PDBsum entry 5hap

Hydrolase

PDB id: 5hap

Contents

Protein chains

241 a.a.

Ligands

1PE

PGE ×2

PEG ×8

MPD

PG4

EDO ×4

Metals

_CL

_NA ×2

Waters ×277

PDB id:

5hap

Name:

Hydrolase

Title:

Oxa-48 beta-lactamase - s70a mutant

Structure:

Beta-lactamase. Chain: a, b. Fragment: unp residues 25-265. Engineered: yes. Mutation: yes

Source:

Klebsiella pneumoniae. Organism_taxid: 573. Gene: bla oxa-48, blaoxa-48, kpe71t_00045. Expressed in: escherichia coli. Expression_system_taxid: 469008.

Resolution:

1.89Å R-factor: 0.192 R-free: 0.226

Authors:

V.Stojanoski,C.J.Adamski,L.Hu,S.C.Mehta,B.Sankaran,B.V.V.Prasad, T.G.Palzkill

Key ref:

V.Stojanoski et al. (2016). Removal of the Side Chain at the Active-Site Serine by a Glycine Substitution Increases the Stability of a Wide Range of Serine β-Lactamases by Relieving Steric Strain. Biochemistry, 55, 2479-2490. PubMed id: 27073009

Date:

30-Dec-15 Release date: 07-Sep-16

Protein chains

Q6XEC0  (Q6XEC0_KLEPN) -  Beta-lactamase from Klebsiella pneumoniae

Seq: 265 a.a.

Struc: 241 a.a.*

* PDB and UniProt seqs differ at 2 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.3.5.2.6 - beta-lactamase.
• Pathway: Penicillin Biosynthesis and Metabolism
• Reaction: a beta-lactam + H2O = a substituted beta-amino acid
• Cofactor: Zn(2+)

Biochemistry 55:2479-2490 (2016)

PubMed id: 27073009

Removal of the Side Chain at the Active-Site Serine by a Glycine Substitution Increases the Stability of a Wide Range of Serine β-Lactamases by Relieving Steric Strain.

V.Stojanoski, C.J.Adamski, L.Hu, S.C.Mehta, B.Sankaran, P.Zwart, B.V.Prasad, T.Palzkill.

ABSTRACT

Serine β-lactamases are bacterial enzymes that hydrolyze β-lactam antibiotics. They utilize an active-site serine residue as a nucleophile, forming an acyl-enzyme intermediate during hydrolysis. In this study, thermal denaturation experiments as well as X-ray crystallography were performed to test the effect of substitution of the catalytic serine with glycine on protein stability in serine β-lactamases. Six different enzymes comprising representatives from each of the three classes of serine β-lactamases were examined, including TEM-1, CTX-M-14, and KPC-2 of class A, P99 of class C, and OXA-48 and OXA-163 of class D. For each enzyme, the wild type and a serine-to-glycine mutant were evaluated for stability. The glycine mutants all exhibited enhanced thermostability compared to that of the wild type. In contrast, alanine substitutions of the catalytic serine in TEM-1, OXA-48, and OXA-163 did not alter stability, suggesting removal of the Cβ atom is key to the stability increase associated with the glycine mutants. The X-ray crystal structures of P99 S64G, OXA-48 S70G and S70A, and OXA-163 S70G suggest that removal of the side chain of the catalytic serine releases steric strain to improve enzyme stability. Additionally, analysis of the torsion angles at the nucleophile position indicates that the glycine mutants exhibit improved distance and angular parameters of the intrahelical hydrogen bond network compared to those of the wild-type enzymes, which is also consistent with increased stability. The increased stability of the mutants indicates that the enzyme pays a price in stability for the presence of a side chain at the catalytic serine position but that the cost is necessary in that removal of the serine drastically impairs function. These findings support the stability-function hypothesis, which states that active-site residues are optimized for substrate binding and catalysis but that the requirements for catalysis are often not consistent with the requirements for optimal stability.