Structure of the prolyl-acyl carrier protein oxidase involved in the biosynthesis of the cyanotoxin anatoxin-a

2013 ◽  
Vol 69 (12) ◽  
pp. 2340-2352 ◽  
Author(s):  
Karine Moncoq ◽  
Leslie Regad ◽  
Stéphane Mann ◽  
Annick Méjean ◽  
Olivier Ploux
Keyword(s):  
Molecular Oxygen ◽  
Acyl Carrier Protein ◽  
Three Dimensional ◽  
Accessible Surface Area ◽  
Polyketide Synthases ◽  
Site Directed Mutagenesis ◽  
Solvent Accessible Surface Area ◽  
Dimensional Structure ◽  
Structural Basis ◽  
Carrier Protein

Anatoxin-a and homoanatoxin-a are two potent cyanobacterial neurotoxins biosynthesized from L-proline by a short pathway involving polyketide synthases. Proline is first loaded onto AnaD, an acyl carrier protein, and prolyl-AnaD is then oxidized to 1-pyrroline-5-carboxyl-AnaD by a flavoprotein, AnaB. Three polyketide synthases then transform this imine into anatoxin-a or homoanatoxin-a. AnaB was crystallized in its holo form and its three-dimensional structure was determined by X-ray diffraction at 2.8 Å resolution. AnaB is a homotetramer and its fold is very similar to that of the acyl-CoA dehydrogenases (ACADs). The active-site base of AnaB, Glu244, superimposed very well with that of human isovaleryl-CoA dehydrogenase, confirming previous site-directed mutagenesis experiments and mechanistic proposals. The substrate-binding site of AnaB is small and is likely to be fitted for the pyrrolidine ring of proline. However, in contrast to ACADs, which use an electron-transport protein, AnaB uses molecular oxygen as the electron acceptor, as in acyl-CoA oxidases. Calculation of the solvent-accessible surface area around the FAD in AnaB and in several homologues showed that it is significantly larger in AnaB than in its homologues. A protonated histidine near the FAD in AnaB is likely to participate in oxygen activation. Furthermore, an array of water molecules detected in the AnaB structure suggests a possible path for molecular oxygen towards FAD. This is consistent with AnaB being an oxidase rather than a dehydrogenase. The structure of AnaB is the first to be described for a prolyl-ACP oxidase and it will contribute to defining the structural basis responsible for oxygen reactivity in flavoenzymes.

scholarly journals Biochemical and Structural Insights Concerning Triclosan Resistance in a Novel YX7K Type Enoyl-Acyl Carrier Protein Reductase from Soil Metagenome

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Raees Khan ◽  
Amir Zeb ◽  
Kihyuck Choi ◽  
Gihwan Lee ◽  
Keun Woo Lee ◽  
...  
Keyword(s):  
Pathogenic Bacteria ◽  
Fatty Acid Biosynthesis ◽  
Catalytic Domain ◽  
Acyl Carrier Protein ◽  
Site Directed Mutagenesis ◽  
Structural Basis ◽  
Carrier Protein ◽  
The Novel ◽  
Triclosan Resistance ◽  
Structural Insights

Abstract Enoyl-acyl carrier protein reductase (ENR) catalyzes the last reduction step in the bacterial type II fatty acid biosynthesis cycle. ENRs include FabI, FabL, FabL2, FabK, and FabV. Previously, we reported a unique triclosan (TCL) resistant ENR homolog that was predominant in obligate intracellular pathogenic bacteria and Apicomplexa. Herein, we report the biochemical and structural basis of TCL resistance in this novel ENR. The purified protein revealed NADH-dependent ENR activity and shared similarity to prototypic FabI. Thus, this metagenome-derived ENR was designated FabI2. Unlike other prototypic bacterial ENRs with the YX6K type catalytic domain, FabI2 possessed a unique YX7K type catalytic domain. Computational modeling followed by site-directed mutagenesis revealed that mild resistance (20 µg/ml of minimum inhibitory concentration) of FabI2 to TCL was confined to the relatively less bulky side chain of A128. Substitution of A128 in FabI2 with bulky valine (V128) elevated TCL resistance. Phylogenetic analysis further suggested that the novel FabI2 and prototypical FabI evolved from a common short-chain dehydrogenase reductase family. To our best knowledge, FabI2 is the only known ENR shared by intracellular pathogenic prokaryotes, intracellular pathogenic lower eukaryotes, and a few higher eukaryotes. This suggests that the ENRs of prokaryotes and eukaryotes diverged from a common ancestral ENR of FabI2.

scholarly journals Biochemical and Structural Basis of Triclosan Resistance in a Novel Enoyl-Acyl Carrier Protein Reductase

2018 ◽  
Vol 62 (8) ◽  
Author(s):  
Raees Khan ◽  
Amir Zeb ◽  
Nazish Roy ◽  
Roniya Thapa Magar ◽  
Hyo Jeong Kim ◽  
...  
Keyword(s):  
Pathogenic Bacteria ◽  
Fatty Acid Biosynthesis ◽  
Acyl Carrier Protein ◽  
Site Directed Mutagenesis ◽  
Structural Basis ◽  
Carrier Protein ◽  
Content Type ◽  
Triclosan Resistance ◽  
Bacterial Type

ABSTRACTEnoyl-acyl carrier protein reductases (ENR), such as FabI, FabL, FabK, and FabV, catalyze the last reduction step in bacterial type II fatty acid biosynthesis. Previously, we reported metagenome-derived ENR homologs resistant to triclosan (TCL) and highly similar to 7-α hydroxysteroid dehydrogenase (7-AHSDH). These homologs are commonly found inEpsilonproteobacteria, a class that contains several human-pathogenic bacteria, including the generaHelicobacterandCampylobacter. Here we report the biochemical and predicted structural basis of TCL resistance in a novel 7-AHSDH-like ENR. The purified protein exhibited NADPH-dependent ENR activity but no 7-AHSDH activity, despite its high homology with 7-AHSDH (69% to 96%). Because this ENR was similar to FabL (41%), we propose that this metagenome-derived ENR be referred to as FabL2. Homology modeling, molecular docking, and molecular dynamic simulation analyses revealed the presence of an extrapolated six-amino-acid loop specific to FabL2 ENR, which prevented the entry of TCL into the active site of FabL2 and was likely responsible for TCL resistance. Elimination of this extrapolated loop via site-directed mutagenesis resulted in the complete loss of TCL resistance but not enzyme activity. Phylogenetic analysis suggested that FabL, FabL2, and 7-AHSDH diverged from a common short-chain dehydrogenase reductase family. This study is the first to report the role of the extrapolated loop of FabL2-type ENRs in conferring TCL resistance. Thus, the FabL2 ENR represents a new drug target specific for pathogenicEpsilonproteobacteria.

Structural Basis of Acyl-Carrier Protein Interactions in Fatty Acid and Polyketide Biosynthesis

2020 ◽  
pp. 61-122 ◽  
Author(s):  
Jeffrey T. Mindrebo ◽  
Ashay Patel ◽  
Laëtitia E. Misson ◽  
Woojoo E. Kim ◽  
Tony D. Davis ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Protein Interactions ◽  
Acyl Carrier Protein ◽  
Structural Basis ◽  
Carrier Protein ◽  
Polyketide Biosynthesis

scholarly journals Structural Basis for Env Incorporation into HIV-1 Particles

Proceedings ◽  
2020 ◽  
Vol 50 (1) ◽  
pp. 114
Author(s):  
R. Elliot Murphy ◽  
Alexandra B. Samal ◽  
Gunnar Eastep ◽  
Ruba H. Ghanam ◽  
Peter E. Prevelige ◽  
...  
Keyword(s):  
Structural Characterization ◽  
Late Phase ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Structural Basis ◽  
Membrane Interactions ◽  
Env Protein ◽  
Gag Assembly ◽  
Hiv 1

During the late phase of the HIV-1 replication cycle, the Gag polyproteins are transported to the plasma membrane (PM) for assembly. Gag targeting and assembly on the PM is dependent on interactions between its matrix (MA) domain and phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2). Subsequent to Gag assembly, the envelope (Env) protein is recruited to the PM for incorporation into virus particles. Evidence suggests that the incorporation of the Env protein is mediated by interactions between the MA domain of Gag and the cytoplasmic tail of the gp41 subunit of Env (gp41CT), a mechanism that remains to be elucidated. Trimerization of the MA domain of Gag appears to be an obligatory step for this interaction. The interplay between gp41CT, the MA trimer, and the membrane has yet to be determined. Our lab has pioneered methods and approaches to investigate, at the molecular level, how the retroviral MA domains of Gag interact with membranes, a key requirement for understanding the Gag assembly and Env incorporation. Herein, we devised innovative approaches that will enable the structural characterization of the gp41CT–MA–membrane interactions. We employed structural biology (NMR and cryo-electron microscopy, biophysical methods, and biochemical tools to generate a macromolecular picture of how the MA domain of Gag binds to the membrane and how it interacts with gp41CT. To this end, we: (i) determined the three-dimensional structure of HIV-1 gp41CT and characterized its interaction with the membrane, (ii) engineered trimeric constructs of gp41CT and the MA to recapitulate the native and functional states of the proteins, and (iii) utilized membrane nanodisc technology to anchor the MA and gp41CT proteins. Our studies will allow for a detailed structural characterization of the gp41CT–MA–membrane interactions, which will advance our knowledge of HIV-1 Gag assembly and Env incorporation.

Elucidation of the paratope of scFv-8H9D4, a PAI-1 neutralizing antibody derivative

2003 ◽  
Vol 89 (01) ◽  
pp. 74-82 ◽  
Author(s):  
Koen Verbeke ◽  
Ann Gils ◽  
Jean-Marie Stassen ◽  
Paul Declerck
Keyword(s):  
Rational Design ◽  
Three Dimensional ◽  
Neutralizing Antibody ◽  
Plasminogen Activator Inhibitor ◽  
Site Directed Mutagenesis ◽  
Dimensional Structure ◽  
Single Chain ◽  
Plasminogen Activator Inhibitor 1 ◽  
Activator Inhibitor ◽  
Pai 1

SummaryInterfering with increased levels of plasminogen activator inhibitor-1 (PAI-1) might offer new therapeutic strategies for a variety of cardiovascular diseases. Inactivation of PAI-1 can be accomplished by a number of monoclonal antibodies (MA), including MA-8H9D4. In a previous study, a single-chain variable fragment (scFv-8H9D4) was cloned and found to have the same properties as the parental MA-8H9D4. In the present study, we identified the residues of scFv-8H9D4 that contribute significantly to the paratope. The complementarity determining region 3 from the heavy (H3) and the light (L3) chain were analysed through site-directed mutagenesis. Out of twelve mutations, only four residues appeared to contribute to the paratope. The affinity of scFv-8H9D4-H3-L97D for PAI-1 was 38-fold decreased (KA = 4.8 ± 0.2 × 107 M–1 vs. 1.8 ± 0.7 × 109 M–1 for scFv-8H9D4) whereas scFv-8H9D4-H3-R98Y did not bind to PAI-1. The affinities of scFv-8H9D4-L3-Y91S and scFv-8H9D4-L3-F94D for PAI-1 were 9- and 5-fold reduced, respectively, whereas the combined mutation resulted in an 86-fold decreased affinity (KA = 2.1 ± 0.2 × 107 M–1).In accordance with the affinity data, these mutants had no, or a reduced, PAI-1 inhibitory capacity, confirming that these four particular residues form the major interaction site of scFv-8H9D4 with PAI-1. In combination with the three-dimensional structure, these data contribute to the rational design of PAI-1 neutralizing compounds.

Protein intrachain contact prediction with most interacting residues (MIR)

2014 ◽  
Vol 10 (4) ◽  
Author(s):  
Ruben Acuña ◽  
Zoé Lacroix ◽  
Nikolaos Papandreou ◽  
Jacques Chomilier
Keyword(s):  
Structural Changes ◽  
Hydrophobic Interactions ◽  
Three Dimensional ◽  
Accessible Surface Area ◽  
Dimensional Structure ◽  
Closed Loops ◽  
Folding Process ◽  
Folding Nucleus ◽  
Accessible Surface ◽  
The Stability

AbstractThe transition state ensemble during the folding process of globular proteins occurs when a sufficient number of intrachain contacts are formed, mainly, but not exclusively, due to hydrophobic interactions. These contacts are related to the folding nucleus, and they contribute to the stability of the native structure, although they may disappear after the energetic barrier of transition states has been passed. A number of structure and sequence analyses, as well as protein engineering studies, have shown that the signature of the folding nucleus is surprisingly present in the native three-dimensional structure, in the form of closed loops, and also in the early folding events. These findings support the idea that the residues of the folding nucleus become buried in the very first folding events, therefore helping the formation of closed loops that act as anchor structures, speed up the process, and overcome the Levinthal paradox. We present here a review of an algorithm intended to simulate in a discrete space the early steps of the folding process. It is based on a Monte Carlo simulation where perturbations, or moves, are randomly applied to residues within a sequence. In contrast with many technically similar approaches, this model does not intend to fold the protein but to calculate the number of non-covalent neighbors of each residue, during the early steps of the folding process. Amino acids along the sequence are categorized as most interacting residues (MIRs) or least interacting residues. The MIR method can be applied under a variety of circumstances. In the cases tested thus far, MIR has successfully identified the exact residue whose mutation causes a switch in conformation. This follows with the idea that MIR identifies residues that are important in the folding process. Most MIR positions correspond to hydrophobic residues; correspondingly, MIRs have zero or very low accessible surface area. Alongside the review of the MIR method, we present a new postprocessing method called smoothed MIR (SMIR), which refines the original MIR method by exploiting the knowledge of residue hydrophobicity. We review known results and present new ones, focusing on the ability of MIR to predict structural changes, secondary structure, and the improved precision with the SMIR method.

scholarly journals Active site labeling of fatty acid and polyketide acyl-carrier protein transacylases

2019 ◽  
Vol 17 (19) ◽  
pp. 4720-4724 ◽  
Author(s):  
Tony D. Davis ◽  
Jennifer M. Michaud ◽  
Michael D. Burkart
Keyword(s):  
Fatty Acid ◽  
Fluorescent Probe ◽  
Active Site ◽  
Polyketide Synthase ◽  
Acyl Carrier Protein ◽  
Site Directed Mutagenesis ◽  
Probe Design ◽  
Directed Mutagenesis ◽  
Carrier Protein

Fluorescent probe design and site-directed mutagenesis unveil new activity-based chemical reporters for fatty acid and polyketide synthase acyl-carrier protein transacylases.

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