Article
Biochemistry & Molecular Biology
Andrea Dali, Thomas Gabler, Federico Sebastiani, Alina Destinger, Paul Georg Furtmueller, Vera Pfanzagl, Maurizio Becucci, Giulietta Smulevich, Stefan Hofbauer
Summary: Coproporphyrin ferrochelatases (CpfCs) are enzymes that catalyze the penultimate step in the coproporphyrin-dependent heme biosynthesis pathway. The discovery of the correct substrate for these ferrochelatases and the characterization of their binding mode provide valuable insights into the iron insertion process. This knowledge is essential for understanding the preconditions and mechanisms of iron insertion in CpfCs.
Review
Chemistry, Organic
Houchao Xu, Jeroen S. Dickschat
Summary: In the past three decades, numerous terpene synthases have been characterized from various life kingdoms. The mechanisms of terpene synthases have been investigated through site-directed mutagenesis, providing valuable insights into their catalysis and resulting in mutants with improved yields.
SYNTHESIS-STUTTGART
(2022)
Article
Biochemistry & Molecular Biology
Christoph P. Sager, Susanne Weber, Matthias Negri, Pauline Banachowicz, Gabriele Moeller, Jerzy Adamski, Rolf W. Hartmann, Sandrine Marchais-Oberwinkler
Summary: This study aimed to determine the topology of 17 beta-Hydroxystemid dehydrogenase type 2 (17β-HSD2) by constructing a homology model in complex with NAD(+) and 17 beta-estradiol, which was confirmed to be of high quality through site-directed mutagenesis. The model identified key amino acids involved in ligand or internal structure stabilization, providing a basis for further experiments and drug design studies.
JOURNAL OF STEROID BIOCHEMISTRY AND MOLECULAR BIOLOGY
(2021)
Article
Microbiology
Zhizeng Sun, Timothy Palzkill
Summary: The study identified essential active-site residues of the MCR-1 enzyme using deep sequencing, which are critical for its polymyxin resistance function. Approximately 75% of the residues examined were found to be essential for the enzyme's function, suggesting inhibitors binding near these sites will broadly inhibit MCR-1 and similar enzymes.
Article
Chemistry, Multidisciplinary
Anwei Hou, Bernd Goldfuss, Jeroen S. Dickschat
Summary: The reinvestigation of linalool synthase from Chryseobacterium polytrichastri revealed its diterpene synthase activity, leading to the discovery of new compounds with unique skeletons. Isotopic labeling experiments and DFT calculations were used to study the enzyme mechanism and explain the formation of an unusual ethyl group. Rational exchanges of active site residues resulted in major functional switches and the production of new compounds.
ANGEWANDTE CHEMIE-INTERNATIONAL EDITION
(2021)
Article
Biochemistry & Molecular Biology
Victoria J. Laye, Shiladitya DasSarma
Summary: In this study, mutations were introduced into an Antarctic haloarchaeon enzyme to investigate its temperature-dependent catalytic activity. The results suggest that a small number of residues, located distant from the active site, mediate complex and subtle effects on the enzyme's kinetic properties at different temperatures.
Article
Chemistry, Organic
Anwei Hou, Jeroen S. Dickschat
Summary: The study conducted site-directed mutagenesis experiments on the sesterterpene synthase SmTS1 from Streptomyces mobaraensis, revealing diverse effects of mutations on catalytic activity and enzyme function switch. Rational explanations were provided for these findings, offering insights into protein engineering of terpene synthases for improved efficiency or altered functions.
BEILSTEIN JOURNAL OF ORGANIC CHEMISTRY
(2021)
Article
Biochemistry & Molecular Biology
Mashael A. Alghamdi, Rania A. Hussien, Yuanzhang Zheng, Hiral P. Patel, Matias D. Asencion Diez, Alberto A. Iglesias, Dali Liu, Miguel A. Ballicora
Summary: The allosteric regulation of ADP-glucose pyrophosphorylase plays a critical role in glycogen and starch biosynthesis. This study investigates the binding and activation of the enzyme by fructose 6-phosphate (Fru6P) and pyruvate (Pyr). It is found that a sulfate ion in the crystal structure of the enzyme acts as a regulatory site similar to Fru6P. Ser72 interacts with this sulfate ion, and its mutation affects the interaction with Fru6P and enzyme activation. Mutations in Ser72 hinder the binding of Fru6P and the inhibitor AMP, while still allowing activation by Pyr. These findings suggest that ADP-glucose pyrophosphorylase in Agrobacterium tumefaciens has distinct sites for Fru6P and Pyr, working together to regulate glycogen biosynthesis.
Review
Biotechnology & Applied Microbiology
Haoran Yu, Shuang Ma, Yiwen Li, Paul A. Dalby
Summary: Directed evolution is a powerful strategy to engineer protein properties, and hot spot prediction is crucial for producing smart libraries. Selection of hot spots based on sequence and structure allows for efficient generation of proteins with desired properties.
BIOTECHNOLOGY ADVANCES
(2022)
Article
Biochemistry & Molecular Biology
Sylvester Hoffmann, Maik Damm, Leonard Roth, Roderich D. Suessmuth
Summary: This study investigates the activation mechanism of hydroxy acid substrates in non-ribosomal peptide synthetases (NRPSs). By homology modelling and molecular docking, it was found that the selection of hydroxy acid is determined by its interaction with the backbone carbonyls rather than a specific side chain. These findings contribute to the understanding of non-amino acid substrate activation and have implications for the engineering of depsipeptide synthetases.
Article
Biochemistry & Molecular Biology
Amy E. Medlock, Wided Najahi-Missaoui, Mesafint T. Shiferaw, Angela N. Albetel, William N. Lanzilotta, Harry A. Dailey
Summary: Ferrochelatase is an important enzyme involved in catalyzing heme synthesis in humans, serving a regulatory and catalytic role in the pathway. Studies on its crystal structures, kinetic properties, and catalytic cycle have provided insights into its functions. However, questions regarding metal ion delivery, active site residue roles, and the catalytic mechanism remain open for further investigation.
BIOCHEMICAL JOURNAL
(2021)
Article
Biochemistry & Molecular Biology
Suresh Pal, Bryce Plapp
Summary: X-ray crystallography has revealed that the threonine residue (Thr-45) in the yeast alcohol dehydrogenase (ADH1) plays an important role in catalysis and its substitution results in a significant decrease in catalytic efficiency. Conformational changes of the enzyme and ligand exchanges on the catalytic zinc are severely impacted by the removal of the hydroxyethyl group of Thr-45.
CHEMICO-BIOLOGICAL INTERACTIONS
(2021)
Article
Immunology
Yue Zhou, Chelsi D. Cassilly, Todd B. Reynolds
Summary: This study identified the role of the CAPT motif in phosphatidylserine synthesis and a unique motif related to serine binding in fungal PS synthases, through alanine substitution mutagenesis. The results suggest that specific residues within these motifs are essential for Cho1 function, and mutants like L184A and R189A show contrasting impacts on PS synthase activity, providing insights for potential Cho1 inhibitors development.
FRONTIERS IN CELLULAR AND INFECTION MICROBIOLOGY
(2021)
Article
Biochemistry & Molecular Biology
Amalie Carnbring Bonde, Sofie Rosenorn Hansen, Eva Johansson, Jais Rose Bjelke, Jacob Lund
Summary: This study investigates the conversion of zymogen Factor X to an active protease and reveals the importance of proline residues in efficient proteolysis. It also identifies essential interaction sites for Factor IXa and the intrinsic tenase complex, as well as the role of a carbohydrate chain in activator specificity.
Article
Microbiology
Emily C. Hoedt, Francesca Bottacini, Nora Cash, Roger S. Bongers, Kees van Limpt, Kaouther Ben Amor, Jan Knol, John MacSharry, Douwe van Sinderen
Summary: Members of the genus Bifidobacterium are difficult to genetically manipulate due to their extensive and variable Restriction-Modification systems. A modified suicide vector, pFREM28, was developed to target specific genes in B. breve by removing known restriction sites, reducing the time, effort, and resources required for generating site-directed mutants. This approach could be applied to other (bifido)bacterial species as well.
FRONTIERS IN MICROBIOLOGY
(2021)
Article
Biochemistry & Molecular Biology
Daniel Schmidt, Nikolaus Falb, Ilenia Serra, Marzia Bellei, Vera Pfanzagl, Stefan Hofbauer, Sabine Van Doorslaer, Gianantonio Battistuzzi, Paul G. Furtmuller, Christian Obinger
Article
Biochemistry & Molecular Biology
Andrea Dali, Thomas Gabler, Federico Sebastiani, Alina Destinger, Paul Georg Furtmueller, Vera Pfanzagl, Maurizio Becucci, Giulietta Smulevich, Stefan Hofbauer
Summary: Coproporphyrin ferrochelatases (CpfCs) are enzymes that catalyze the penultimate step in the coproporphyrin-dependent heme biosynthesis pathway. The discovery of the correct substrate for these ferrochelatases and the characterization of their binding mode provide valuable insights into the iron insertion process. This knowledge is essential for understanding the preconditions and mechanisms of iron insertion in CpfCs.
Article
Biochemistry & Molecular Biology
Federico Sebastiani, Chiara Baroni, Gaurav Patil, Andrea Dali, Maurizio Becucci, Stefan Hofbauer, Giulietta Smulevich
Summary: Monoderm bacteria accumulate heme b through the coproporphyrin-dependent biosynthesis pathway. The decarboxylation of propionate groups in coproheme by coproheme decarboxylase (ChdC) is a stepwise process. H-bond interactions in the pocket of ChdCs play a crucial role in stabilizing the active site and enzyme functionality, which were evaluated through characterization of mutants complexed with coproheme and heme b via spectroscopies.
Article
Biochemistry & Molecular Biology
Daniel R. Ramos, Paul G. Furtmueller, Christian Obinger, Angeles Pena-Gallego, Ignacio Perez-Juste, J. Arturo Santaballa
Summary: Electronic structure calculations using DFT were conducted to examine the influence of water molecules and protonation on the heme group of peroxidases in different redox and spin states. The study discusses shared geometries, spectroscopic properties, and thermodynamics of peroxidases. Computed Gibbs free energies suggest that the corresponding aquo complexes are not thermodynamically stable, supporting the five-coordinate Fe(III) center in native ferric peroxidases with a non-bonding water molecule. Protonation of the ferryl oxygen of compound II is found to be necessary and computed Gibbs free energies reveal pK(a) values of approximately 8.5-9.0 for compound II.
Article
Chemistry, Analytical
Jürgen Beck, Matthias Biechele, Christoph Repik, Petra Gruber, Paul G. Furtmueller, Rainer Hahn
Summary: This study systematically compared the elution behavior of plasmid DNA on three common anion exchange resins. It was found that plasmid DNA consistently elutes at a characteristic salt concentration in linear gradient elution, and elutes only above this concentration in isocratic elution. Structural analysis supported this explanation.
JOURNAL OF SEPARATION SCIENCE
(2023)
Article
Biochemistry & Molecular Biology
Gaurav Patil, Hanna Michlits, Paul G. Furtmueller, Stefan Hofbauer
Summary: Coproheme decarboxylases (ChdCs) are enzymes involved in the biosynthesis of heme. This study focuses on the second part of the decarboxylation reaction catalyzed by ChdCs, which has not been previously studied. The researchers optimized the production and purification of a intermediate compound called monovinyl, monopropionate deuteroheme (MMD), and used it to study the reaction mechanism. The results indicate that the second part of the reaction is similar to the first part, with slight differences in the active site architecture and H-bonding network.
Article
Biochemistry & Molecular Biology
Federico Sebastiani, Andrea Dali, Diego Javier Alonso de Armino, Lorenzo Campagni, Gaurav Patil, Maurizio Becucci, Stefan Hofbauer, Dario A. Estrin, Giulietta Smulevich
Summary: This study focuses on the carbon monoxide adducts of the wild-type and selected variants of the coproheme decarboxylase from actinobacterial Corynebacterium diphtheriae complexed with coproheme, monovinyl monopropionyl deuteroheme (MMD), and heme b. The results show that the wild-type coproheme-CO adduct is characterized by two CO conformers, hydrogen-bonded and weak polar interaction with the distal cavity. The absence of the H118 residue leads to the formation of non-H-bonded CO species. In addition, CO binding to reversed heme b and heme d is also investigated in this work.
JOURNAL OF INORGANIC BIOCHEMISTRY
(2023)
Article
Biochemistry & Molecular Biology
Thomas Gabler, Andrea Dali, Federico Sebastiani, Paul Georg Furtmueller, Maurizio Becucci, Stefan Hofbauer, Giulietta Smulevich
Summary: Understanding the reaction mechanism of enzymes is challenging, but studying model substrates can provide valuable information. In this study, we investigated the mechanism of ferrous iron incorporation in a bacterial enzyme complex and discovered the role of hydrogen bond interactions in this process.
Review
Biochemistry & Molecular Biology
Nikolaus Falb, Gaurav Patil, Paul G. Furtmueller, Thomas Gabler, Stefan Hofbauer
Summary: The coproporphyrin dependent heme biosynthesis pathway is predominantly used by Gram-positive bacteria. This pathway is of interest for basic research as it relates to medical biotechnology and the development of new antibiotic targets against Gram-positive superbugs. A review of the accumulated structural data on the enzymes involved in this pathway is provided, highlighting the need for further analysis and future research to gain a comprehensive understanding of prokaryotic heme biosynthesis.
COMPUTATIONAL AND STRUCTURAL BIOTECHNOLOGY JOURNAL
(2023)