A Noncanonical Metal Center Drives the Activity of the Sediminispirochaeta smaragdinae Metallo-β-lactamase SPS-1

Abstract

In an effort to evaluate whether a recently reported putative metallo-β-lactamase (MβL) contains a novel MβL active site, SPS-1 from Sediminispirochaeta smaragdinae was overexpressed, purified, and characterized using spectroscopic and crystallographic studies. Metal analyses demonstrate that recombinant SPS-1 binds nearly 2 equiv of Zn(II), and steady-state kinetic studies show that the enzyme hydrolyzes carbapenems and certain cephalosporins but not β-lactam substrates with bulky substituents at the 6/7 position. Spectroscopic studies of Co(II)-substituted SPS-1 suggest a novel metal center in SPS-1, with a reduced level of spin coupling between the metal ions and a novel Zn metal binding site. This site was confirmed with a crystal structure of the enzyme. The structure shows a Zn site that is similar to that in NDM-1 and other subclass B1 MβLs; however, the Zn metal ion is coordinated by two histidine residues and a water molecule, which is held in position by a hydrogen bond network. The Zn metal is displaced nearly 1 Å from the position reported in other MβLs. The structure also shows extended helices above the active site, which create a binding pocket that precludes the binding of substrates with large, bulky substituents at the 6/7 position of β-lactam antibiotics. This study reveals a novel metal binding site in MβLs and suggests that the targeting of metal binding sites in MβLs with inhibitors is now more challenging with the identification of this new MβL.

Current trends in drug metabolism and pharmacokinetics

“…Uptake transporters such as OATP2B1, PEPT1 and sodium-dependent bile acid transporter (ASBT/SLC10A2) are involved in the intestinal uptake of drugs across the brush border membrane 75 . For example, PEPT1 transports di/tripeptides-like anticancer drugs such as bestatin and β -lactam antibiotics into enterocytes76, 77, 78. Efflux transporters expressed on the brush border membrane of the intestine, are considered as the barriers for intestinal drug absorption.…”

Characterization of PAN-1, a Carbapenem-Hydrolyzing Class B β-Lactamase From the Environmental Gram-Negative Pseudobacteriovorax antillogorgiicola

“…Despite some significant sequence heterogeneity with SPM-1, the protein alignment between the two enzymes revealed conserved amino acid residues known to be crucial for the hydrolytic activity of MβLs, such as the histidine residues 116, 118, 196, and 263, the aspartic acid residue 120, and the cysteine residue 221, respectively (Figure 1; Meini et al, 2014). Phylogenetic analysis using the alignment software SeaView showed that PAN-1 is the closest relative enzyme to SPM-1 (Figure 2) and to the recently reported SPS-1 B1 MβL (Cheng et al, 2018). Interestingly, PAN-1 and SPS-1 exhibited a central inserted sequence that was considered as a unique feature of the SPM-1 MβL (Figure 1; Murphy et al, 2006; Meini et al, 2014; González et al, 2018).…”

“…Our study characterized a novel MβL from an environmental isolate recovered from a coral. This enzyme showed the highest amino acid identity with the class B carbapenemase SPM-1 being its closest relative after the recently reported SPS-1 enzyme (Figure 2; Cheng et al, 2018)(. Interestingly, the amino acid sequence of PAN-1 exhibits an inserted sequence that was also described in SPM-1 (Murphy et al, 2003), being an original feature comparing to other subclass B1 MβLs (Figure 1).…”

“…Interestingly, the amino acid sequence of PAN-1 exhibits an inserted sequence that was also described in SPM-1 (Murphy et al, 2003), being an original feature comparing to other subclass B1 MβLs (Figure 1). Several studies hypothesized that this inserted sequence might be involved in the restriction of substrates spectrum of subclass B2 MβLs, suggesting that SPM-1, SPS-1, and PAN-1 may have evolved from a common ancestor compared to subclass B2 MβLs (Cheng et al, 2018; González et al, 2018).…”

Comprehensive Transcriptomic Profiling of Murine Osteoclast Differentiation Reveals Novel Differentially Expressed Genes and LncRNAs

“…The proteins encoded by these genes are primarily associated with biochemical or signaling pathways at the cellular level and defects in expression may lead to certain disorders. For instance, anomalies in PHETA1/2 have been associated with abnormal bone formation resulting in craniofacial defects ( Ates et al, 2020 ), HAGH has been associated with skin, bone and joint infections in Yaws disease ( Cheng et al, 2018 ) and mutations in GFPT1 have been associated with muscle weakness in congenital myasthenic syndrome ( Helman et al, 2019 ). In addition, histone-related genes H2B Clustered Histone 21 ( H2bc21 ) and H2bc4 were also significantly upregulated during osteoclast differentiation.…”