PDBsum entry 4r3b

Hydrolase

PDB id: 4r3b

Contents

Protein chain

265 a.a.

Ligands

MA4 ×2

3GE

Waters ×269

PDB id:

4r3b

Name:

Hydrolase

Title:

Crystal structure of shv-1 b-lactamase in complex with 6b- (hydroxymethyl)penicillanic acid sulfone psr-283a

Structure:

Beta-lactamase shv-1. Chain: a. Fragment: shv-1 beta-lactamase. Synonym: pit-2. Engineered: yes

Source:

Klebsiella pneumoniae. Organism_taxid: 573. Gene: bla, shv1. Expressed in: escherichia coli. Expression_system_taxid: 562

Resolution:

1.37Å R-factor: 0.132 R-free: 0.172

Authors:

E.A.Rodkey,F.Van Den Akker

Key ref:

T.Che et al. (2015). Detecting a quasi-stable imine species on the reaction pathway of SHV-1 β-lactamase and 6β-(hydroxymethyl)penicillanic acid sulfone. Biochemistry, 54, 734-743. PubMed id: 25536850 DOI: 10.1021/bi501197t

Date:

14-Aug-14 Release date: 21-Jan-15

Protein chain

P0AD64  (BLA1_KLEPN) -  Beta-lactamase SHV-1 from Klebsiella pneumoniae

Seq: 286 a.a.

Struc: 265 a.a.

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+)

DOI no: 10.1021/bi501197t

Biochemistry 54:734-743 (2015)

PubMed id: 25536850

Detecting a quasi-stable imine species on the reaction pathway of SHV-1 β-lactamase and 6β-(hydroxymethyl)penicillanic acid sulfone.

T.Che, E.A.Rodkey, C.R.Bethel, S.Shanmugam, Z.Ding, M.Pusztai-Carey, M.Nottingham, W.Chai, J.D.Buynak, R.A.Bonomo, F.van den Akker, P.R.Carey.

ABSTRACT

For the class A β-lactamase SHV-1, the kinetic and mechanistic properties of the clinically used inhibitor sulbactam are compared with the sulbactam analog substituted in its 6β position by a CH2OH group (6β-(hydroxymethyl)penicillanic acid). The 6β substitution improves both in vitro and microbiological inhibitory properties of sulbactam. Base hydrolysis of both compounds was studied by Raman and NMR spectroscopies and showed that lactam ring opening is followed by fragmentation of the dioxothiazolidine ring leading to formation of the iminium ion within 3 min. The iminium ion slowly loses a proton and converts to cis-enamine (which is a β-aminoacrylate) in 1 h for sulbactam and in 4 h for 6β-(hydroxymethyl) sulbactam. Rapid mix-rapid freeze Raman spectroscopy was used to follow the reactions between the two sulfones and SHV-1. Within 23 ms, a 10-fold excess of sulbactam was entirely hydrolyzed to give a cis-enamine product. In contrast, the 6β-(hydroxymethyl) sulbactam formed longer-lived acyl-enzyme intermediates that are a mixture of imine and enamines. Single crystal Raman studies, soaking in and washing out unreacted substrates, revealed stable populations of imine and trans-enamine acyl enzymes. The corresponding X-ray crystallographic data are consonant with the Raman data and also reveal the role played by the 6β-hydroxymethyl group in retarding hydrolysis of the acyl enzymes. The 6β-hydroxymethyl group sterically hinders approach of the water molecule as well as restraining the side chain of E166 that facilitates hydrolysis.