Catalytic properties of the metal ion variants of mandelate racemase reveal alterations in the apparent electrophilicity of the metal cofactor

Metallomics ◽  
2019 ◽  
Vol 11 (3) ◽  
pp. 707-723
Author(s):  
Matthew L. Harty ◽  
Amar Nath Sharma ◽  
Stephen L. Bearne
Keyword(s):  
Active Site ◽  
Metal Ion ◽  
Catalytic Properties ◽  
Mandelate Racemase ◽  
Metal Cofactor ◽  
Leveling Effect

The apparent electrophilicity of the metal cofactor is altered at the active site of mandelate racemase, causing a “leveling effect” of the catalytic properties of the metalloenzyme variants.

scholarly journals Structural and biochemical characteristics of two Staphylococcus epidermidis RNase J paralogs RNase J1 and RNase J2

2020 ◽  
Vol 295 (49) ◽  
pp. 16863-16876
Author(s):  
Rishi Raj ◽  
Savitha Nadig ◽  
Twinkal Patel ◽  
Balasubramanian Gopal
Keyword(s):  
Conformational Changes ◽  
Active Site ◽  
Staphylococcus Epidermidis ◽  
Metal Ion ◽  
Quaternary Structure ◽  
Structural Similarity ◽  
Endonuclease Activity ◽  
Metal Cofactor ◽  
Rna Maturation ◽  
Metal Cofactors

RNase J enzymes are metallohydrolases that are involved in RNA maturation and RNA recycling, govern gene expression in bacteria, and catalyze both exonuclease and endonuclease activity. The catalytic activity of RNase J is regulated by multiple mechanisms which include oligomerization, conformational changes to aid substrate recognition, and the metal cofactor at the active site. However, little is known of how RNase J paralogs differ in expression and activity. Here we describe structural and biochemical features of two Staphylococcus epidermidis RNase J paralogs, RNase J1 and RNase J2. RNase J1 is a homodimer with exonuclease activity aided by two metal cofactors at the active site. RNase J2, on the other hand, has endonuclease activity and one metal ion at the active site and is predominantly a monomer. We note that the expression levels of these enzymes vary across Staphylococcal strains. Together, these observations suggest that multiple interacting RNase J paralogs could provide a strategy for functional improvisation utilizing differences in intracellular concentration, quaternary structure, and distinct active site architecture despite overall structural similarity.

scholarly journals Molecular mechanism for catalysis by a new zinc-enzyme, dopachrome tautomerase

1996 ◽  
Vol 313 (2) ◽  
pp. 447-453 ◽  
Author(s):  
Francisco SOLANO ◽  
Celia JIMÉNEZ-CERVANTES ◽  
José H. MARTÍNEZ-LIARTE ◽  
José C. GARCÍA-BORRÓN ◽  
José R. JARA ◽  
...  
Keyword(s):  
Molecular Mechanism ◽  
Active Site ◽  
Binding Sites ◽  
Metal Ion ◽  
Copper Binding ◽  
Enzyme Active Site ◽  
Dopachrome Tautomerase ◽  
Theoretical Predictions ◽  
Metal Cofactor ◽  
Related Proteins

Dopachrome tautomerase (DCT; EC 5.3.3.12) catalyses the conversion of L-dopachrome into 5,6-dihydroxyindole-2-carboxylic acid in the mammalian eumelanogenic biosynthetic pathway. This enzyme, also named TRP2, belongs to a family of three metalloenzymes termed the tyrosinase-related proteins (TRPs). It is well known that tyrosinase has copper in its active site. However, the nature of the metal ion in the active site of DCT is under discussion. Whereas theoretical predictions based on similarity between the protein sequences of the TRPs suggest the presence of copper, the different inhibition pattern of DCT with some metal chelators compared with that of tyrosinase suggests that the nature of the metal ion could differ. Direct estimations of the metal content in purified DCT preparations show the presence of around 1.5 Zn atoms/molecule and the absence of copper. Apoenzyme preparation by treatment of DCT with cyanide or o-phenanthroline followed by reconstitution experiments of tautomerase activity in the presence of different ions confirmed that the metal cofactor for the DCT active site is zinc. Our results are consistent with Zn2+ chelation by the highly conserved histidine residues homologous to the histidines at the classical copper-binding sites in tyrosinase. This finding accounts for the reaction catalysed by DCT, i.e. a tautomerization, versus the copper-mediated oxidations catalysed by tyrosinase. Based on the predicted tetrahedrical co-ordination of the zinc ions in the enzyme active site, a molecular mechanism for the catalysis of L-dopachrome tautomerization is proposed. From the present data, the existence of additional ligands for metal ions other than zinc in the DCT molecule, such as the proposed cysteine iron-binding sites, cannot be completely ruled out. However, if such sites exist, they could be subsidiary binding sites, whose function would be likely to stabilize the protein.

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

2019 ◽  
Vol 476 (21) ◽  
pp. 3333-3353 ◽  
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.

scholarly journals Non-Conventional Metal Ion Cofactor Requirement of Dinoflagellate Alkaline Phosphatase and Translational Regulation by Phosphorus Limitation

2019 ◽  
Vol 7 (8) ◽  
pp. 232 ◽  
Author(s):  
Xin Lin ◽  
Chentao Guo ◽  
Ling Li ◽  
Tangcheng Li ◽  
Senjie Lin
Keyword(s):  
Alkaline Phosphatase ◽  
Metal Ion ◽  
Translational Regulation ◽  
Organic Phosphorus ◽  
Niche Differentiation ◽  
Phosphorus Limitation ◽  
Marine Phytoplankton ◽  
Phosphate Limitation ◽  
Dissolved Organic Phosphorus ◽  
Metal Cofactor

Alkaline phosphatase (AP) enables marine phytoplankton to utilize dissolved organic phosphorus (DOP) when dissolved inorganic phosphate (DIP) is depleted in the ocean. Dinoflagellate AP (Dino-AP) represents a newly classified atypical type of AP, PhoAaty. Despite While being a conventional AP, PhoAEC is known to recruit Zn2+ and Mg2+ in the active center, and the cofactors required by PhoAaty have been contended and remain unclear. In this study, we investigated the metal ion requirement of AP in five dinoflagellate species. After AP activity was eliminated by using EDTA to chelate metal ions, the enzymatic activity could be recovered by the supplementation of Ca2+, Mg2+ and Mn2+ in all cases but not by that of Zn2+. Furthermore, the same analysis conducted on the purified recombinant ACAAP (AP of Amphidinium carterae) verified that the enzyme could be activated by Ca2+, Mg2+, and Mn2+ but not Zn2+. We further developed an antiserum against ACAAP, and a western blot analysis using this antibody showed a remarkable up-regulation of ACAAP under a phosphate limitation, consistent with elevated AP activity. The unconventional metal cofactor requirement of Dino-AP may be an adaptation to trace metal limitations in the ocean, which warrants further research to understand the niche differentiation between dinoflagellates and other phytoplankton that use Zn–Mg AP in utilizing DOP.

scholarly journals The Electronic Structure of FeV-cofactor in Vanadium-Dependent Nitrogenase

2021 ◽  
Author(s):  
Zhi-yong Yang ◽  
Emilio Jimenez-Vicente ◽  
Hayden Kallas ◽  
Dmitriy A Lukoyanov ◽  
Hao Yang ◽  
...  
Keyword(s):  
Electronic Structure ◽  
Active Site ◽  
Resting State ◽  
Metal Cofactor ◽  
Open Question

The electronic structure of the active-site metal cofactor (FeV-cofactor) of resting-state V-dependent nitrogenase has been an open question, with earlier studies indicating that it exhibits a broad S = 3/2...

scholarly journals A comparative study of class-D β-lactamases

1993 ◽  
Vol 292 (2) ◽  
pp. 555-562 ◽  
Author(s):  
P Ledent ◽  
X Raquet ◽  
B Joris ◽  
J Van Beeumen ◽  
J M Frère
Keyword(s):  
Active Site ◽  
Gene Sequence ◽  
Catalytic Properties ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Beta Lactamase ◽  
Serine Residue ◽  
Beta Lactamases ◽  
Class D ◽  
Burst Kinetics

Three class-D beta-lactamases (OXA2, OXA1 and PSE2) were produced and purified to protein homogeneity. 6 beta-Iodopenicillanate inactivated the OXA2 enzyme without detectable turnover. Labelling of the same beta-lactamase with 6 beta-iodo[3H]penicillanate allowed the identification of Ser-70 as the active-site serine residue. In agreement with previous reports, the apparent M(r) of the OXA2 enzyme as determined by molecular-sieve filtration, was significantly higher than that deduced from the gene sequence, but this was not due to an equilibrium between a monomer and a dimer. The heterogeneity of the OXA2 beta-lactamase on ion-exchange chromatography contrasted with the similarity of the catalytic properties of the various forms. A first overview of the enzymic properties of the three ‘oxacillinases’ is presented. With the OXA2 enzyme, ‘burst’ kinetics, implying branched pathways, seemed to prevail with many substrates.

scholarly journals How cyanophage S-2L rejects adenine and incorporates 2-aminoadenine to saturate hydrogen bonding in its DNA

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Dariusz Czernecki ◽  
Pierre Legrand ◽  
Mustafa Tekpinar ◽  
Sandrine Rosario ◽  
Pierre-Alexandre Kaminski ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Hydrogen Bonding ◽  
Crystal Structures ◽  
Active Site ◽  
Metal Ion ◽  
Restriction Endonucleases ◽  
Structural Element

AbstractBacteriophages have long been known to use modified bases in their DNA to prevent cleavage by the host’s restriction endonucleases. Among them, cyanophage S-2L is unique because its genome has all its adenines (A) systematically replaced by 2-aminoadenines (Z). Here, we identify a member of the PrimPol family as the sole possible polymerase of S-2L and we find it can incorporate both A and Z in front of a T. Its crystal structure at 1.5 Å resolution confirms that there is no structural element in the active site that could lead to the rejection of A in front of T. To resolve this contradiction, we show that a nearby gene is a triphosphohydolase specific of dATP (DatZ), that leaves intact all other dNTPs, including dZTP. This explains the absence of A in S-2L genome. Crystal structures of DatZ with various ligands, including one at sub-angstrom resolution, allow to describe its mechanism as a typical two-metal-ion mechanism and to set the stage for its engineering.

1.85 Å Resolution Structure of the ZincII β-Lactamase from Bacillus cereus

1998 ◽  
Vol 54 (3) ◽  
pp. 313-323 ◽  
Author(s):  
Andrea Carfi ◽  
Emile Duée ◽  
Moreno Galleni ◽  
Jean-Marie Frère ◽  
Otto Dideberg
Keyword(s):  
Bacillus Cereus ◽  
Active Site ◽  
Metal Ion ◽  
Wide Spectrum ◽  
Zinc Ion ◽  
Beta Lactamases ◽  
Carbonate Ion ◽  
Class B ◽  
Bivalent Metal Ions ◽  
Using Data

Class B \beta-lactamases are wide spectrum enzymes which require bivalent metal ions for activity. The structure of the class B zinc-ion-dependent β-lactamase from Bacillus cereus (BCII) has been refined at 1.85 Å resolution using data collected on cryocooled crystals (100 K). The enzyme from B. cereus has a molecular mass of 24 946 Da and is folded into a \beta-sandwich structure with helices on the external faces. The active site is located in a groove running between the two \beta-sheets [Carfi et al. (1995). EMBO J. 14, 4914–4921]. The 100 K high-resolution BCII structure shows one fully and one partially occupied zinc site. The zinc ion in the fully occupied site (the catalytic zinc) is coordinated by three histidines and one water molecule. The second zinc ion is at 3.7 Å from the first one and is coordinated by one histidine, one cysteine, one aspartate and one unknown molecule (which is most likely to be a carbonate ion). In the B. cereus zinc \beta-lactamase the affinity for the second metal ion is low at the pH of crystallization (Kd = 25 mM, 293 K; [Baldwin et al. (1978). Biochem. J. 175, 441–447] and the dissociation constant of the second zinc ion thus apparently decreased at the cryogenic temperature. In addition, the structure of the apo enzyme was determined at 2.5 Å resolution. The removal of the zinc ion by chelating agents causes small changes in the active-site environment.

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