PDBsum entry 3brm

Hydrolase

PDB id: 3brm

Contents

Protein chains

290 a.a. *

Ligands

ONL ×2

Waters ×295

* Residue conservation analysis

PDB id:

3brm

Name:

Hydrolase

Title:

Crystal structure of the covalent complex between the bacillus subtilis glutaminase ybgj and 5-oxo-l-norleucine formed by reaction of the protein with 6-diazo-5-oxo-l-norleucine

Structure:

Glutaminase 1. Chain: a, b. Engineered: yes

Source:

Bacillus subtilis. Strain: 168. Gene: glsa1, glsa, ybgj, bsu02430. Expressed in: escherichia coli.

Resolution:

2.29Å R-factor: 0.178 R-free: 0.248

Authors:

A.U.Singer,Y.Kim,I.Dementieva,E.Vinokour,A.Joachimiak,A.Savchenko, A.Yakunin

Key ref:

G.Brown et al. (2008). Functional and structural characterization of four glutaminases from Escherichia coli and Bacillus subtilis. Biochemistry, 47, 5724-5735. PubMed id: 18459799

Date:

21-Dec-07 Release date: 20-May-08

Protein chains

O31465  (GLSA1_BACSU) -  Glutaminase 1 from Bacillus subtilis (strain 168)

Seq: 327 a.a.

Struc: 290 a.a.

Enzyme reactions

• Enzyme class: E.C.3.5.1.2 - glutaminase.
• Reaction: L-glutamine + H2O = L-glutamate + NH4⁺

L-glutamine (Bound ligand (Het Group name = ONL) matches with 81.82% similarity) + H2O = L-glutamate + NH4(+)

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

Added reference

Biochemistry 47:5724-5735 (2008)

PubMed id: 18459799

Functional and structural characterization of four glutaminases from Escherichia coli and Bacillus subtilis.

G.Brown, A.Singer, M.Proudfoot, T.Skarina, Y.Kim, C.Chang, I.Dementieva, E.Kuznetsova, C.F.Gonzalez, A.Joachimiak, A.Savchenko, A.F.Yakunin.

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.