scholarly journals Characterization of the Enzymes Encoded by the Anthrose Biosynthetic Operon of Bacillus anthracis

2010 ◽  
Vol 192 (19) ◽  
pp. 5053-5062 ◽  
Author(s):  
Shengli Dong ◽  
Sylvia A. McPherson ◽  
Yun Wang ◽  
Mei Li ◽  
Pengfei Wang ◽  
...  
Keyword(s):  
Bacillus Anthracis ◽  
Biosynthetic Pathway ◽  
Basal Layer ◽  
Side Chain ◽  
Side Chains ◽  
Aminotransferase Activity ◽  
Acyltransferase Activity ◽  
Hydratase Activity ◽  
Etiological Agents

ABSTRACT Bacillus anthracis spores, the etiological agents of anthrax, possess a loosely fitting outer layer called the exosporium that is composed of a basal layer and an external hairlike nap. The filaments of the nap are formed by trimers of the collagenlike glycoprotein BclA. Multiple pentasaccharide and trisaccharide side chains are O linked to BclA. The nonreducing terminal residue of the pentasaccharide side chain is the unusual sugar anthrose. A plausible biosynthetic pathway for anthrose biosynthesis has been proposed, and an antABCD operon encoding four putative anthrose biosynthetic enzymes has been identified. In this study, we genetically and biochemically characterized the activities of these enzymes. We also used mutant B. anthracis strains to determine the effects on BclA glycosylation of individually inactivating the genes of the anthrose operon. The inactivation of antA resulted in the appearance of BclA pentasaccharides containing anthrose analogs possessing shorter side chains linked to the amino group of the sugar. The inactivation of antB resulted in BclA being replaced with only trisaccharides, suggesting that the enzyme encoded by the gene is a dTDP-β-l-rhamnose α-1,3-l-rhamnosyl transferase that attaches the fourth residue of the pentasaccharide side chain. The inactivation of antC and antD resulted in the disappearance of BclA pentasaccharides and the appearance of a tetrasaccharide lacking anthrose. These phenotypes are entirely consistent with the proposed roles for the antABCD-encoded enzymes in anthrose biosynthesis. Purified AntA was then shown to exhibit β-methylcrotonyl-coenzyme A (CoA) hydratase activity, as we predicted. Similarly, we confirmed that purified AntC had aminotransferase activity and that purified AntD displayed N-acyltransferase activity.

scholarly journals Identification of the UDP-N-Acetylglucosamine 4-Epimerase Involved in Exosporium Protein Glycosylation in Bacillus anthracis

2009 ◽  
Vol 191 (22) ◽  
pp. 7094-7101 ◽  
Author(s):  
Shengli Dong ◽  
Olga N. Chesnokova ◽  
Charles L. Turnbough ◽  
David G. Pritchard
Keyword(s):  
Escherichia Coli ◽  
Bacillus Anthracis ◽  
Basal Layer ◽  
Amino Sugar ◽  
Protein Glycosylation ◽  
Side Chains ◽  
Protein Backbone ◽  
Gene Encoding ◽  
Multiple Copies ◽  
Late Stages

ABSTRACT Spores of Bacillus anthracis, the causative agent of anthrax, are enclosed by a loosely fitting exosporium composed of a basal layer and an external hair-like nap. The filaments of the nap are formed by trimers of the collagen-like glycoprotein BclA. The side chains of BclA include multiple copies of two linear rhamnose-containing oligosaccharides, a trisaccharide and a pentasaccharide. The pentasaccharide terminates with the unusual deoxyamino sugar anthrose. Both oligosaccharide side chains are linked to the BclA protein backbone through an N-acetylgalactosamine (GalNAc) residue. To identify the gene encoding the epimerase required to produce GalNAc for BclA oligosaccharide biosynthesis, three annotated UDP-glucose 4-epimerase genes of B. anthracis were cloned and expressed in Escherichia coli. The candidate proteins were purified, and their enzymatic activities were assessed. Only two proteins, encoded by the BAS5114 and BAS5304 genes (B. anthracis Sterne designations), exhibited epimerase activity. Both proteins were able to convert UDP-glucose (Glc) to UDP-Gal, but only the BAS5304-encoded protein could convert UDP-GlcNAc to UDP-GalNAc, indicating that BAS5304 was the gene sought. Surprisingly, spores produced by a mutant strain lacking the BAS5304-encoded enzyme still contained normal levels of BclA-attached oligosaccharides. However, monosaccharide analysis of the oligosaccharides revealed that GlcNAc had replaced GalNAc. Thus, while GalNAc appears to be the preferred amino sugar for the linkage of oligosaccharides to the BclA protein backbone, in its absence, GlcNAc can serve as a substitute linker. Finally, we demonstrated that the expression of the BAS5304 gene occurred in a biphasic manner during both the early and late stages of sporulation.

scholarly journals Identification and characterization of the 2-enoyl-CoA hydratases involved in peroxisomal β-oxidation in rat liver

1997 ◽  
Vol 321 (1) ◽  
pp. 253-259 ◽  
Author(s):  
Martine DIEUAIDE-NOUBHANI ◽  
Dmitry NOVIKOV ◽  
Joël VANDEKERCKHOVE ◽  
Paul P. Van VELDHOVEN ◽  
Guy P. MANNAERTS
Keyword(s):  
Rat Liver ◽  
Hydroxysteroid Dehydrogenase ◽  
Acid Synthesis ◽  
Bile Acid Synthesis ◽  
Side Chain ◽  
Multifunctional Protein ◽  
Hydratase Activity ◽  
Hydroxyacyl Coa Dehydrogenase ◽  
Enoyl Coa Hydratase

In this study we attempted to determine the number of 2-enoyl-CoA hydratases involved in peroxisomal β-oxidation. We therefore separated peroxisomal proteins from rat liver on several chromatographic columns and measured hydratase activities on the eluates with different substrates. The results indicate that rat liver peroxisomes contain two hydratase activities: (1) a hydratase activity associated with multifunctional protein 1 (MFP-1) (2-enoyl-CoA hydratase/Δ3,Δ2-enoyl-CoA isomerase/l-3-hydroxyacyl-CoA dehydrogenase) and (2) a hydratase activity associated with MFP-2 (17β-hydroxysteroid dehydrogenase/d-3-hydroxyacyl-CoA dehydrogenase/2-enoyl-CoA hydratase). MFP-1 forms and dehydrogenates l-3-hydroxyacyl-CoA species, whereas MFP-2 forms and dehydrogenates d-3-hydroxyacyl-CoA species. A portion of MFP-2 is proteolytically cleaved, most probably in the peroxisome, into a 34 kDa 17β-hydroxysteroid dehydrogenase/d-3-hydroxyacyl-CoA dehydrogenase and a 45 kDa d-specific 2-enoyl-CoA hydratase. Finally, the results confirm that MFP-1 is involved in the degradation of straight-chain fatty acids, whereas MFP-2 and its cleavage products seem to be involved in the degradation of the side chain of cholesterol (bile acid synthesis)

Synthesis and characterization of chiral PEDOT enantiomers bearing chiral moieties in side chains: chiral recognition and its mechanism using electrochemical sensing technology

RSC Advances ◽  
2016 ◽  
Vol 6 (14) ◽  
pp. 11536-11545 ◽  
Author(s):  
Liqi Dong ◽  
Long Zhang ◽  
Xuemin Duan ◽  
Daize Mo ◽  
Jingkun Xu ◽  
...  
Keyword(s):  
Chiral Recognition ◽  
Electrochemical Sensing ◽  
Side Chain ◽  
Synthesis And Characterization ◽  
Side Chains ◽  
Sensing Technology

This manuscript reports a couple of novel polymers of side-chain functionalized PEDOT. The new polymers can be employed to successfully recognize 3,4-dihydroxyphenylalanine enantiomers and we also discuss the mechanism of chiral recognition.

Controlled RAFT synthesis of side-chain oleic acid containing polymers and their post-polymerization functionalization

RSC Advances ◽  
2014 ◽  
Vol 4 (99) ◽  
pp. 56415-56423 ◽  
Author(s):  
Binoy Maiti ◽  
Sonu Kumar ◽  
Priyadarsi De
Keyword(s):  
Oleic Acid ◽  
Renewable Resource ◽  
Side Chain ◽  
Synthesis And Characterization ◽  
Cross Linking ◽  
Side Chains ◽  
Double Bonds ◽  
Ene Reaction

We report the synthesis and characterization of well-defined polymers from oleic acid as the bio-renewable resource. Double bonds in oleate side-chains in the polymer are further modified by thiol-ene reaction, epoxidation, and cross-linking.

scholarly journals An Unusual Mechanism of Isopeptide Bond Formation Attaches the Collagenlike Glycoprotein BclA to the Exosporium of Bacillus anthracis

mBio ◽  
2011 ◽  
Vol 2 (3) ◽  
Author(s):  
Li Tan ◽  
Mei Li ◽  
Charles L. Turnbough
Keyword(s):  
Bacillus Anthracis ◽  
Bond Formation ◽  
Basal Layer ◽  
Side Chain ◽  
Cross Linking ◽  
Amino Group ◽  
Content Type ◽  
Isopeptide Bond ◽  
Isopeptide Bonds

ABSTRACTThe outermost exosporium layer of spores ofBacillus anthracis, the causative agent of anthrax, is comprised of a basal layer and an external hairlike nap. The nap includes filaments composed of trimers of the collagenlike glycoprotein BclA. Essentially all BclA trimers are tightly attached to the spore in a process requiring the basal layer protein BxpB (also called ExsFA). Both BclA and BxpB are incorporated into stable, high-molecular-mass complexes, suggesting that BclA is attached directly to BxpB. The 38-residue amino-terminal domain of BclA, which is normally proteolytically cleaved between residues 19 and 20, is necessary and sufficient for basal layer attachment. In this study, we demonstrate that BclA attachment occurs through the formation of isopeptide bonds between the free amino group of BclA residue A20 and a side chain carboxyl group of an acidic residue of BxpB. Ten of the 13 acidic residues of BxpB can participate in isopeptide bond formation, and at least three BclA polypeptide chains can be attached to a single molecule of BxpB. We also demonstrate that similar cross-linking occursin vitrobetween purified recombinant BclA and BxpB, indicating that the reaction is spontaneous. The mechanism of BclA attachment, specifically, the formation of a reactive amino group by proteolytic cleavage and the promiscuous selection of side chain carboxyl groups of internal acidic residues, appears to be different from other known mechanisms for protein cross-linking through isopeptide bonds. Analogous mechanisms appear to be involved in the cross-linking of other spore proteins and could be found in unrelated organisms.IMPORTANCEIsopeptide bonds are protein modifications found throughout nature in which amide linkages are formed between functional groups of two amino acids, with at least one of the functional groups provided by an amino acid side chain. Isopeptide bonds generate cross-links within and between proteins that are necessary for proper protein structure and function. In this study, we discovered that BclA, the dominant structural protein of the external nap ofBacillus anthracisspores, is attached to the underlying exosporium basal layer protein BxpB via isopeptide bonds formed through a mechanism fundamentally different from previously described mechanisms of isopeptide bond formation. The most unusual features of this mechanism are the generation of a reactive amino group by proteolytic cleavage and promiscuous selection of acidic side chains. This mechanism, which apparently relies only on short peptide sequences in protein substrates, could be a general mechanismin vivoand adapted for protein cross-linkingin vitro.

scholarly journals Characterization of Novel Acyl Coenzyme A Dehydrogenases Involved in Bacterial Steroid Degradation

2015 ◽  
Vol 197 (8) ◽  
pp. 1360-1367 ◽  
Author(s):  
Amanda Ruprecht ◽  
Jaymie Maddox ◽  
Alexander J. Stirling ◽  
Nicole Visaggio ◽  
Stephen Y. K. Seah
Keyword(s):  
Active Site ◽  
Coenzyme A ◽  
Side Chain ◽  
Side Chains ◽  
Content Type ◽  
Carbon Side Chain ◽  
Acyl Coa Dehydrogenases ◽  
Acyl Coenzyme A ◽  
Rhodococcus Jostii

ABSTRACTThe acyl coenzyme A (acyl-CoA) dehydrogenases (ACADs) FadE34 and CasC, encoded by the cholesterol and cholate gene clusters ofMycobacterium tuberculosisandRhodococcus jostiiRHA1, respectively, were successfully purified. Both enzymes differ from previously characterized ACADs in that they contain two fused acyl-CoA dehydrogenase domains in a single polypeptide. Site-specific mutagenesis showed that only the C-terminal ACAD domain contains the catalytic glutamate base required for enzyme activity, while the N-terminal ACAD domain contains an arginine required for ionic interactions with the pyrophosphate of the flavin adenine dinucleotide (FAD) cofactor. Therefore, the two ACAD domains must associate to form a single active site. FadE34 and CasC were not active toward the 3-carbon side chain steroid metabolite 3-oxo-23,24-bisnorchol-4-en-22-oyl-CoA (4BNC-CoA) but were active toward steroid CoA esters containing 5-carbon side chains. CasC has similar specificity constants for cholyl-CoA, deoxycholyl-CoA, and 3β-hydroxy-5-cholen-24-oyl-CoA, while FadE34 has a preference for the last compound, which has a ring structure similar to that of cholesterol metabolites. Knockout of thecasCgene inR. jostiiRHA1 resulted in a reduced growth on cholate as a sole carbon source and accumulation of a 5-carbon side chain cholate metabolite. FadE34 and CasC represent unique members of ACADs with primary structures and substrate specificities that are distinct from those of previously characterized ACADs.IMPORTANCEWe report here the identification and characterization of acyl-CoA dehydrogenases (ACADs) involved in the metabolism of 5-carbon side chains of cholesterol and cholate. The two homologous enzymes FadE34 and CasC, fromM. tuberculosisandRhodococcus jostiiRHA1, respectively, contain two ACAD domains per polypeptide, and we show that these two domains interact to form a single active site. FadE34 and CasC are therefore representatives of a new class of ACADs with unique primary and quaternary structures. The bacterial steroid degradation pathway is important for the removal of steroid waste in the environment and for survival of the pathogenM. tuberculosiswithin host macrophages. FadE34 is a potential target for development of new antibiotics against tuberculosis.

scholarly journals Anthrose Biosynthetic Operon of Bacillus anthracis

2008 ◽  
Vol 190 (7) ◽  
pp. 2350-2359 ◽  
Author(s):  
Shengli Dong ◽  
Sylvia A. McPherson ◽  
Li Tan ◽  
Olga N. Chesnokova ◽  
Charles L. Turnbough ◽  
...  
Keyword(s):  
Bacillus Anthracis ◽  
Basal Layer ◽  
Side Chain ◽  
Biosynthetic Enzymes ◽  
Multiple Copies

ABSTRACT The exosporium of Bacillus anthracis spores consists of a basal layer and an external hair-like nap. The nap is composed primarily of the glycoprotein BclA, which contains a collagen-like region with multiple copies of a pentasaccharide side chain. This oligosaccharide possesses an unusual terminal sugar called anthrose, followed by three rhamnose residues and a protein-bound N-acetylgalactosamine. Based on the structure of anthrose, we proposed an enzymatic pathway for its biosynthesis. Examination of the B. anthracis genome revealed six contiguous genes that could encode the predicted anthrose biosynthetic enzymes. These genes are transcribed in the same direction and appear to form two operons. We introduced mutations into the B. anthracis chromosome that either delete the promoter of the putative upstream, four-gene operon or delete selected genes in both putative operons. Spores produced by strains carrying mutations in the upstream operon completely lacked or contained much less anthrose, indicating that this operon is required for anthrose biosynthesis. In contrast, inactivation of the downstream, two-gene operon did not alter anthrose content. Additional experiments confirmed the organization of the anthrose operon and indicated that it is transcribed from a σE-specific promoter. Finally, we demonstrated that anthrose biosynthesis is not restricted to B. anthracis as previously suggested.

scholarly journals Characterization of the Exosporium Basal Layer Protein BxpB of Bacillus anthracis

2005 ◽  
Vol 187 (17) ◽  
pp. 5868-5876 ◽  
Author(s):  
Christopher T. Steichen ◽  
John F. Kearney ◽  
Charles L. Turnbough
Keyword(s):  
Electron Microscopy ◽  
Monoclonal Antibody ◽  
Bacillus Anthracis ◽  
Basal Layer ◽  
Immunogold Electron Microscopy ◽  
Stable Complex ◽  
Wild Type

ABSTRACT Bacillus anthracis spores, the cause of anthrax, are enclosed by a prominent loose-fitting structure called the exosporium. The exosporium is composed of a basal layer and an external hair-like nap. The filaments of the hair-like nap are apparently formed by a single collagen-like glycoprotein called BclA, whereas several different proteins form or are tightly associated with the basal layer. In this study, we used immunogold electron microscopy to demonstrate that BxpB (also called ExsF) is a component of the exosporium basal layer. Binding to the basal layer by an anti-BxpB monoclonal antibody was greatly increased by the loss of BclA. We found that BxpB and BclA are part of a stable complex that appears to include the putative basal layer protein ExsY and possibly other proteins. Previous results suggested that BxpB was glycosylated; however, our results indicate that it is not a glycoprotein. We showed that ΔbxpB spores, which lack BxpB, contain an exosporium devoid of hair-like nap even though the ΔbxpB strain produces normal levels of BclA. These results indicated that BxpB is required for the attachment of BclA to the exosporium. Finally, we found that the efficiency of production of ΔbxpB spores and their resistance properties were similar to those of wild-type spores. However, ΔbxpB spores germinate faster than wild-type spores, indicating that BxpB suppresses germination. This effect did not appear to be related to the absence from ΔbxpB spores of a hair-like nap or of enzymes that degrade germinants.

Synthesis and Characterization of Urethane Side Chain Substituted Diketopyrrolopyrrole

2021 ◽  
Vol 13 (2) ◽  
pp. 635-642
Author(s):  
S. Dharmapurikar ◽  
J. Aher ◽  
A. Puyad ◽  
G. K. Kakade ◽  
V. G. Kalalawe ◽  
...  
Keyword(s):  
Optical Properties ◽  
Hydrogen Bonding ◽  
Alkyl Chain ◽  
Side Chain ◽  
Synthesis And Characterization ◽  
Side Chains ◽  
Easy Method ◽  
Secondary Interaction ◽  
Sensing Applications

The synthesis of Diketopyrrolopyrrole (DPP) having secondary interaction in the side chain explores its possibility to use in electronic and sensing applications. Herein we report easy method to engineer side chains of DPP. The hydrogen bonding is introduced on the side chain by substitution of urethane side chains on Diketopyrrolopyrrole (DPPurethane).The urethane side chain comprises a branched alkyl chain with good yields and purities. The DPPurethane characterized by NMR and IR, optical properties along with energy minimized structure were studied.  

Influence of Flexible Side-Chains on the Breathing Phase Transition of Pillared Layer MOFs: A Force Field Investigation

2020 ◽  
Author(s):  
Julian Keupp ◽  
Johannes P. Dürholt ◽  
Rochus Schmid
Keyword(s):  
First Principles ◽  
Good Accuracy ◽  
Field Investigation ◽  
Square Lattice ◽  
Side Chain ◽  
Second Step ◽  
Side Chains ◽  
Dynamics Simulations ◽  
Complex Phenomena ◽  
Closed Pore

The prototypical pillared layer MOFs, formed by a square lattice of paddle-<br>wheel units and connected by dinitrogen pillars, can undergo a breathing phase<br>transition by a “wine-rack” type motion of the square lattice. We studied this not<br>yet fully understood behavior using an accurate first principles parameterized force<br>field (MOF-FF) for larger nanocrystallites on the example of Zn 2 (bdc) 2 (dabco) [bdc:<br>benzenedicarboxylate, dabco: (1,4-diazabicyclo[2.2.2]octane)] and found clear indi-<br>cations for an interface between a closed and an open pore phase traveling through<br>the system during the phase transformation [Adv. Theory Simul. 2019, 2, 11]. In<br>conventional simulations in small supercells this mechanism is prevented by periodic<br>boundary conditions (PBC), enforcing a synchronous transformation of the entire<br>crystal. Here, we extend this investigation to pillared layer MOFs with flexible<br>side-chains, attached to the linker. Such functionalized (fu-)MOFs are experimen-<br>tally known to have different properties with the side-chains acting as fixed guest<br>molecules. First, in order to extend the parameterization for such flexible groups,<br>1a new parametrization strategy for MOF-FF had to be developed, using a multi-<br>structure force based fit method. The resulting parametrization for a library of<br>fu-MOFs is then validated with respect to a set of reference systems and shows very<br>good accuracy. In the second step, a series of fu-MOFs with increasing side-chain<br>length is studied with respect to the influence of the side-chains on the breathing<br>behavior. For small supercells in PBC a systematic trend of the closed pore volume<br>with the chain length is observed. However, for a nanocrystallite model a distinct<br>interface between a closed and an open pore phase is visible only for the short chain<br>length, whereas for longer chains the interface broadens and a nearly concerted trans-<br>formation is observed. Only by molecular dynamics simulations using accurate force<br>fields such complex phenomena can be studied on a molecular level.

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