EBR-1, a novel Ambler subclass B1 beta-lactamase from Empedobacter brevis

Abstract

Empedobacter brevis (formerly designated Flavobacterium breve) is a gram-negative aerobe involved in nosocomial infections. The Ambler class B beta-lactamase gene bla(EBR-1) was cloned and expressed in Escherichia coli from E. brevis clinical strain ASS-1, which had reduced susceptibility to expanded-spectrum cephalosporins and carbapenems. Purified beta-lactamase EBR-1 hydrolyzed penicillins, cephalosporins, and carbapenems efficiently but not aztreonam. Kinetic parameters of EBR-1 were similar to those of class B enzymes such as BlaB, IND-2, and GOB-1 identified from other Flavobacteriaceae species, except for meropenem, which was more hydrolyzed by beta-lactamase GOB-1. EBR-1, with a pI of 8.0 and a relative molecular mass of ca. 25 kDa, was classified in functional subgroup 3a, which includes most of the class B beta-lactamases. EBR-1, which belongs to molecular subclass B1 of metalloenzymes, shares 58, 57, and 42% amino acid identity with the most closely related beta-lactamases, IND-1/IND-2 from Chryseobacterium indologenes, CGB-1 from Chryseobacterium gleum, and BlaB from Chryseobacterium meningosepticum, respectively.

FIG. 1.

Comparison of the amino acid sequence of EBR-1 with those of other β-lactamases of subclass B1 of metalloenzymes. The origin of the β-lactamases is as follows: IND-1 from C. indologenes 001 (2), CGB-1 from C. gleum (4), BlaB from C. meningosepticum CIP 6058 (32), CcrA from Bacteroides fragilis (31), VIM-1 from Pseudomonas aeruginosa VR-143/97 (18), BcII from Bacillus cereus 569/H (16), and IMP-1 from Serratia marcescens TN9106 (26). The BBL numbering scheme is indicated above the sequences (12). Broken lines indicate gaps introduced in the alignment. The vertical arrow indicates putative cleavage of the peptide leader site for EBR-1. Amino acid residues that may be involved in Zn²⁺ or/and water molecule binding are boxed. Identical amino acids in subclass B1 enzymes are indicated by an asterisk.

FIG. 2.

SDS-PAGE analysis of the purified β-lactamase EBR-1 from a culture of E. coli BL21(pET-EBR-1). Lane A, Coomassie blue staining. The horizontal arrow on the left side indicates the migration position of β-lactamase EBR-1. Lane M, size of relative molecular masses expressed in kilodaltons is indicated on the right. Lane B, zymogram of EBR-1.