Corrigendum to: “Mutational and spectroscopic studies of the significance of the active site glutamine to metal ion specificity in superoxide dismutase [Journal of Inorganic Biochemistry 80 (2000) 247]”

"The synthesis, structural characterization, as well as the chemical activity studies of a Cu(II) ""bowl complex", based on the resorcin[4]arene scaffold with three imidazole-containing coordinating arms grafted at the large rim, is presented. This complex is a biomimetic model of a metalloenzyme active site where a cofacial triade of amino-acid residues holds the metal ion in the active site [1]. The trisimidazole ligand reacts with a stoichiometric amount of copper(II) perchlorate to produce a Cu(II) diperchloratocomplex 1. Spectroscopic studies revealed a 5-coordinate SBP environment for the Cu(II) center provided by three imidazole arms, and two extra donors, one embedded in the resorcinarene cavity, the other exposed to the solvent, in exo position. These two labile sites are occupied by either coordinating solvent molecules or residual water, and are readily displaced by carboxylate donors, the position of which (endo or exo) is under tight control of the bowl-cavity. The reaction of 1 with CH3COONa led to a formation of the Cu(II) acetatocomplex 2. Molecular structure of 2 features a rigidified resorcinarene bowl, which was constructed by the addition of the four methylene bridges between the eight hydroxyl groups of the octol precursor [2]. The isolated resorcinarene basket reveals an approximate, non-crystallographic, 4mm point symmetry, and can easily host small guest molecules. Three methylimidazole-containing coordination arms at the large rim coordinate the Cu (II) ion. Its coordination sphere is completed by two O atoms from the intra-cavity bound acetate. The electron donors form a distorted square pyramide, where one of the nitrogens is at the appical position. The endo-coordination of the acetate is supported by an extensive network of intramolecular C-H···O and C-H···π interactions. Complex 2 crystallizes in P21/c space group; a=32.3310 (4)Å, b=11.5490 (1)Å, c=21.6020 (2)Å, beta=102.281(3)0."

Download Full-text

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

10.1101/336024 ◽

2018 ◽

Author(s):

Zishuo Cheng ◽

Jamie VanPelt ◽

Alexander Bergstrom ◽

Christopher Bethel ◽

Andrew Katko ◽

...

Keyword(s):

Binding Site ◽

Active Site ◽

Metal Binding ◽

Metal Ion ◽

Kinetic Studies ◽

Binding Pocket ◽

Spectroscopic Studies ◽

Metal Center ◽

Spin Coupling ◽

Metal Binding Site

ABSTRACTIn 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 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 β-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 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 in 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.

Download Full-text

Structures of c-di-GMP/cGAMP degrading phosphodiesterase VcEAL: identification of a novel conformational switch and its implication

Biochemical Journal ◽

10.1042/bcj20190399 ◽

2019 ◽

Vol 476 (21) ◽

pp. 3333-3353 ◽

Cited By ~ 3

Author(s):

Malti Yadav ◽

Kamalendu Pal ◽

Udayaditya Sen

Keyword(s):

Active Site ◽

Metal Binding ◽

Metal Ion ◽

Triosephosphate Isomerase ◽

Catalytic Mechanism ◽

Degradation Mechanism ◽

Structural Basis ◽

Conserved Residues ◽

Phosphodiester Bond ◽

Cyclic Dinucleotides

Cyclic dinucleotides (CDNs) have emerged as the central molecules that aid bacteria to adapt and thrive in changing environmental conditions. Therefore, tight regulation of intracellular CDN concentration by counteracting the action of dinucleotide cyclases and phosphodiesterases (PDEs) is critical. Here, we demonstrate that a putative stand-alone EAL domain PDE from Vibrio cholerae (VcEAL) is capable to degrade both the second messenger c-di-GMP and hybrid 3′3′-cyclic GMP–AMP (cGAMP). To unveil their degradation mechanism, we have determined high-resolution crystal structures of VcEAL with Ca2+, c-di-GMP-Ca2+, 5′-pGpG-Ca2+ and cGAMP-Ca2+, the latter provides the first structural basis of cGAMP hydrolysis. Structural studies reveal a typical triosephosphate isomerase barrel-fold with substrate c-di-GMP/cGAMP bound in an extended conformation. Highly conserved residues specifically bind the guanine base of c-di-GMP/cGAMP in the G2 site while the semi-conserved nature of residues at the G1 site could act as a specificity determinant. Two metal ions, co-ordinated with six stubbornly conserved residues and two non-bridging scissile phosphate oxygens of c-di-GMP/cGAMP, activate a water molecule for an in-line attack on the phosphodiester bond, supporting two-metal ion-based catalytic mechanism. PDE activity and biofilm assays of several prudently designed mutants collectively demonstrate that VcEAL active site is charge and size optimized. Intriguingly, in VcEAL-5′-pGpG-Ca2+ structure, β5–α5 loop adopts a novel conformation that along with conserved E131 creates a new metal-binding site. This novel conformation along with several subtle changes in the active site designate VcEAL-5′-pGpG-Ca2+ structure quite different from other 5′-pGpG bound structures reported earlier.

Download Full-text