scholarly journals Crystal Structure of an Iron-Dependent Group III Dehydrogenase That Interconverts l-Lactaldehyde and l-1,2-Propanediol in Escherichia coli

2005 ◽  
Vol 187 (14) ◽  
pp. 4957-4966 ◽  
Author(s):  
Cristina Montella ◽  
Lluis Bellsolell ◽  
Rosa Pérez-Luque ◽  
Josefa Badía ◽  
Laura Baldoma ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Metal Ion ◽  
Catalytic Mechanism ◽  
Protein A ◽  
Close Approach ◽  
Site Directed Mutagenesis ◽  
Dimensional Structure ◽  
Group Iii ◽  
Terminal Domain ◽  
Α Helix

ABSTRACT The FucO protein, a member of the group III “iron-activated” dehydrogenases, catalyzes the interconversion between l-lactaldehyde and l-1,2-propanediol in Escherichia coli. The three-dimensional structure of FucO in a complex with NAD+ was solved, and the presence of iron in the crystals was confirmed by X-ray fluorescence. The FucO structure presented here is the first structure for a member of the group III bacterial dehydrogenases shown experimentally to contain iron. FucO forms a dimer, in which each monomer folds into an α/β dinucleotide-binding N-terminal domain and an all-α-helix C-terminal domain that are separated by a deep cleft. The dimer is formed by the swapping (between monomers) of the first chain of the β-sheet. The binding site for Fe2+ is located at the face of the cleft formed by the C-terminal domain, where the metal ion is tetrahedrally coordinated by three histidine residues (His200, His263, and His277) and an aspartate residue (Asp196). The glycine-rich turn formed by residues 96 to 98 and the following α-helix is part of the NAD+ recognition locus common in dehydrogenases. Site-directed mutagenesis and enzyme kinetic assays were performed to assess the role of different residues in metal, cofactor, and substrate binding. In contrast to previous assumptions, the essential His267 residue does not interact with the metal ion. Asp39 appears to be the key residue for discriminating against NADP+. Modeling l-1,2-propanediol in the active center resulted in a close approach of the C-1 hydroxyl of the substrate to C-4 of the nicotinamide ring, implying that there is a typical metal-dependent dehydrogenation catalytic mechanism.

scholarly journals Mutational analysis of the catalytic centre of the Citrobacter freundii AmpD N-acetylmuramyl-l-alanine amidase

2004 ◽  
Vol 377 (1) ◽  
pp. 111-120 ◽  
Author(s):  
Catherine GÉNÉREUX ◽  
Dominique DEHARENG ◽  
Bart DEVREESE ◽  
Jozef VAN BEEUMEN ◽  
Jean-Marie FRÈRE ◽  
...  
Keyword(s):  
Molecular Modelling ◽  
Metal Ion ◽  
Catalytic Mechanism ◽  
Mutational Analysis ◽  
Hydrolytic Enzymes ◽  
Site Directed Mutagenesis ◽  
Dimensional Structure ◽  
Citrobacter Freundii ◽  
Wild Type ◽  
Type Activity

Citrobacter freundii AmpD is an intracellular 1,6-anhydro-N-acetylmuramyl-l-alanine amidase involved in both peptidoglycan recycling and β-lactamase induction. AmpD exhibits a strict specificity for 1,6-anhydromuropeptides and requires zinc for enzymic activity. The AmpD three-dimensional structure exhibits a fold similar to that of another Zn2+N-acetylmuramyl-l-alanine amidase, the T7 lysozyme, and these two enzymes define a new family of Zn-amidases which can be related to the eukaryotic PGRP (peptidoglycan-recognition protein) domains. In an attempt to assign the different zinc ligands and to probe the catalytic mechanism of AmpD amidase, molecular modelling based on the NMR structure and site-directed mutagenesis were performed. Mutation of the two residues presumed to act as zinc ligands into alanine (H34A and D164A) yielded inactive proteins which had also lost their ability to bind zinc. By contrast, the active H154N mutant retained the capacity to bind the metal ion. Three other residues which could be involved in the AmpD catalytic mechanism have been mutated (Y63F, E116A, K162H and K162Q). The E116A mutant was inactive, but on the basis of the molecular modelling this residue is not directly involved in the catalytic mechanism, but rather in the binding of the zinc by contributing to the correct orientation of His-34. The K162H and K162Q mutants retained very low activity (0.7 and 0.2% of the wild-type activity respectively), whereas the Y63F mutant showed 16% of the wild-type activity. These three latter mutants exhibited a good affinity for Zn ions and the substituted residues are probably involved in the binding of the substrate. We also describe a new method for generating the N-acetylglucosaminyl-1,6-anhydro-N-acetylmuramyl-tripeptide AmpD substrate from purified peptidoglycan by the combined action of two hydrolytic enzymes.

scholarly journals Structural and biochemical characterization of bifunctional XynA

2020 ◽  
Author(s):  
Wei Xie ◽  
Qi Yu ◽  
Yun Liu ◽  
Ruoting Cao ◽  
Ruiqing Zhang ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Enzyme Activity ◽  
Catalytic Mechanism ◽  
Biochemical Characterization ◽  
Cellulase Activity ◽  
Cost Savings ◽  
Site Directed Mutagenesis ◽  
Enzyme Activity Assay ◽  
Great Cost ◽  
Terminal Domain

AbstractXylan and cellulose are the two major constituents in numerous types of lignocellulosic biomass, representing a promising resource for biofuels and other biobased industries. The efficient degradation of lignocellulose requires the synergistic actions of cellulase and xylanase. Thus, bifunctional enzyme incorporated xylanase/cellulase activity has attracted considerable attention since it has great cost savings potential. Recently, a novel GH10 family enzyme XynA identified from Bacillus sp. is found to degrade both cellulose and xylan. To understand its molecular catalytic mechanism, here we first solve the crystal structure of XynA at 2.3 Å. XynA is characterized with a classic (α/β)8 TIM-barrel fold (GH10 domain) flanked by the flexible N-terminal domain and C-terminal domain. Circular dichroism, protein thermal shift and enzyme activity assays reveal that conserved residues Glu182 and Glu280 are both important for catalytic activities of XynA, which is verified by the crystal structure of XynA with E182A/E280A double mutant. Molecular docking studies of XynA with xylohexaose and cellohexaose as well as site-directed mutagenesis and enzyme activity assay demonstrat that Gln250 and His252 are indispensible to cellulase and bifunctional activity, separately. These results elucidate the structural and biochemical features of XynA, providing clues for further modification of XynA for industrial application.

Crystal structure of the complex of the interaction domains of Escherichia coli DnaB helicase and DnaC helicase loader: structural basis implying a distortion-accumulation mechanism for the DnaB ring opening caused by DnaC binding

2019 ◽  
Vol 167 (1) ◽  
pp. 1-14
Author(s):  
Koji Nagata ◽  
Akitoshi Okada ◽  
Jun Ohtsuka ◽  
Takatoshi Ohkuri ◽  
Yusuke Akama ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Escherichia Coli ◽  
Molecular Model ◽  
Structural Basis ◽  
Terminal Domain ◽  
Helicase Dnab ◽  
Accumulation Mechanism ◽  
Interaction Domains ◽  
Α Helix ◽  
Available Information

Abstract Loading the bacterial replicative helicase DnaB onto DNA requires a specific loader protein, DnaC/DnaI, which creates the loading-competent state by opening the DnaB hexameric ring. To understand the molecular mechanism by which DnaC/DnaI opens the DnaB ring, we solved 3.1-Å co-crystal structure of the interaction domains of Escherichia coli DnaB–DnaC. The structure reveals that one N-terminal domain (NTD) of DnaC interacts with both the linker helix of a DnaB molecule and the C-terminal domain (CTD) of the adjacent DnaB molecule by forming a three α-helix bundle, which fixes the relative orientation of the two adjacent DnaB CTDs. The importance of the intermolecular interface in the crystal structure was supported by the mutational data of DnaB and DnaC. Based on the crystal structure and other available information on DnaB–DnaC structures, we constructed a molecular model of the hexameric DnaB CTDs bound by six DnaC NTDs. This model suggested that the binding of a DnaC would cause a distortion in the hexameric ring of DnaB. This distortion of the DnaB ring might accumulate by the binding of up to six DnaC molecules, resulting in the DnaB ring to open.

Dimerization of the RelA protein of Escherichia coli

2001 ◽  
Vol 79 (6) ◽  
pp. 729-736 ◽  
Author(s):  
Xiaoming Yang ◽  
Edward E Ishiguro
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Cellular Localization ◽  
Stringent Response ◽  
Site Directed Mutagenesis ◽  
Ribosome Binding ◽  
Terminal Domain ◽  
Proposed Model ◽  
Mutagenesis Study

The RelA protein of Escherichia coli is a ribosome-associated (p)ppGpp synthetase that is activated by amino acid deprivation. It was recently reported that the activity of RelA is regulated by oligomerization mediated by the C-terminal domain of RelA. The oligomerization of RelA is further characterized in this study. The C-terminal domain consisting of amino acids 455–744, designated 'RelA, formed homooligomers as well as heterooligomers with RelA as demonstrated by copurification of RelA and 'RelA and by an affinity blotting assay. Glutaraldehyde-induced cross-linking indicated that the oligomer was a dimer. The functional analysis of 'RelA was based on a combination of yeast two-hybrid analysis, the determination of the effects of overexpression of 'RelA derivatives on the stringent response, and the cellular localization of the overexpressed 'RelA derivatives. These studies indicated that two regions, designated 'RelA-1 (amino acids 455–538) and 'RelA-2 (amino acids 550–682), were involved in dimerization. The involvement of one of these two regions, RelA-2, is consistent with a previous site-directed mutagenesis study. In addition to dimerization, 'RelA-2 apparently contained the main ribosome-binding domain of RelA. The third region, 'RelA-3 (amino acids 682–744), was not involved in either dimerization or ribosome binding. The overexpression of 'RelA-1 and 'RelA-2, but not 'RelA-3, inhibited the stringent response. These results support the previously proposed model which suggests a role for oligomerization in the regulation of (p)ppGpp synthetase.Key words: RelA, Escherichia coli, stringent response.

scholarly journals Role of αArg145 and βArg263 in the active site of penicillin acylase of Escherichia coli

2002 ◽  
Vol 365 (1) ◽  
pp. 303-309 ◽  
Author(s):  
Wynand B.L. ALKEMA ◽  
Antoon K. PRINS ◽  
Erik de VRIES ◽  
Dick B. JANSSEN
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Penicillin Acylase ◽  
Precursor Protein ◽  
Site Directed Mutagenesis ◽  
Penicillin G ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Arginine Residues

The active site of penicillin acylase of Escherichia coli contains two conserved arginine residues. The function of these arginines, αArg145 and βArg263, was studied by site-directed mutagenesis and kinetic analysis of the mutant enzymes. The mutants αArg145→Leu (αArg145Leu), αArg145Cys and αArg145Lys were normally processed and exported to the periplasm, whereas expression of the mutants βArg263Leu, βArg263Asn and βArg263Lys yielded large amounts of precursor protein in the periplasm, indicating that βArg263 is crucial for efficient processing of the enzyme. Either modification of both arginine residues by 2,3-butanedione or replacement by site-directed mutagenesis yielded enzymes with a decreased specificity (kcat/Km) for 2-nitro-5-[(phenylacetyl)amino]benzoic acid, indicating that both residues are important in catalysis. Compared with the wild type, the αArg145 mutants exhibited a 3–6-fold-increased preference for 6-aminopenicillanic acid as the deacylating nucleophile compared with water. Analysis of the steady-state parameters of these mutants for the hydrolysis of penicillin G and phenylacetamide indicated that destabilization of the Michaelis—Menten complex accounts for the improved activity with β-lactam substrates. Analysis of pH—activity profiles of wild-type enzyme and the βArg263Lys mutant showed that βArg263 has to be positively charged for catalysis, but is not involved in substrate binding. The results provide an insight into the catalytic mechanism of penicillin acylase, in which αArg145 is involved in binding of β-lactam substrates and βArg263 is important both for stabilizing the transition state in the reaction and for correct processing of the precursor protein.

scholarly journals Functional Analysis of the Carbohydrate-Binding Domains of Erwinia chrysanthemi Cel5 (Endoglucanase Z) and an Escherichia coli Putative Chitinase

1999 ◽  
Vol 181 (15) ◽  
pp. 4611-4616 ◽  
Author(s):  
Helen D. Simpson ◽  
Frederic Barras
Keyword(s):  
Escherichia Coli ◽  
Catalytic Domain ◽  
Three Dimensional ◽  
Site Directed Mutagenesis ◽  
Erwinia Chrysanthemi ◽  
Dimensional Structure ◽  
Carbohydrate Binding ◽  
Binding Domains ◽  
Cellulose Binding

ABSTRACT The Cel5 cellulase (formerly known as endoglucanase Z) fromErwinia chrysanthemi is a multidomain enzyme consisting of a catalytic domain, a linker region, and a cellulose binding domain (CBD). A three-dimensional structure of the CBDCel5 has previously been obtained by nuclear magnetic resonance. In order to define the role of individual residues in cellulose binding, site-directed mutagenesis was performed. The role of three aromatic residues (Trp18, Trp43, and Tyr44) in cellulose binding was demonstrated. The exposed potential hydrogen bond donors, residues Gln22 and Glu27, appeared not to play a role in cellulose binding, whereas residue Asp17 was found to be important for the stability of Cel5. A deletion mutant lacking the residues Asp17 to Pro23 bound only weakly to cellulose. The sequence of CBDCel5 exhibits homology to a series of five repeating domains of a putative large protein, referred to as Yheb, from Escherichia coli. One of the repeating domains (Yheb1), consisting of 67 amino acids, was cloned from the E. coli chromosome and purified by metal chelating chromatography. While CBDCel5 bound to both cellulose and chitin, Yheb1 bound well to chitin, but only very poorly to cellulose. The Yheb protein contains a region that exhibits sequence homology with the catalytic domain of a chitinase, which is consistent with the hypothesis that the Yheb protein is a chitinase.

scholarly journals Structure of Plasmodium falciparum TRAP (thrombospondin-related anonymous protein) A domain highlights distinct features in apicomplexan von Willebrand factor A homologues

2013 ◽  
Vol 450 (3) ◽  
pp. 469-476 ◽  
Author(s):  
Tero Pihlajamaa ◽  
Tommi Kajander ◽  
Juho Knuuti ◽  
Kaisa Horkka ◽  
Amit Sharma ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Von Willebrand Factor ◽  
Metal Ion ◽  
Cell Attachment ◽  
Protein A ◽  
Site Directed Mutagenesis ◽  
Mammalian Host ◽  
Functional Roles ◽  
Von Willebrand ◽  
Willebrand Factor

TRAP (thrombospondin-related anonymous protein), localized in the micronemes and on the surface of sporozoites of the notorious malaria parasite Plasmodium, is a key molecule upon infection of mammalian host hepatocytes and invasion of mosquito salivary glands. TRAP contains two adhesive domains responsible for host cell recognition and invasion, and is known to be essential for infectivity. In the present paper, we report high-resolution crystal structures of the A domain of Plasmodium falciparum TRAP with and without bound Mg2+. The structure reveals a vWA (von Willebrand factor A)-like fold and a functional MIDAS (metal-ion-dependent adhesion site), as well as a potential heparan sulfate-binding site. Site-directed mutagenesis and cell-attachment assays were used to investigate the functional roles of the surface epitopes discovered. The reported structures are the first determined for a complete vWA domain of parasitic origin, highlighting unique features among homologous domains from other proteins characterized hitherto. Some of these are conserved among Plasmodiae exclusively, whereas others may be common to apicomplexan organisms in general.

scholarly journals The Structure of Bypass of Forespore C, an Intercompartmental Signaling Factor during Sporulation in Bacillus

2005 ◽  
Vol 280 (43) ◽  
pp. 36214-36220 ◽  
Author(s):  
Hayley M. Patterson ◽  
James A. Brannigan ◽  
Simon M. Cutting ◽  
Keith S. Wilson ◽  
Anthony J. Wilkinson ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Mother Cell ◽  
Asymmetric Cell Division ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Coat Proteins ◽  
Terminal Domain ◽  
Spore Coat Proteins ◽  
Α Helix ◽  
Β Sheet

Sporulation in Bacillus subtilis begins with an asymmetric cell division giving rise to smaller forespore and larger mother cell compartments. Different programs of gene expression are subsequently directed by compartment-specific RNA polymerase σ-factors. In the final stages, spore coat proteins are synthesized in the mother cell under the control of RNA polymerase containing σK, (EσK). σK is synthesized as an inactive zymogen, pro-σK, which is activated by proteolytic cleavage. Processing of pro-σK is performed by SpoIVFB, a metalloprotease that resides in a complex with SpoIVFA and bypass of forespore (Bof)A in the outer forespore membrane. Ensuring coordination of events taking place in the two compartments, pro-σK processing in the mother cell is delayed until appropriate signals are received from the forespore. Cell-cell signaling is mediated by SpoIVB and BofC, which are expressed in the forespore and secreted to the intercompartmental space where they regulate pro-σK processing by mechanisms that are not yet fully understood. Here we present the three-dimensional structure of BofC determined by solution state NMR. BofC is a monomer made up of two domains. The N-terminal domain, containing a four-stranded β-sheet onto one face of which an α-helix is packed, closely resembles the third immunoglobulin-binding domain of protein G from Streptococcus. The C-terminal domain contains a three-stranded β-sheet and three α-helices in a novel domain topology. The sequence connecting the domains contains a conserved DISP motif to which mutations that affect BofC activity map. Possible roles for BofC in the σK checkpoint are discussed in the light of sequence and structure comparisons.

Identification of the ferroxidase centre of Escherichia coli bacterioferritin

1995 ◽  
Vol 312 (2) ◽  
pp. 385-392 ◽  
Author(s):  
N E Le Brun ◽  
S C Andrews ◽  
J R Guest ◽  
P M Harrison ◽  
G R Moore ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Metal Ion ◽  
Oxidation Mechanism ◽  
Site Directed Mutagenesis ◽  
Ion Binding ◽  
Metal Ion Binding ◽  
Dinuclear Iron ◽  
Ferroxidase Activity ◽  
A Site ◽  
R2 Subunit

The bacterioferritin (BFR) of Escherichia coli takes up iron in the ferrous form and stores it within its central cavity as a hydrated ferric oxide mineral. The mechanism by which oxidation of iron (II) occurs in BFR is largely unknown, but previous studies indicated that there is ferroxidase activity associated with a site capable of forming a dinuclear-iron centre within each subunit [Le Brun, Wilson, Andrews, Harrison, Guest, Thomson and Moore (1993) FEBS Lett. 333, 197-202]. We now report site-directed mutagenesis experiments based on a putative dinuclear-metal-ion-binding site located within the BFR subunit. The data reveal that this dinuclear-iron centre is located at a site within the four-alpha-helical bundle of each subunit of BFR, thus identified as the ferroxidase centre of BFR. The metal-bound form of the centre bears a remarkable similarity to the dinuclear-iron sites of the hydroxylase subunit of methane mono-oxygenase and the R2 subunit of ribonucleotide reductase. Details of how the dinuclear centre of BFR is involved in the oxidation mechanism were investigated by studying the inhibition of iron (II) oxidation by zinc (II) ions. Data indicate that zinc (II) ions bind at the ferroxidase centre of apo-BFR in preference to iron (II), resulting in a dramatic reduction in the rate of oxidation. The mechanism of iron (II) oxidation is discussed in the light of this and previous work.

scholarly journals Probing the mechanistic role of the long α‐helix in subunit L of respiratory Complex I from Escherichia coli by site‐directed mutagenesis

2011 ◽  
Vol 82 (5) ◽  
pp. 1086-1095 ◽  
Author(s):  
Galina Belevich ◽  
Juho Knuuti ◽  
Michael I. Verkhovsky ◽  
Mårten Wikström ◽  
Marina Verkhovskaya
Keyword(s):  
Escherichia Coli ◽  
Complex I ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Respiratory Complex ◽  
Respiratory Complex I ◽  
Α Helix
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