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

Francisco SOLANO; Celia JIMÉNEZ-CERVANTES; José H. MARTÍNEZ-LIARTE; José C. GARCÍA-BORRÓN; José R. JARA; José A. LOZANO

doi:10.1042/bj3130447

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

Biochemical Journal ◽

10.1042/bj3130447 ◽

1996 ◽

Vol 313 (2) ◽

pp. 447-453 ◽

Cited By ~ 37

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.

Download Full-text

A thiono-β-lactam substrate for the β-lactamase II of Bacillus cereus. Evidence for direct interaction between the essential metal ion and substrate

Biochemical Journal ◽

10.1042/bj2580765 ◽

1989 ◽

Vol 258 (3) ◽

pp. 765-768 ◽

Cited By ~ 7

Author(s):

B P Murphy ◽

R F Pratt

Keyword(s):

Bacillus Cereus ◽

Active Site ◽

Metal Ion ◽

Direct Interaction ◽

Beta Lactamase ◽

Beta Lactam ◽

Enzyme Active Site ◽

Lactam Carbonyl ◽

Beta Lactamase Ii ◽

Hydrolysis Of

An 8-thionocephalosporin was shown to be a substrate of the beta-lactamase II of Bacillus cereus, a zinc metalloenzyme. Although it is a poorer substrate, as judged by the Kcat./Km parameter, than the corresponding 8-oxocephalosporin, the discrimination against sulphur decreased when the bivalent metal ion in the enzyme active site was varied in the order Mn2+ (the manganese enzyme catalysed the hydrolysis of the oxo compound but not that of the thiono compound), Zn2+, Co2+ and Cd2+. This result is taken as evidence for kinetically significant direct contact between the active-site metal ion of beta-lactamase II and the beta-lactam carbonyl heteroatom. No evidence was obtained, however, for accumulation of an intermediate with such co-ordination present.

Download Full-text

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

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.014876 ◽

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.

Download Full-text

Calcium-binding properties of human erythrocyte calpain

Biochemical Journal ◽

10.1042/bj3250721 ◽

1997 ◽

Vol 325 (3) ◽

pp. 721-726 ◽

Cited By ~ 28

Author(s):

Mauro MICHETTI ◽

Franca SALAMINO ◽

Roberto MINAFRA ◽

Edon MELLONI ◽

Sandro PONTREMOLI

Keyword(s):

Active Site ◽

Human Erythrocyte ◽

Binding Sites ◽

Calcium Binding ◽

Metal Ion ◽

Primary Event ◽

Binding Properties ◽

Physiological Concentration ◽

Physiological Conditions ◽

Sequential Mechanism

The results presented provide more information on the sequential mechanism that promotes the Ca2+-induced activation of human erythrocyte μ-calpain under physiological conditions. The primary event in this process corresponds to the binding of Ca2+ to eight interacting sites, of which there are four in each of the two calpain subunits. Progressive binding of this metal ion is linearly correlated with the dissociation of the proteinase, which reaches completion when all eight binding sites are occupied. The affinity for Ca2+ in the native heterodimeric calpain is increased 2-fold in the isolated 80 kDa catalytic subunit, but it reaches a Kd consistent with the physiological concentration of Ca2+ only in the active autoproteolytically derived 75 kDa form. Binding of Ca2+ in physiological conditions, and thus the formation of the 75 kDa subunit, can occur only in the presence of positive modulators. These are represented by the natural activator protein, found to be a Ca2+-binding protein, and by highly digestible substrates. The former produces a very large increase in the affinity of calpain for Ca2+, and the latter a smaller but still consistent decrease in the Kd of the proteinase for the metal ion. As a result, both dissociation into the constituent subunits and the autoproteolytic conversion of the native 80 kDa subunit into the active 75 kDa form can occur within the physiological fluctuations in Ca2+ concentration. The delay in the expression of the proteolytic activity with respect to Ca2+ binding to native calpain, no longer detectable in the 75 kDa form, can be attributed to a Ca2+-induced functional conformational change, which is correlated with the accessibility of the active site of the enzyme.

Download Full-text

Metal cofactor requirement of β-lactamase II

Biochemical Journal ◽

10.1042/bj1430129 ◽

1974 ◽

Vol 143 (1) ◽

pp. 129-135 ◽

Cited By ~ 61

Author(s):

Richard B. Davies ◽

E. P. Abraham

Keyword(s):

Binding Sites ◽

Metal Ion ◽

Equilibrium Dialysis ◽

Thiol Group ◽

High Yield ◽

Absorption Bands ◽

Nitrobenzoic Acid ◽

Metal Cofactor ◽

Group 3 ◽

Ellman's Reagent

1. The apoenzyme obtained on removal of Zn2+from β-lactamase II from Bacillus cereus 569/H/9 showed less than 0.001% of the activity of the Zn2+-containing enzyme. 2. Removal of Zn2+led to a conformational change in the enzyme and partial unmasking of a thiol group. 3. Replacement of Zn2+by Co2+, Cd2+, Mn2+or Hg2+gave enzymes with significant, but lower, β-lactamase activity. No activity was detected in the presence of Cu2+, Ni2+, Mg2+or Ca2+. 4. Equilibrium dialysis indicated that the enzyme had at least two Zn2+binding sites. With benzylpenicillin as substrate the variation in activity with concentration of Zn2+indicated that activity paralleled binding of Zn2+to the site of highest affinity. 5. Replacement of Zn2+by Co2+and Cd2+gave enzymes with absorption bands at 340 and 245nm respectively, and raised the question of whether the thiol group in the enzyme is a metal-ion ligand. 6. Reduction of the product obtained by reaction of denatured β-lactamase II with Ellman's reagent [5,5′-dithiobis-(2-nitrobenzoic acid)] gave a protein which could refold to produce β-lactamase II activity in high yield.

Download Full-text

Model complexes of the active site of galactose oxidase. Effects of the metal ion binding sites

Inorganica Chimica Acta ◽

10.1016/j.ica.2004.04.008 ◽

2004 ◽

Vol 357 (11) ◽

pp. 3369-3381 ◽

Cited By ~ 29

Author(s):

Masayasu Taki ◽

Haruna Hattori ◽

Takao Osako ◽

Shigenori Nagatomo ◽

Motoo Shiro ◽

...

Keyword(s):

Active Site ◽

Binding Sites ◽

Metal Ion ◽

Galactose Oxidase ◽

Ion Binding ◽

Metal Ion Binding ◽

Model Complexes ◽

Ion Binding Sites ◽

Metal Ion Binding Sites

Download Full-text

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

Metallomics ◽

10.1039/c8mt00330k ◽

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.

Download Full-text

Carbonic Anhydrase Interaction With Lipothioars Enites: A Novel Class of Isozymes I and II Inhibitors

Metal-Based Drugs ◽

10.1155/mbd.1996.263 ◽

1996 ◽

Vol 3 (6) ◽

pp. 263-268 ◽

Cited By ~ 15

Author(s):

Despina Timotheatou ◽

Panayiotis V. Ioannou ◽

Andrea Scozzafava ◽

Fabrizio Briganti ◽

Claudiu T. Supuran

Keyword(s):

Carbonic Anhydrase ◽

Active Site ◽

Electronic Spectra ◽

Metal Ion ◽

Carboxyl Groups ◽

Substitution Pattern ◽

Active Compounds ◽

Enzyme Active Site

The interaction of carbonic anhydrase (CA) isozymes I and II with a series of As(III) derivatives, dialkyl and diaryl rac-2,3-dimyristoyloxypropyldithioarsonites, was investigated kinetically and spectrophotometrically, utilizing the native and Co(II)-substituted enzymes. Depending on the substitution pattern at the -As(SR)2 moiety of the investigated derivatives, inactive compounds were found for R = phenyl or naphthyl, and active ones for derivatives containing carboxyl groups (R = CH2COOH, cysteinyl and glutathionyl). Together with the arsonolipids previously investigated, the active compounds of this series - the "lipothioarsenites"- constitute a novel class of CA inhibitors that bind to the metal ion within the enzyme active site, as proved by changes in the electronic spectra of adducts of such inhibitors with Co(II)CA.

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