Structural and Biochemical Characterization of Rm3, a Subclass B3 Metallo-β-Lactamase Identified from a Functional Metagenomic Study

Abstract

c ␤-Lactamase production increasingly threatens the effectiveness of ␤-lactams, which remain a mainstay of antimicrobial chemotherapy. New activities emerge through both mutation of previously known ␤-lactamases and mobilization from environmental reservoirs. The spread of metallo-␤-lactamases (MBLs) represents a particular challenge because of their typically broad-spectrum activities encompassing carbapenems, in addition to other ␤-lactam classes. Increasingly, genomic and metagenomic studies have revealed the distribution of putative MBLs in the environment, but in most cases their activity against clinically relevant ␤-lactams and, hence, the extent to which they can be considered a resistance reservoir remain uncharacterized. Here we characterize the product of one such gene, bla Rm3 , identified through functional metagenomic sampling of an environment with high levels of biocide exposure. bla Rm3 encodes a subclass B3 MBL that, when expressed in a recombinant Escherichia coli strain, is exported to the bacterial periplasm and hydrolyzes clinically used penicillins, cephalosporins, and carbapenems with an efficiency limited by high K m values. An Rm3 crystal structure reveals the MBL superfamily ␣␤/␤␣ fold, which more closely resembles that in mobilized B3 MBLs (AIM-1 and SMB-1) than other chromosomal enzymes (L1 or FEZ-1). A binuclear zinc site sits in a deep channel that is in part defined by a relatively extended N terminus. Structural comparisons suggest that the steric constraints imposed by the N terminus may limit its affinity for ␤-lactams. Sequence comparisons identify Rm3-like MBLs in numerous other environmental samples and species. Our data suggest that Rm3-like enzymes represent a distinct group of B3 MBLs with a wide distribution and can be considered an environmental reservoir of determinants of ␤-lactam resistance.T he continued efficacy of ␤-lactam antibiotics is threatened by the dissemination of ␤-lactamases, hydrolytic enzymes that inactivate these important drugs by cleavage of the scissile ␤-lactam amide bond (1). In the 70 years since ␤-lactams were first introduced into the clinic, repeated mobilizations of ␤-lactamase genes from a variety of bacterial sources have led to their rapid propagation in opportunistic Gram-negative pathogens, such as the Enterobacteriaceae and nonfermenting species, including Pseudomonas aeruginosa and Acinetobacter baumannii (2). Notably, some of the most successful ␤-lactamases, in particular, the CTX-M extended-spectrum ␤-lactamase (ESBL), which is associated with resistance to third-generation cephalosporins, such as cefotaxime, and which is now distributed worldwide (3), find their origins in environmental organisms, illustrating how transfer of antibiotic resistance genes from environmental to pathogenic species can have profound clinical consequences (4). In the case of CTX-M enzymes, it is now accepted that these originated in Kluyvera spp. (5, 6), which are Gram-negative rod bacteria that are found in both the human intestinal microbiome and the ...

Structural and biochemical analysis of the metallo‐β‐lactamase L1 from emerging pathogen Stenotrophomonas maltophilia revealed the subtle but distinct di‐metal scaffold for catalytic activity

“…S. maltophilia L1 is a subclass B3 MBL, one of two chromosome‐encoded β‐lactamases (L2 belongs to a class A) . Several crystal structures of L1 and other B3 MBLs have been reported in the complexes with hydrolyzed antibiotics and inhibitors, which are supported by multiple biochemical analysis . B3 MBLs share a relatively low sequence identity of 23–35%; however, they are structurally well‐conserved particularly their di‐nuclear zinc active sites.…”

“…16 Several crystal structures of L1 [17][18][19][20][21] and other B3 MBLs have been reported in the complexes with hydrolyzed antibiotics and inhibitors, which are supported by multiple biochemical analysis. [17][18][19][20][21][22][23][24][25][26][27][28][29][30] B3 MBLs share a relatively low sequence identity of 23-35%; however, they are structurally well-conserved particularly their di-nuclear zinc active sites. Here, we extend the L1 studies and report several high-resolution structures of L1, the two zinc bound form (native form), the metal-free form (apo form), the captopril inhibitor bound form, several complexes with hydrolyzed antibiotics (imipenem, moxalactam, meropenem, and penicillin G) and with different metal ions (Zn +2 , Cd +2 , and Cu +2 ).…”

Structure and mechanism of potent bifunctional β-lactam- and homoserine lactone-degrading enzymes from marine microorganisms

“…AIM-1 and SMB-1) tend to have higher MBL activity compared to those whose N-terminus enters or imposes on the space available within the active site (i.e. L1, BJP and Rm3) 44,45,49,51 . In agreement with this interpretation, it could be demonstrated, that the catalytic efficiency (i.e.…”

Kinetic, mechanistic, structural and spectroscopic investigations of Bimetallic Metallohydrolases

“…Examples of enzymes form this subclass include: L1 from S. maltophilia (76,(121)(122)(123)(124)(125)(126)(127)(128) , SMB from S. marcescens (70,71) , BJP from B. japonicum (129,130) , FEZ from F. gormanii (131)(132)(133)(134) , LRA and Rm3 from functional metagenomic studies (135,136) , AIM from P. aeroginosa (7,72,137) , MIM-1 from N. pentaromativorans and MIM-2 from S. agarivorans (26) ; LRA, AIM and the two MIMs will be discussed in more detail in subsequent chapters (i.e. Chapters 4, 5 and 6).…”

“…Similar B3 MBL that share this feature (Eg AIM-1, SMB-1) tend to have higher activity than those whose N-terminus enters or imposes on the space available within the active site (eg. BJP and RM3) (70,71,130,135) . With this theme in mind, it has been shown that the L1 protein also gained a reduction of the Kcat/km by at least 20-fold when its extended N-terminus was truncated such that it no longer imposed on the active site (126) .…”