A non-canonical metal center drives activity of the Sediminispirochaeta smaragdinae metallo-β- lactamase SPS-1

Abstract

In an effort to evaluate whether a recently reported putative metallo-b-lactamase (MbL) contains a novel MbL active site, SPS-1 from Sediminispirochaeta smaragdinae was over-expressed, purified, and characterized using spectroscopic and crystallographic studies. Metal analyses demonstrate that recombinant SPS-1 binds nearly 2 equivalents of Zn(II), and steady-state kinetic studies show that the enzyme hydrolyzes carbapenems and certain cephalosporins but not b-lactam substrates with bulky substituents in the 6/7 position. Spectroscopic studies on Co(II)-substituted SPS-1 suggest a novel metal center in SPS-1, with reduced spin coupling between the metal ions and a novel Zn1 metal binding site.This site was confirmed with a crystal structure of the enzyme. The structure shows a Zn2 site that is similar that that in NDM-1 and other subclass B1 MβLs; however, the Zn1 metal ion is coordinated by 2 histidine residues and a water molecule, which is held in position by a hydrogen bond network. The Zn1 metal is displaced nearly 1 Å from the position reported in other MbLs. 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 in the 6/7 position of b-lactam antibiotics. This study reveals a novel metal binding site in MβLs, and suggests that the targeting of metal binding sites in MbLs with inhibitors is now more challenging with the identification of this new MbL.Recently, with the use of the Markov model and a bioinformatic study, Berglund et al. identified 279 unique, potential subclass B1 MβL genes, and 76 of the corresponding gene products were novel and able to hydrolyze imipenem (6). One gene, SPS-1, was found in the bacterium Sediminispirochata smaragdinae, (NCBI Reference sequence WP_013255389.1). The S. smaragdinae strain SEBR 4228 T was discovered in a water sample near a Congo offshore oilfield (7). SEBR 4228 T is Gram-negative, chemoorganotrophic and anaerobic bacterium that adopts a spiral, corkscrew-like structure. This organism is halophilic and able to grow in high-salt environments. SPS-1 was predicted to be a MβL; however, SPS-1 lacked the typical H-X-H-Y-D motif found in all MβLs except the B2 MβLs. In SPS-1, the first consensus histidine residue was substituted with a glycine (6). In an effort to determine if SPS-1 is indeed a MβL and how the histidine to glycine substitution affected the consensus Zn1 site, we cloned, over-expressed, and purified the enzyme, and characterized the recombinant protein with kinetic, spectroscopic, and crystallographic studies. RESULTS Phylogenetic analysis of SPS-1.MβLs can be grouped into three subclasses phylogenetically (1). Subclass B1 MβLs contain a Zn1 binding site with three histidine residues (His116, His118, His196) and a Zn2 site with three other residues (Asp120, Cys221, His263) (1-3). Subclass B2 MβLs contain a Zn1 site with one altered residue (Asn116, His118, His196) and Zn2 site identical to that of the subclass B1 MβLs; the altered Zn1 site r...

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.…”

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“…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.…”

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“…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.…”