CsGS for all the three substrates are higher compared to rice, Arabidopsis, maize and human. This suggests lower affinity of CsGS for substrates. Further, kinetic mechanism of CsGS catalysis was investigated using initial velocity analysis and product inhibition studies. Initial velocity data eliminate the possibility of ping-pong mechanism and favor the random mechanism of catalysis. Through product inhibition studies, ADP was found to be a competitive inhibitor with respect to ATP and noncompetitive inhibitor versus both glutamate and ammonium. While, glutamine and inorganic phosphate were found to be non-competitive inhibitors of ATP, glutamate and ammonia. Taken together, these observations are consistent with a random catalysis mechanism for the CsGS where the binding order of certain substrates is kinetically preferred by the enzyme.

0 Followers
 · 
5 Reads
  • [Show abstract] [Hide abstract]
    ABSTRACT: Steviol glycosides are the major secondary metabolites synthesized through steviol glycoside biosynthesis pathway operating in the leaves of Stevia rebaudiana. Present article documents the structural analysis of enzymes specific to steviol glycoside biosynthesis pathway, kaurenoic acid-13 hydoxylase (KAH) and three UDP-glycosyltransferases (UGT85C2, UGT74G1 and UGT76G1). The in silico protein structure prediction server SWISS MODEL was used to predict and evaluate the models. The secondary structure data of predicted model for KAH was in accordance to that of cytochrome P450s suggesting its nativeness to the respective superfamily. Similarly, the secondary structure data of target UGTs also showed conservation with the structural information of glycosyltransferases superfamily. PROCHECK and QMEAN Z-score evaluations suggested that the models predicted for the 4 query enzymatic proteins were of good quality. In addition, Ligand binding site analysis and molecular docking analysis was carried out for the predicted models. The following data suggested a possibility of the presence of an alternate pathway for the synthesis of steviol glycosides.
    No preview · Article · Jan 2013
  • [Show abstract] [Hide abstract]
    ABSTRACT: Tea is an important non-alcoholic beverage plant of the world. Cultivation of tea is very important as it earns revenue for the tea growing nations especially the developing countries such as India. Although conventional breeding is well-established and has contributed significantly for varietal improvement of this plant and other Camellia species with ornamental value, yet applications of biotechnology are required to intervene some of the issues where conventional breeding is restricted particularly for woody plants such as tea. It is note-worthy to mention that some amounts of biotechnology works in several facets of tea and its wild species have also been done. In the present book, a state-of-the-art on various aspects of breeding and biotechnology has been complied in eight chapters. They are: i) Origin and descriptions of health benefits as well as morphological classification as first chapter, ii) Breeding and cytogenetics that comprise with various conventional approaches of varietal improvement of tea along with their genetic resources, iii) Micropropagation which deals with in-depth study of clonal propagation, iv) Somatic embryogenesis along with alternative techniques such as suspension culture, cry-preservation etc. v) Molecular breeding that deals with application of various DNA-based markers, linkage map etc., vi) Genetic transformation and associated factors, vii) Stress physiology complied with various works done in tea along with its wild relatives on abiotic as well as biotic stress, and viii) Functional genomics that describe the various works of molecular cloning and characterizations, differential gene expression, high-throughput sequencing, bioinformatics etc. Importantly, the author has made exclusive tables in most of the chapters that include the summary of the works in particular topic. In a nutshell, the book compiles the work already been done, identifies the problems, analyzes the gaps on breeding and biotechnological works of tea as well as its wild species and discusses the future scope as conclusion. Every effort has been made to include all the published works till June 2013. The book will be a useful resource for post-graduate, doctoral as well post-doctoral students working on tea as well as other woody plants. This will also be useful for the scientists working in the areas of life sciences, genomics, biotechnology and molecular biology.
    No preview · Article · Nov 2013