Molecular and Biochemical Heterogeneity of Class B Carbapenem-Hydrolyzing β-Lactamases in Chryseobacterium meningosepticum

Abstract

Although the carbapenem-hydrolyzing ␤-lactamase (CH␤L) BlaB-1 is known to be in Chryseobacterium meningosepticum NCTC 10585, a second CH␤L gene, bla GOB-1 , was cloned from another C. meningosepticum clinical isolate (PINT). The G؉C content of bla GOB-1 (36%) indicated the likely chromosomal origin of this gene. Its expression in Escherichia coli DH10B yields a mature CH␤L with a pI of 8.7 and a relative molecular mass of 28.2 kDa. In E. coli, GOB-1 conferred resistance to narrow-spectrum cephalosporins and reduced susceptibility to ureidopenicillins, broad-spectrum cephalosporins, and carbapenems. GOB-1 had a broadspectrum hydrolysis profile including penicillins and cephalosporins (but not aztreonam). The catalytic efficiency for meropenem was higher than for imipenem. GOB-1 had low amino acid identity with the class B CH␤Ls, sharing 18% with the closest, L-1 from Stenotrophomonas maltophilia, and only 11% with BlaB-1. Most of the conserved amino acids that may be involved in the active site of CH␤Ls (His-101, Asp-103, His-162, and His-225) were identified in GOB-1. Sequence heterogeneity was found for GOB-1-like and BlaB-1-like ␤-lactamases, having 90 to 100% and 86 to 100% amino acid identity, respectively, among 10 unrelated C. meningosepticum isolates. Each isolate had a GOB-1-like and a BlaB-1-like gene. The same combination of GOB-1-like and BlaB-1-like ␤-lactamases was not found in two different isolates. C. meningosepticum is a bacterial species with two types of unrelated chromosome-borne class B CH␤Ls that can be expressed in E. coli and, thus, may represent a clinical threat if spread in gram-negative aerobes.

The Metallo-β-lactamase GOB Is a Mono-Zn(II) Enzyme with a Novel Active Site

“…The complete coding sequence of the gob-18 gene was amplified by employing primers 1 and 2 described in Ref. 32. The DNA fragment was sequenced and two new primers were designed (see supplemental material) to clone the mature GOB-18 coding sequence cloned into BamHI-HindIII sites of pETGEXTerm vector (13) in frame to the 3Ј-end of Schistosoma japonicus glutathione S-transferase gene, for expression purposes.…”

“…Subclass B2 includes the CphA (25,26) and ImiS (27) lactamases from Aeromonas species. Subclass B3, originally represented only by L1 from Stenotrophomonas maltophilia (28 -30), now includes enzymes from other opportunistic pathogens like FEZ-1 from Legionella gormanii (31) and GOB from E. meningoseptica (32), as well as from environmental bacteria such as CAU-1 from Caulobacter crescentus (33) and THIN-B from Janthinobacterium lividum (34).…”

Role of Zinc Content on the Catalytic Efficiency of B1 Metallo β-Lactamases

“…Although, as already stated, this conformation is present for about 10% of the MD simulated time, it points to a non-crucial role of Asn233 for the function, which in turn is consistent with an increase of K M in the N233A, N233L and N233E, 15,64 without largely affecting k cat /K M . In this respect, it is worth noting that so far Asn233-Tyr is the only mutation which significant improves the catalytic power in another MβL (BcII) (unpublished results), and it is the only alternative residue found in MβL's at position 233 (namely, in B1 MβL blaB, from E. meningoseptica 39 ), that can perform a similar role. Moreover, a Tyr residue is found in glyoxalase II (a dizinc enzyme belonging to the metallo β-lactamase superfamily 65 ) at the same topological position, and might have a similar binding function.…”

“…27-29 Subclass B3, along with the extensively characterized enzyme L1 from Stenotrophomonas maltophilia ,30-33 includes enzymes from environmental bacteria, such as CAU-1 from Caulobacter crescentus 34 and THIN-B from Janthinobacterium lividum , 35 and from opportunistic pathogens like FEZ-1 from Legionella gormanii ,36-38 and GOB from E. meningoseptica. 39 Atomistic structures of MβL's spanning all the three subclasses have been solved by X-ray crystallography (B1: BcII, 40,41 BlaB, 18 CcrA, 42 IMP-1, 43 SPM-1, 44 (B1); B2: CphA; 27 and B3: L1 31 and FEZ- 1 37 ). The folding frame is characterized in all cases by a compact αβ/ βα sandwich (two core β-sheets surrounded by α-helices), with an active site that can allocate one or two Zn 2+ metal ions.…”

Catalytic Role of the Metal Ion in the Metallo-β-lactamase GOB

“…Subclass B2 includes the CphA (24,25) and ImiS (26) lactamases from Aeromonas species and Sfh-I from Serratia fonticola (27). Subclass B3, originally represented only by L1 from Stenotrophomonas maltophilia (28 -30), now includes enzymes from other opportunistic pathogens like FEZ-1 from Legionella gormanii (31) and GOB from E. meningoseptica (32,33) as well as from environmental bacteria, such as CAU-1 from Caulobacter crescentus (34) and THIN-B from Janthinobacterium lividum (35).…”