Kuznetsova E

References (2)

Title : Functional and structural characterization of four glutaminases from Escherichia coli and Bacillus subtilis - Brown_2008_Biochemistry_47_5724
Author(s) : Brown G , Singer A , Proudfoot M , Skarina T , Kim Y , Chang C , Dementieva I , Kuznetsova E , Gonzalez CF , Joachimiak A , Savchenko A , Yakunin AF
Ref : Biochemistry , 47 :5724 , 2008
Abstract : Glutaminases belong to the large superfamily of serine-dependent beta-lactamases and penicillin-binding proteins, and they catalyze the hydrolytic deamidation of L-glutamine to L-glutamate. In this work, we purified and biochemically characterized four predicted glutaminases from Escherichia coli (YbaS and YneH) and Bacillus subtilis (YlaM and YbgJ). The proteins demonstrated strict specificity to L-glutamine and did not hydrolyze D-glutamine or L-asparagine. In each organism, one glutaminase showed higher affinity to glutamine ( E. coli YbaS and B. subtilis YlaM; K m 7.3 and 7.6 mM, respectively) than the second glutaminase ( E. coli YneH and B. subtilis YbgJ; K m 27.6 and 30.6 mM, respectively). The crystal structures of the E. coli YbaS and the B. subtilis YbgJ revealed the presence of a classical beta-lactamase-like fold and conservation of several key catalytic residues of beta-lactamases (Ser74, Lys77, Asn126, Lys268, and Ser269 in YbgJ). Alanine replacement mutagenesis demonstrated that most of the conserved residues located in the putative glutaminase catalytic site are essential for activity. The crystal structure of the YbgJ complex with the glutaminase inhibitor 6-diazo-5-oxo- l-norleucine revealed the presence of a covalent bond between the inhibitor and the hydroxyl oxygen of Ser74, providing evidence that Ser74 is the primary catalytic nucleophile and that the glutaminase reaction proceeds through formation of an enzyme-glutamyl intermediate. Growth experiments with the E. coli glutaminase deletion strains revealed that YneH is involved in the assimilation of l-glutamine as a sole source of carbon and nitrogen and suggested that both glutaminases (YbaS and YneH) also contribute to acid resistance in E. coli.
ESTHER : Brown_2008_Biochemistry_47_5724
PubMedSearch : Brown_2008_Biochemistry_47_5724
PubMedID: 18459799

Title : Enzyme genomics: Application of general enzymatic screens to discover new enzymes - Kuznetsova_2005_FEMS.Microbiol.Rev_29_263
Author(s) : Kuznetsova E , Proudfoot M , Sanders SA , Reinking J , Savchenko A , Arrowsmith CH , Edwards AM , Yakunin AF
Ref : FEMS Microbiology Reviews , 29 :263 , 2005
Abstract : In all sequenced genomes, a large fraction of predicted genes encodes proteins of unknown biochemical function and up to 15% of the genes with "known" function are mis-annotated. Several global approaches are routinely employed to predict function, including sophisticated sequence analysis, gene expression, protein interaction, and protein structure. In the first coupling of genomics and enzymology, Phizicky and colleagues undertook a screen for specific enzymes using large pools of partially purified proteins and specific enzymatic assays. Here we present an overview of the further developments of this approach, which involve the use of general enzymatic assays to screen individually purified proteins for enzymatic activity. The assays have relaxed substrate specificity and are designed to identify the subclass or sub-subclasses of enzymes (phosphatase, phosphodiesterase/nuclease, protease, esterase, dehydrogenase, and oxidase) to which the unknown protein belongs. Further biochemical characterization of proteins can be facilitated by the application of secondary screens with natural substrates (substrate profiling). We demonstrate here the feasibility and merits of this approach for hydrolases and oxidoreductases, two very broad and important classes of enzymes. Application of general enzymatic screens and substrate profiling can greatly speed up the identification of biochemical function of unknown proteins and the experimental verification of functional predictions produced by other functional genomics approaches.
ESTHER : Kuznetsova_2005_FEMS.Microbiol.Rev_29_263
PubMedSearch : Kuznetsova_2005_FEMS.Microbiol.Rev_29_263
PubMedID: 15808744
Gene_locus related to this paper: ecoli-yafa , ecoli-ybff , ecoli-ycjy , ecoli-yeiG , ecoli-YFBB , ecoli-yjfp , ecoli-ypfh , ecoli-yqia , ecoli-yuar