Structural and Biochemical Analysis of Protein-Protein Interactions Between the Acyl-Carrier Protein and Product Template Domain

2016 ◽  
Vol 128 (42) ◽  
pp. 13199-13203 ◽  
Author(s):  
Jesus F. Barajas ◽  
Kara Finzel ◽  
Timothy R. Valentic ◽  
Gaurav Shakya ◽  
Nathan Gamarra ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Biochemical Analysis ◽  
Acyl Carrier Protein ◽  
Carrier Protein ◽  
Protein Protein Interactions

scholarly journals Determination of the Protein-Protein Interactions within Acyl Carrier Protein (MmcB)-Dependent Modifications in the Biosynthesis of Mitomycin

Molecules ◽  
2021 ◽  
Vol 26 (22) ◽  
pp. 6791
Author(s):  
Dongjin Leng ◽  
Yong Sheng ◽  
Hengyu Wang ◽  
Jianhua Wei ◽  
Yixin Ou ◽  
...  
Keyword(s):  
Mitomycin C ◽  
Protein Interactions ◽  
Biochemical Analysis ◽  
Genetic Manipulation ◽  
Acyl Carrier Protein ◽  
Early Stage ◽  
Carrier Protein ◽  
Protein Protein Interactions ◽  
Enzymatic Mechanisms

Mitomycin has a unique chemical structure and contains densely assembled functionalities with extraordinary antitumor activity. The previously proposed mitomycin C biosynthetic pathway has caused great attention to decipher the enzymatic mechanisms for assembling the pharmaceutically unprecedented chemical scaffold. Herein, we focused on the determination of acyl carrier protein (ACP)-dependent modification steps and identification of the protein–protein interactions between MmcB (ACP) with the partners in the early-stage biosynthesis of mitomycin C. Based on the initial genetic manipulation consisting of gene disruption and complementation experiments, genes mitE, mmcB, mitB, and mitF were identified as the essential functional genes in the mitomycin C biosynthesis, respectively. Further integration of biochemical analysis elucidated that MitE catalyzed CoA ligation of 3-amino-5-hydroxy-bezonic acid (AHBA), MmcB-tethered AHBA triggered the biosynthesis of mitomycin C, and both MitB and MitF were MmcB-dependent tailoring enzymes involved in the assembly of mitosane. Aiming at understanding the poorly characterized protein–protein interactions, the in vitro pull-down assay was carried out by monitoring MmcB individually with MitB and MitF. The observed results displayed the clear interactions between MmcB and MitB and MitF. The surface plasmon resonance (SPR) biosensor analysis further confirmed the protein–protein interactions of MmcB with MitB and MitF, respectively. Taken together, the current genetic and biochemical analysis will facilitate the investigations of the unusual enzymatic mechanisms for the structurally unique compound assembly and inspire attempts to modify the chemical scaffold of mitomycin family antibiotics.

scholarly journals Structural and Biochemical Analysis of Protein-Protein Interactions Between the Acyl-Carrier Protein and Product Template Domain

2016 ◽  
Vol 55 (42) ◽  
pp. 13005-13009 ◽  
Author(s):  
Jesus F. Barajas ◽  
Kara Finzel ◽  
Timothy R. Valentic ◽  
Gaurav Shakya ◽  
Nathan Gamarra ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Biochemical Analysis ◽  
Acyl Carrier Protein ◽  
Carrier Protein ◽  
Protein Protein Interactions

scholarly journals Elucidation of transient protein-protein interactions within carrier protein-dependent biosynthesis

2020 ◽  
Author(s):  
Michael Burkart ◽  
Thomas Bartholow ◽  
Terra Sztain ◽  
Ashay Patel ◽  
D Lee ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Protein Interactions ◽  
Fatty Acid Biosynthesis ◽  
De Novo ◽  
Acyl Carrier Protein ◽  
Protein Docking ◽  
Carrier Protein ◽  
Protein Protein Interactions ◽  
Acid Biosynthesis ◽  
E Coli

Abstract Fatty acid biosynthesis (FAB) is an essential and highly conserved metabolic pathway. In bacteria, this process is mediated by an elaborate network of protein•protein interactions (PPIs) involving a small, dynamic acyl carrier protein that interacts with dozens of other partner proteins (PPs). These PPIs have remained poorly characterized due to their dynamic and transient nature. Using a combination of solution-phase NMR spectroscopy and protein-protein docking simulations, we report a comprehensive residue-by-residue comparison of the PPIs formed during FAB in Escherichia coli. This work reveals the molecular basis of six discrete binding events responsible for E. coli FAB and offers insights into a method to characterize these events and those in related carrier protein-dependent pathways. ONE SENTENCE SUMMARY: Through a combination of structural and computational analysis, a comparative evaluation of protein-protein interactions in de novo fatty acid biosynthesis in E. coli is performed.

scholarly journals Elucidation of transient protein-protein interactions within carrier protein-dependent biosynthesis

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Thomas G. Bartholow ◽  
Terra Sztain ◽  
Ashay Patel ◽  
D. John Lee ◽  
Megan A. Young ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Fatty Acid Biosynthesis ◽  
Acyl Carrier Protein ◽  
Protein Docking ◽  
Carrier Protein ◽  
Protein Protein Interactions ◽  
Docking Simulations ◽  
E Coli ◽  
Transient Nature ◽  
Partner Proteins

AbstractFatty acid biosynthesis (FAB) is an essential and highly conserved metabolic pathway. In bacteria, this process is mediated by an elaborate network of protein•protein interactions (PPIs) involving a small, dynamic acyl carrier protein that interacts with dozens of other partner proteins (PPs). These PPIs have remained poorly characterized due to their dynamic and transient nature. Using a combination of solution-phase NMR spectroscopy and protein-protein docking simulations, we report a comprehensive residue-by-residue comparison of the PPIs formed during FAB in Escherichia coli. This technique describes and compares the molecular basis of six discrete binding events responsible for E. coli FAB and offers insights into a method to characterize these events and those in related carrier protein-dependent pathways.

scholarly journals Decoding allosteric regulation by the acyl carrier protein

2021 ◽  
Vol 118 (16) ◽  
pp. e2025597118
Author(s):  
Terra Sztain ◽  
Thomas G. Bartholow ◽  
D. John Lee ◽  
Lorenzo Casalino ◽  
Andrew Mitchell ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Metabolic Pathways ◽  
Acyl Carrier Protein ◽  
Allosteric Regulation ◽  
Structural Features ◽  
Carrier Protein ◽  
Protein Protein Interactions ◽  
Fatty Acid Chain ◽  
Four Helical Bundle ◽  
Chain Lengths

Enzymes in multistep metabolic pathways utilize an array of regulatory mechanisms to maintain a delicate homeostasis [K. Magnuson, S. Jackowski, C. O. Rock, J. E. Cronan, Jr, Microbiol. Rev. 57, 522–542 (1993)]. Carrier proteins in particular play an essential role in shuttling substrates between appropriate enzymes in metabolic pathways. Although hypothesized [E. Płoskoń et al., Chem. Biol. 17, 776–785 (2010)], allosteric regulation of substrate delivery has never before been demonstrated for any acyl carrier protein (ACP)-dependent pathway. Studying these mechanisms has remained challenging due to the transient and dynamic nature of protein–protein interactions, the vast diversity of substrates, and substrate instability [K. Finzel, D. J. Lee, M. D. Burkart, ChemBioChem 16, 528–547 (2015)]. Here we demonstrate a unique communication mechanism between the ACP and partner enzymes using solution NMR spectroscopy and molecular dynamics to elucidate allostery that is dependent on fatty acid chain length. We demonstrate that partner enzymes can allosterically distinguish between chain lengths via protein–protein interactions as structural features of substrate sequestration are translated from within the ACP four-helical bundle to the protein surface, without the need for stochastic chain flipping. These results illuminate details of cargo communication by the ACP that can serve as a foundation for engineering carrier protein-dependent pathways for specific, desired products.

scholarly journals Structure-based analysis of the molecular interactions between acyltransferase and acyl carrier protein in vicenistatin biosynthesis

2016 ◽  
Vol 113 (7) ◽  
pp. 1802-1807 ◽  
Author(s):  
Akimasa Miyanaga ◽  
Shohei Iwasawa ◽  
Yuji Shinohara ◽  
Fumitaka Kudo ◽  
Tadashi Eguchi
Keyword(s):  
Crystal Structure ◽  
Protein Interactions ◽  
Acyl Carrier Protein ◽  
Complex Structure ◽  
Binding Pocket ◽  
Carrier Protein ◽  
Protein Protein Interactions ◽  
Substrate Binding Pocket ◽  
Insight Into

Acyltransferases (ATs) are key determinants of building block specificity in polyketide biosynthesis. Despite the importance of protein–protein interactions between AT and acyl carrier protein (ACP) during the acyltransfer reaction, the mechanism of ACP recognition by AT is not understood in detail. Herein, we report the crystal structure of AT VinK, which transfers a dipeptide group between two ACPs, VinL and VinP1LdACP, in vicenistatin biosynthesis. The isolated VinK structure showed a unique substrate-binding pocket for the dipeptide group linked to ACP. To gain greater insight into the mechanism of ACP recognition, we attempted to crystallize the VinK–ACP complexes. Because transient enzyme–ACP complexes are difficult to crystallize, we developed a covalent cross-linking strategy using a bifunctional maleimide reagent to trap the VinK–ACP complexes, allowing the determination of the crystal structure of the VinK–VinL complex. In the complex structure, Arg-153, Met-206, and Arg-299 of VinK interact with the negatively charged helix II region of VinL. The VinK–VinL complex structure allows, to our knowledge, the first visualization of the interaction between AT and ACP and provides detailed mechanistic insights into ACP recognition by AT.

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