scholarly journals Structural analysis ofClostridium acetobutylicumATCC 824 glycoside hydrolase from CAZy family GH105

2015 ◽  
Vol 71 (8) ◽  
pp. 1100-1108 ◽  
Author(s):  
Katherine L. Germane ◽  
Matthew D. Servinsky ◽  
Elliot S. Gerlach ◽  
Christian J. Sund ◽  
Margaret M. Hurley
Keyword(s):  
Bacillus Subtilis ◽  
Active Site ◽  
Molecular Replacement ◽  
Tertiary Structures ◽  
Aspartic Acid Residue ◽  
Rhamnogalacturonan I ◽  
Protein Sequence Identity ◽  
Unsaturated Disaccharide ◽  
Β Sheet ◽  
Surface Electrostatic Potential

Clostridium acetobutylicumATCC 824 gene CA_C0359 encodes a putative unsaturated rhamnogalacturonyl hydrolase (URH) with distant amino-acid sequence homology to YteR ofBacillus subtilisstrain 168. YteR, like other URHs, has core structural homology to unsaturated glucuronyl hydrolases, but hydrolyzes the unsaturated disaccharide derivative of rhamnogalacturonan I. The crystal structure of the recombinant CA_C0359 protein was solved to 1.6 Å resolution by molecular replacement using the phase information of the previously reported structure of YteR (PDB entry 1nc5) fromBacillus subtilisstrain 168. The YteR-like protein is a six-α-hairpin barrel with two β-sheet strands and a small helix overlaying the end of the hairpins next to the active site. The protein has low primary protein sequence identity to YteR but is structurally similar. The two tertiary structures align with a root-mean-square deviation of 1.4 Å and contain a highly conserved active pocket. There is a conserved aspartic acid residue in both structures, which has been shown to be important for hydration of the C=C bond during the release of unsaturated galacturonic acid by YteR. A surface electrostatic potential comparison of CA_C0359 and proteins from CAZy families GH88 and GH105 reveals the make-up of the active site to be a combination of the unsaturated rhamnogalacturonyl hydrolase and the unsaturated glucuronyl hydrolase fromBacillus subtilisstrain 168. Structural and electrostatic comparisons suggests that the protein may have a slightly different substrate specificity from that of YteR.

scholarly journals Crystal structures of non-oxidative decarboxylases reveal a new mechanism of action with a catalytic dyad and structural twists

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Matthias Zeug ◽  
Nebojsa Markovic ◽  
Cristina V. Iancu ◽  
Joanna Tripp ◽  
Mislav Oreb ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Active Site ◽  
Enzyme Engineering ◽  
Base Catalysis ◽  
Oxidative Decarboxylation ◽  
Transition State Stabilization ◽  
Major Bottleneck ◽  
Hydroxybenzoic Acids ◽  
Potassium Binding ◽  
Β Sheet

AbstractHydroxybenzoic acids, like gallic acid and protocatechuic acid, are highly abundant natural compounds. In biotechnology, they serve as critical precursors for various molecules in heterologous production pathways, but a major bottleneck is these acids’ non-oxidative decarboxylation to hydroxybenzenes. Optimizing this step by pathway and enzyme engineering is tedious, partly because of the complicating cofactor dependencies of the commonly used prFMN-dependent decarboxylases. Here, we report the crystal structures (1.5–1.9 Å) of two homologous fungal decarboxylases, AGDC1 from Arxula adenivorans, and PPP2 from Madurella mycetomatis. Remarkably, both decarboxylases are cofactor independent and are superior to prFMN-dependent decarboxylases when heterologously expressed in Saccharomyces cerevisiae. The organization of their active site, together with mutational studies, suggests a novel decarboxylation mechanism that combines acid–base catalysis and transition state stabilization. Both enzymes are trimers, with a central potassium binding site. In each monomer, potassium introduces a local twist in a β-sheet close to the active site, which primes the critical H86-D40 dyad for catalysis. A conserved pair of tryptophans, W35 and W61, acts like a clamp that destabilizes the substrate by twisting its carboxyl group relative to the phenol moiety. These findings reveal AGDC1 and PPP2 as founding members of a so far overlooked group of cofactor independent decarboxylases and suggest strategies to engineer their unique chemistry for a wide variety of biotechnological applications.

scholarly journals The δ subunit and NTPase HelD institute a two-pronged mechanism for RNA polymerase recycling

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Hao-Hong Pei ◽  
Tarek Hilal ◽  
Zhuo A. Chen ◽  
Yong-Heng Huang ◽  
Yuan Gao ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Nucleic Acids ◽  
Rna Polymerase ◽  
Active Site ◽  
Genome Stability ◽  
Slow Growth ◽  
Coiled Coil ◽  
Main Channel ◽  
Dependent Manner ◽  
Secondary Channel

AbstractCellular RNA polymerases (RNAPs) can become trapped on DNA or RNA, threatening genome stability and limiting free enzyme pools, but how RNAP recycling into active states is achieved remains elusive. In Bacillus subtilis, the RNAP δ subunit and NTPase HelD have been implicated in RNAP recycling. We structurally analyzed Bacillus subtilis RNAP-δ-HelD complexes. HelD has two long arms: a Gre cleavage factor-like coiled-coil inserts deep into the RNAP secondary channel, dismantling the active site and displacing RNA, while a unique helical protrusion inserts into the main channel, prying the β and β′ subunits apart and, aided by δ, dislodging DNA. RNAP is recycled when, after releasing trapped nucleic acids, HelD dissociates from the enzyme in an ATP-dependent manner. HelD abundance during slow growth and a dimeric (RNAP-δ-HelD)2 structure that resembles hibernating eukaryotic RNAP I suggest that HelD might also modulate active enzyme pools in response to cellular cues.

scholarly journals Structure of undecaprenyl pyrophosphate synthase from Acinetobacter baumannii

2018 ◽  
Vol 74 (12) ◽  
pp. 765-769 ◽  
Author(s):  
Tzu-Ping Ko ◽  
Chi-Hung Huang ◽  
Shu-Jung Lai ◽  
Yeh Chen
Keyword(s):  
Acinetobacter Baumannii ◽  
Active Site ◽  
Multidrug Resistant ◽  
Structural Basis ◽  
X Ray Diffraction ◽  
X Ray ◽  
Peptidoglycan Synthesis ◽  
C Terminus ◽  
Dimeric Protein ◽  
Β Sheet

Undecaprenyl pyrophosphate (UPP) is an important carrier of the oligosaccharide component in peptidoglycan synthesis. Inhibition of UPP synthase (UPPS) may be an effective strategy in combating the pathogen Acinetobacter baumannii, which has evolved to be multidrug-resistant. Here, A. baumannii UPPS (AbUPPS) was cloned, expressed, purified and crystallized, and its structure was determined by X-ray diffraction. Each chain of the dimeric protein folds into a central β-sheet with several surrounding α-helices, including one at the C-terminus. In the active site, two molecules of citrate interact with the side chains of the catalytic aspartate and serine. These observations may provide a structural basis for inhibitor design against AbUPPS.

Identification of the active site and characterization of a novel sporulation-specific cysteine protease YabG from Bacillus subtilis

2021 ◽  
Author(s):  
Ryuji Yamazawa ◽  
Ritsuko Kuwana ◽  
Kenji Takeuchi ◽  
Hiromu Takamatsu ◽  
Yoshitaka Nakajima ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Chemical Modification ◽  
Cysteine Protease ◽  
Active Site ◽  
Amino Acid Sequences ◽  
Site Directed Mutagenesis ◽  
Protease Gene ◽  
Active Site Residues ◽  
Protein Determination ◽  
35 Kda Protein

Abstract In order to characterize the probable protease gene yabG found in the genomes of spore-forming bacteria, Bacillus subtilis yabG was expressed as a 35 kDa His-tagged protein (BsYabG) in Escherichia coli cells. During purification using Ni-affinity chromatography, the 35 kDa protein was degraded via several intermediates to form a 24 kDa protein. Furthermore, it was degraded after an extended incubation period. The effect of protease inhibitors, including certain chemical modification reagents, on the conversion of the 35 kDa protein to the 24 kDa protein was investigated. Reagents reacting with sulfhydryl groups exerted significant effects, strongly suggesting that the yabG gene product is a cysteine protease with autolytic activity. Site-directed mutagenesis of the conserved Cys and His residues indicated that Cys218 and His172 are active site residues. No degradation was observed in the C218A/S and H172A mutants. In addition to the chemical modification reagents, benzamidine inhibited the degradation of the 24 kDa protein. Determination of the N-terminal amino acid sequences of the intermediates revealed trypsin-like specificity for YabG protease. Based on the relative positions of His172 and Cys218 and their surrounding sequences, we propose the classification of YabG as a new family of clan CD in the Merops peptidase database.

scholarly journals Inter-Active Site Communication Mediated by the Dimer Interface β-Sheet in the Half-the-Sites Enzyme, Thymidylate Synthase

Biochemistry ◽  
2019 ◽  
Vol 58 (30) ◽  
pp. 3302-3313 ◽  
Author(s):  
Paul J. Sapienza ◽  
Konstantin I. Popov ◽  
David D. Mowrey ◽  
Bradley T. Falk ◽  
Nikolay V. Dokholyan ◽  
...  
Keyword(s):  
Thymidylate Synthase ◽  
Active Site ◽  
Dimer Interface ◽  
Β Sheet

scholarly journals Atomic structures of the RNA end-healing 5′-OH kinase and 2′,3′-cyclic phosphodiesterase domains of fungal tRNA ligase: conformational switches in the kinase upon binding of the GTP phosphate donor

2019 ◽  
Author(s):  
Ankan Banerjee ◽  
Yehuda Goldgur ◽  
Beate Schwer ◽  
Stewart Shuman
Keyword(s):  
Crystal Structure ◽  
Active Site ◽  
Fungal Pathogens ◽  
Pathogenic Fungus ◽  
Atomic Structures ◽  
Polynucleotide Kinase ◽  
Phosphate Donor ◽  
Guanine Base ◽  
Conformational Switches ◽  
Β Sheet

Abstract Fungal tRNA ligase (Trl1) rectifies RNA breaks with 2′,3′-cyclic-PO4 and 5′-OH termini. Trl1 consists of three catalytic modules: an N-terminal ligase (LIG) domain; a central polynucleotide kinase (KIN) domain; and a C-terminal cyclic phosphodiesterase (CPD) domain. Trl1 enzymes found in all human fungal pathogens are untapped targets for antifungal drug discovery. Here we report a 1.9 Å crystal structure of Trl1 KIN-CPD from the pathogenic fungus Candida albicans, which adopts an extended conformation in which separate KIN and CPD domains are connected by an unstructured linker. CPD belongs to the 2H phosphotransferase superfamily by dint of its conserved central concave β sheet and interactions of its dual HxT motif histidines and threonines with phosphate in the active site. Additional active site motifs conserved among the fungal CPD clade of 2H enzymes are identified. We present structures of the Candida Trl1 KIN domain at 1.5 to 2.0 Å resolution—as apoenzyme and in complexes with GTP•Mg2+, IDP•PO4, and dGDP•PO4—that highlight conformational switches in the G-loop (which recognizes the guanine base) and lid-loop (poised over the nucleotide phosphates) that accompany nucleotide binding.

scholarly journals Site-directed mutagenesis of dicarboxylic acids near the active site of Bacillus cereus 5/B/6 β-lactamase II

1991 ◽  
Vol 276 (2) ◽  
pp. 401-404 ◽  
Author(s):  
H M Lim ◽  
R K Iyer ◽  
J J Pène
Keyword(s):  
Bacillus Cereus ◽  
Aspartic Acid ◽  
Carboxy Group ◽  
Active Site ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Beta Lactamase ◽  
Beta Lactam ◽  
Aspartic Acid Residue ◽  
Beta Lactamase Ii

An amino acid residue functioning as a general base has been proposed to assist in the hydrolysis of beta-lactam antibiotics by the zinc-containing Bacillus cereus beta-lactamase II [Bicknell & Waley (1985) Biochemistry 24, 6876-6887]. Oligonucleotide-directed mutagenesis of cloned Bacillus cereus 5/B/6 beta-lactamase II was used in an ‘in vivo’ study to investigate the role of carboxy-group-containing amino acids near the active site of the enzyme. Substitution of asparagine for the wild-type aspartic acid residue at position 81 resulted in fully functional enzyme. An aspartic acid residue at position 90 is essential for beta-lactamase II to confer any detectable ampicillin and cephalosporin C resistance to Escherichia coli. Conversion of Asp90 into Asn90 or Glu90 lead to the synthesis of inactive enzyme, suggesting that the spatial position of the beta-carboxy group of Asp90 is critical for enzyme function.

scholarly journals An aspartic acid residue at the active site of pepsin. The isolation and sequence of the heptapeptide

1969 ◽  
Vol 113 (2) ◽  
pp. 377-386 ◽  
Author(s):  
R. S. Bayliss ◽  
J. R. Knowles ◽  
Grith B. Wybrandt
Keyword(s):  
Methyl Ester ◽  
Aspartic Acid ◽  
Active Site ◽  
Irreversible Inhibitor ◽  
Aspartic Acid Residue ◽  
Phenylalanine Methyl Ester

Pepsin reacts stoicheiometrically with the active-site-directed irreversible inhibitor N-diazoacetyl-l-phenylalanine methyl ester, with concomitant loss of all proteolytic and peptidolytic activity. The reagent esterifies a unique aspartic acid residue in pepsin, which is in the sequence:Ile-Val-Asp-Thr-Gly-Thr-Ser

scholarly journals New properties of Bacillus subtilis succinate dehydrogenase altered at the active site. The apparent active site thiol of succinate oxidoreductases is dispensable for succinate oxidation

1989 ◽  
Vol 260 (2) ◽  
pp. 491-497 ◽  
Author(s):  
L Hederstedt ◽  
L O Hedén
Keyword(s):  
Bacillus Subtilis ◽  
Succinate Dehydrogenase ◽  
Active Site ◽  
Amino Acid Position ◽  
Biochemical Properties ◽  
Cysteine Residue ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Succinate Oxidation ◽  
E Coli

Mammalian and Escherichia coli succinate dehydrogenase (SDH) and E. coli fumarate reductase apparently contain an essential cysteine residue at the active site, as shown by substrate-protectable inactivation with thiol-specific reagents. Bacillus subtilis SDH was found to be resistant to this type of reagent and contains an alanine residue at the amino acid position equivalent to the only invariant cysteine in the flavoprotein subunit of E. coli succinate oxidoreductases. Substitution of this alanine, at position 252 in the flavoprotein subunit of B. subtilis SDH, by cysteine resulted in an enzyme sensitive to thiol-specific reagents and protectable by substrate. Other biochemical properties of the redesigned SDH were similar to those of the wild-type enzyme. It is concluded that the invariant cysteine in the flavoprotein of E. coli succinate oxidoreductases corresponds to the active site thiol. However, this cysteine is most likely not essential for succinate oxidation and seemingly lacks an assignable specific function. An invariant arginine in juxtaposition to Ala-252 in the flavoprotein of B. subtilis SDH, and to the invariant cysteine in the E. coli homologous enzymes, is probably essential for substrate binding.

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