The polyketide cyclase RemF from <i>Streptomyces resistomycificus</i> contains an unusual octahedral zinc binding site

Silvennoinen, Laura; Sandalova, T.; Schneider, Günter

doi:10.1016/j.febslet.2009.07.061

Cited by 25 publications

(29 citation statements)

References 33 publications

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“…The protein data bank contains few examples of first-row transition metals bound to native His 4 sites, which include a manganese-containing photochemical reaction center from Rhodobacter spaeroides Y , 53 a manganese-containing cupin from Thermotoga maritima , 54 and RemF, a polyketide cyclase from Streptomyces resistomycificus crystallized in the zinc-bound form. 55 These structures exhibit six-coordinate M(II) centers. Recent crystallographic studies of S100B revealed pH-dependent ligand swapping and tetrahedral Zn(II) coordination to four His residues at pH 9 whereas the metal ion was coordinated to a His 3 Asp motif at lower and higher pH values.…”

Section: Discussionmentioning

confidence: 99%

Calcium Ion Gradients Modulate the Zinc Affinity and Antibacterial Activity of Human Calprotectin

2012

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Calprotectin (CP) is an antimicrobial protein produced and released by neutrophils that inhibits the growth of pathogenic microorganisms by sequestering essential metal nutrients in the extracellular space. In this work, spectroscopic and thermodynamic metal-binding studies are presented to delineate the zinc-binding properties of CP. Unique optical absorption and EPR spectroscopic signatures for the interfacial His3Asp and His4 sites of human calprotectin are identified by using Co(II) as a spectroscopic probe. Zinc competition titrations employing chromophoric Zn(II) indicators provide a 2:1 Zn(II):CP stoichiometry, confirm that the His3Asp and His4 sites of CP coordinate Zn(II), and reveal that the Zn(II) affinity of both sites is calcium-dependent. The calcium-insensitive Zn(II) competitor ZP4 afforded dissociation constants of Kd1 = 133 ± 58 pM and Kd2 = 185 ± 219 nM for CP in the absence of Ca(II). These values decreased to Kd1 ≤ 10 pM and Kd2 ≤ 240 pM in the presence of excess Ca(II). The Kd1 and Kd2 values are assigned to the His3Asp and His4 sites, respectively. In vitro antibacterial activity assays indicate that the metal-binding sites and Ca(II)-replete conditions are required for CP to inhibit the growth of both Gram-negative and –positive bacteria. Taken together, these data provide a working model whereby calprotectin responds to physiological Ca(II) gradients to become a potent Zn(II) chelator in the extracellular space.

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Section: Discussionmentioning

confidence: 99%

Calcium Ion Gradients Modulate the Zinc Affinity and Antibacterial Activity of Human Calprotectin

2012

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“…However, it has only 40% sequence identity to GtHNL. The other structures showed even lower similarity [1VJ2: Z-score 14.5, RMSD 1.9, 20% sequence identity; 1O4T: Z-score 14.5, RMSD 2.0, sequence identity 21% (both uncharacterized monocupins from Thermotoga maritima); 3HT2: Z-score 14.5, RMSD 2.4, 14% sequence identity (a zinc-containing polyketide cyclase RemF from Streptomyces resistomycificus [22])]. The gene corresponding to 2F4P was obtained as a synthetic gene and re-cloned, and the protein was expressed as described for GtHNL, but showed only very weak hydroxynitrile lyase activity, which was not investigated any further (data not shown).…”

Section: Overall Three-dimensional Structure Of Gthnlmentioning

confidence: 99%

Biochemical and structural characterization of a novel bacterial manganese‐dependent hydroxynitrile lyase

et al. 2013

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Hydroxynitrile lyases (HNLs), which catalyse the decomposition of cyanohydrins, are found mainly in plants. In vitro, they are able to catalyse the synthesis of enantiopure cyanohydrins, which are versatile building blocks in the chemical industry. Recently, HNLs have also been discovered in bacteria. Here, we report on the detailed biochemical and structural characterization of a hydroxynitrile lyase from Granulicella tundricola (GtHNL), which was successfully heterologously expressed in Escherichia coli. The crystal structure was solved at a crystallographic resolution of 2.5 Å and exhibits a cupin fold. As GtHNL does not show any sequence or structural similarity to any other HNL and does not contain conserved motifs typical of HNLs, cupins represent a new class of HNLs. GtHNL is metal‐dependent, as confirmed by inductively coupled plasma/optical emission spectroscopy, and in the crystal structure, manganese is bound to three histidine and one glutamine residue. GtHNL displayed a specific activity of 1.74 U·mg−1 at pH 6 with (R)‐mandelonitrile, and synthesized (R)‐mandelonitrile with 90% enantiomeric excess at 80% conversion using 0.5 m benzaldehyde in a biphasic reaction system with methyl tertiary butyl ether.

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“…Other enzymes with a "reduction" of the 3His-1Glu motif are the gentisate 1,2-dioxygenases (GDOs) and 3-hydroxyanthranilate 3,4-dioxygenases (HADs) (see below). Most remarkably, the metal binding site in polyketide cyclase RemF, a monocupin protein, contains an octahedral zinc site, with four histidine residues as protein ligands to the Zn 2ϩ ion (110).…”

Section: Fig 2 Ribbon Diagram Of the Monomer Of Quercetinase Of A Jamentioning

confidence: 99%

Ring-Cleaving Dioxygenases with a Cupin Fold

Fetzner

2012

Appl Environ Microbiol

166

141

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ABSTRACTRing-cleaving dioxygenases catalyze key reactions in the aerobic microbial degradation of aromatic compounds. Many pathways converge to catecholic intermediates, which are subject toorthoormetacleavage by intradiol or extradiol dioxygenases, respectively. However, a number of degradation pathways proceed via noncatecholic hydroxy-substituted aromatic carboxylic acids like gentisate, salicylate, 1-hydroxy-2-naphthoate, or aminohydroxybenzoates. The ring-cleaving dioxygenases active toward these compounds belong to the cupin superfamily, which is characterized by a six-stranded β-barrel fold and conserved amino acid motifs that provide the 3His or 2- or 3His-1Glu ligand environment of a divalent metal ion. Most cupin-type ring cleavage dioxygenases use an FeIIcenter for catalysis, and the proposed mechanism is very similar to that of the canonical (type I) extradiol dioxygenases. The metal ion is presumed to act as an electron conduit for single electron transfer from the metal-bound substrate anion to O2, resulting in activation of both substrates to radical species. The family of cupin-type dioxygenases also involves quercetinase (flavonol 2,4-dioxygenase), which opens up two C-C bonds of the heterocyclic ring of quercetin, a wide-spread plant flavonol. Remarkably, bacterial quercetinases are capable of using different divalent metal ions for catalysis, suggesting that the redox properties of the metal are relatively unimportant for the catalytic reaction. The major role of the active-site metal ion could be to correctly position the substrate and to stabilize transition states and intermediates rather than to mediate electron transfer. The tentative hypothesis that quercetinase catalysis involves direct electron transfer from metal-bound flavonolate to O2is supported by model chemistry.

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The polyketide cyclase RemF from Streptomyces resistomycificus contains an unusual octahedral zinc binding site

Cited by 25 publications

References 33 publications

Calcium Ion Gradients Modulate the Zinc Affinity and Antibacterial Activity of Human Calprotectin

Calcium Ion Gradients Modulate the Zinc Affinity and Antibacterial Activity of Human Calprotectin

Biochemical and structural characterization of a novel bacterial manganese‐dependent hydroxynitrile lyase

Ring-Cleaving Dioxygenases with a Cupin Fold

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