Article
Multidisciplinary Sciences
Galen J. Correy, Daniel W. Kneller, Gwyndalyn Phillips, Swati Pant, Silvia Russi, Aina E. Cohen, George Meigs, James M. Holton, Stefan Gahbauer, Michael C. Thompson, Alan Ashworth, Leighton Coates, Andrey Kovalevsky, Flora Meilleur, James S. Fraser
Summary: This study determined the crystal structures of the nonstructural protein 3 (NSP3) macrodomain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), revealing the reorganization of water networks upon binding of ADP-ribose and other ligands. The catalytic mechanism of coronavirus macrodomains was found to be distinct from that of human macrodomains, providing insights for the development of inhibitors.
Article
Biochemistry & Molecular Biology
Stavros Azinas, Marta Carroni
Summary: Cryo-EM is a major tool for determining protein structures, while AlphaFold2 enables high-confidence atomic models for polypeptide chains. Cryo-EM retains unique characteristics for determining structures of macromolecular complexes, allowing for near-atomic structures and exploring conformational panoramas. It also has the potential to develop a structural proteomic approach using ex vivo specimens.
CURRENT OPINION IN STRUCTURAL BIOLOGY
(2023)
Article
Biochemistry & Molecular Biology
Bharath Srinivasan
Summary: The rise of macromolecular crystallography has greatly impacted various scientific fields, including molecular pharmacology, drug discovery, and biotechnology, by allowing atomic resolution visualization of protein structures. However, the teaching of this subject in universities worldwide has been suboptimal due to its interdisciplinary nature and the complex concepts and terminology involved. Additionally, the use of robotics and advanced software algorithms has diminished the incentive for students to understand the foundational concepts of macromolecular crystallography. This article proposes a comprehensive framework for teaching and learning this subject, advocating for interdisciplinary approaches, visual tools, computational resources, and historical context to make it more accessible and relatable to students.
Article
Chemistry, Multidisciplinary
Takeshi Murakawa, Kazuo Kurihara, Motoyasu Adachi, Katsuhiro Kusaka, Katsuyuki Tanizawa, Toshihide Okajima
Summary: Protein neutron crystallography is an important technique for determining the positions of hydrogen atoms in proteins, providing crucial biochemical information. By comparing X-ray and neutron diffraction data, it was found that X-ray/neutron joint refinement is crucial for determining the real chemical structure of the catalytic site of the enzyme.
Article
Chemistry, Multidisciplinary
Vinardas Kelpsas, Octav Caldararu, Matthew P. Blakeley, Nicolas Coquelle, Rikkert K. Wierenga, Ulf Ryde, Claes von Wachenfeldt, Esko Oksanen
Summary: This study investigates the neutron structures of Leishmania mexicana TIM with inhibitors, revealing the protonation states of active-site residues. Quantum calculations were used to study the reaction mechanism based on the identified protonation states.
Article
Biochemical Research Methods
Kai Deng, Xi Wang, Nicole Ing, Paul Opgenorth, Markus de Raad, Jinho Kim, Blake A. Simmons, Paul D. Adams, Anup K. Singh, Taek Soon Lee, Trent R. Northen
Summary: We developed a novel mass spectrometry-based assay that allows rapid quantification of primary alcohols produced by cell cultures. This assay combines TEMPO-based oxidation chemistry and oxime ligation, followed by product analysis using Nanostructure-Initiator Mass Spectrometry. It can accurately monitor C5 to C18 alcohols, as well as glucose and gluconate in the growth medium, which is crucial for strain characterization and optimization. The assay shows comparable results to gas chromatography for isoprenol production but requires significantly less acquisition time per sample. By applying this assay, we gained new insights into the utilization of alcohols by P. Putida and found that this strain had limited growth on heptanol and octanol.
ANALYTICAL BIOCHEMISTRY
(2023)
Article
Biochemical Research Methods
Dorothee Liebschner, Nigel W. Moriarty, Billy K. Poon, Paul D. Adams
Summary: In macromolecular crystallographic structure refinement, the creation of geometric restraints for ligands is challenging due to their chemical variability, novel nature, and specific interactions with protein binding pockets. Quantum-mechanical approaches can accurately determine ligand geometries, but are hindered by the flexibility of molecules with multiple minima. To overcome these issues, the Quantum Mechanical Restraints (QMR) procedure optimizes ligand geometry in situ, considering the influence of the macromolecule on ligand energy minima. QMR restraints generally result in lower deviations from target stereochemistry compared to conventionally generated restraints, especially for accurate torsion restraints.
ACTA CRYSTALLOGRAPHICA SECTION D-STRUCTURAL BIOLOGY
(2023)
Article
Chemistry, Physical
Brandon J. Bloomer, Sean N. Natoli, Marc Garcia-Borras, Jose H. Pereira, Derek B. B. Hu, Paul D. Adams, K. N. Houk, Douglas S. Clark, John F. Hartwig
Summary: Artificial metalloenzymes (ArMs), containing synthetic metal cofactors, are widely used as biocatalysts for unnatural reactions. However, detailed mechanistic information on the effects of protein scaffold on ArM structure and reactivity is limited. This study presents the structure of an unnatural P450 analogue and kinetic analysis of its reaction, revealing a mechanism initiated by cofactor conformational change and highlighting the influence of cofactor dynamics on the catalytic reaction of ArMs.
Article
Chemistry, Multidisciplinary
Elias Englund, Matthias Schmidt, Alberto A. Nava, Anna Lechner, Kai Deng, Renee Jocic, Yingxin Lin, Jacob Roberts, Veronica T. Benites, Ramu Kakumanu, Jennifer W. Gin, Yan Chen, Yuzhong Liu, Christopher J. Petzold, Edward E. K. Baidoo, Trent R. Northen, Paul D. Adams, Leonard Katz, Satoshi Yuzawa, Jay D. Keasling
Summary: This study successfully altered the extension substrates of modular polyketide synthases (PKSs) by exchanging acyltransferase (AT) domains, resulting in the synthesis of 13 structurally different polyketides in vitro. The results greatly enhance our understanding of rare AT domains and demonstrate the benefit of using PKS engineering strategy to produce novel chemicals in vitro.
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
(2023)
Article
Multidisciplinary Sciences
Jing Huang, Andrew Quest, Pablo Cruz-Morales, Kai Deng, Jose Henrique Pereira, Devon Van Cura, Ramu Kakumanu, Edward E. K. Baidoo, Qingyun Dan, Yan Chen, Christopher J. Petzold, Trent R. Northen, Paul D. Adams, Douglas S. Clark, Emily P. Balskus, John F. Hartwig, Aindrila Mukhopadhyay, Jay D. Keasling
Summary: Biosynthesis is a renewable approach for producing natural and new-to-nature products, but it has a narrower scope compared to synthetic chemistry. Recent research has shown that carbene-transfer reactions can be performed in cells, but exogenous carbene donors and unnatural cofactors are needed, limiting the scalability of the biosynthesis process. This study presents a microbial platform for intracellularly conducting abiological carbene-transfer reactions, expanding the range of organic products that can be produced. Evaluation: 8 out of 10.
Article
Multidisciplinary Sciences
Asmit Bhowmick, Rana Hussein, Isabel Bogacz, Philipp S. Simon, Mohamed Ibrahim, Ruchira Chatterjee, Margaret D. Doyle, Mun Hon Cheah, Thomas Fransson, Petko Chernev, In-Sik Kim, Hiroki Makita, Medhanjali Dasgupta, Corey J. Kaminsky, Miao Zhang, Julia Gaetcke, Stephanie Haupt, Isabela I. Nangca, Stephen M. Keable, A. Orkun Aydin, Kensuke Tono, Shigeki Owada, Leland B. Gee, Franklin D. Fuller, Alexander Batyuk, Roberto Alonso-Mori, James M. Holton, Daniel W. Paley, Nigel W. Moriarty, Fikret Mamedov, Paul D. Adams, Aaron S. Brewster, Holger Dobbek, Nicholas K. Sauter, Uwe Bergmann, Athina Zouni, Johannes Messinger, Jan Kern, Junko Yano, Vittal K. Yachandra
Summary: In natural photosynthesis, the light-driven splitting of water is the first step in converting solar energy into chemical energy. The reaction occurs in photosystem II, where the Mn4CaO5 cluster plays a crucial role in storing oxidizing equivalents and catalyzing the O-O bond formation. By using X-ray crystallography, we observed the structural changes during the S-3 ->[S-4]-> S-0 transition, which is the final step of the Kok's photosynthetic water oxidation cycle. Our findings provide insights into the complex events that occur during this process, including changes in the Mn4CaO5 cluster, ligands, water pathways, and proton release mechanism.
Article
Chemistry, Multidisciplinary
Bo Sun, Xingchu Zhao, Birui Xu, Erzheng Su, Andrey Kovalevsky, Qirong Shen, Dongyang Liu, Qun Wan
Summary: In this study, a hyperthermophilic chitinase (ActChi) was discovered, which can directly hydrolyze crystalline chitin at its optimal temperature of 80°C. By introducing a heterogeneous and hyperthermophilic chitin binding domain (ChBD) at the N-terminus of CDchi, the activity and thermostability of the enzyme were significantly enhanced. This strategy provides a good example of green sustainable degradation for crystalline biopolymers in nature.
ACS SUSTAINABLE CHEMISTRY & ENGINEERING
(2023)
Article
Biochemical Research Methods
Thomas C. Terwilliger, Pavel Afonine, Dorothee Liebschner, Tristan Croll, Airlie J. McCoy, Robert D. Oeffner, Christopher J. Williams, Billy K. Poon, Jane S. Richardson, Randy J. Read, Paul D. Adams
Summary: Experimental structure determination can be accelerated with AI-based structure-prediction methods like AlphaFold, which can produce electron-density maps and structural models using sequence information and crystallographic data. By iterating through cycles of structure prediction, a predicted model can be used as a template for prediction in subsequent cycles. This study applied the procedure to X-ray data for 215 structures and achieved a high accuracy, with 87% of the models matching those in the database. It is concluded that AlphaFold predictions based on sequence information alone are usually accurate enough for solving the crystallographic phase problem, and a general strategy for structure determination that includes AI-based prediction is suggested.
ACTA CRYSTALLOGRAPHICA SECTION D-STRUCTURAL BIOLOGY
(2023)
Correction
Biochemical Research Methods
P. V. Afonine, R. W. Grosse-Kunstleve, P. D. Adams, A. Urzhumtsev
Summary: Equations in Sections 2.3 and 2.4 of the article by Afonine et al. have been corrected.
ACTA CRYSTALLOGRAPHICA SECTION D-STRUCTURAL BIOLOGY
(2023)
Article
Biotechnology & Applied Microbiology
Shilva Shrestha, Deepika Awasthi, Yan Chen, Jennifer Gin, Christopher J. Petzold, Paul D. Adams, Blake A. Simmons, Steven W. Singer
Summary: This study investigated the carbon catabolite repression (CCR) functioning in P. putida M2, a strain capable of consuming sugars and aromatic compounds. The results showed that incomplete substrate consumption occurred during cultivation with a mixture of glucose and aromatic compounds, while transient intermediate accumulation and complete aromatic consumption were observed during xylose-aromatic consumption. Proteomics analysis revealed stronger repression of aromatic catabolic proteins by glucose than xylose. Additionally, CRISPRi-based gene repression experiments showed that reduction of crc expression led to faster growth and improved glucose and p-coumarate uptake. Small RNA sequencing results indicated lower levels of CrcY and CrcZ homologues under strong CCR conditions.
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
(2023)
Article
Biochemistry & Molecular Biology
Taichi E. Takasuka, Hoon Kim, Kai Deng, Christopher M. Bianchetti, Kaho Yamashita, Emily T. Beebe, Lai F. Bergeman, Kirk A. Vander Meulen, Samuel Deutsch, John Ralph, Paul D. Adams, Trent R. Northen, Brian G. Fox
Summary: This study demonstrates that a combination of a broad-specificity β-1,3-exoglucanase and a polysaccharide lyase from family 18 can efficiently hydrolyze untreated kelp into soluble sugars, such as glucose, gentiobiose, mannitol-end glucose, and mannuronic and guluronic acids and their soluble oligomers. The data suggest that enzyme combinations targeted to the unique polysaccharide composition of marine biomass are sufficient to deconstruct kelp into soluble sugars for microbial fermentation.
Article
Biochemical Research Methods
Pavel V. Afonine, Oleg V. Sobolev, Nigel W. Moriarty, Thomas C. Terwilliger, Paul D. Adams
Summary: Refining atomic models at low resolution is challenging due to lack of detailed experimental data. Using restraints on Ramachandran plots or residue side-chain rotameric states as refinement targets is not ideal. Therefore, finding alternative model-validation criteria is desirable.
ACTA CRYSTALLOGRAPHICA SECTION D-STRUCTURAL BIOLOGY
(2023)
Article
Chemistry, Multidisciplinary
Victoria N. N. Drago, Claudia Campos, Mattea Hooper, Aliyah Collins, Oksana Gerlits, Kevin L. L. Weiss, Matthew P. P. Blakeley, Robert S. S. Phillips, Andrey Kovalevsky
Summary: The authors determined the structure of Thermus thermophilus SHMT using X-ray/neutron crystallography and proposed its catalytic mechanism. This is important for drug design and enzyme engineering.
COMMUNICATIONS CHEMISTRY
(2023)
Article
Biochemical Research Methods
Noel S. Ha, Jenny R. Onley, Kai Deng, Peter Andeer, Benjamin P. Bowen, Kshitiz Gupta, Peter W. Kim, Nathaniel Kuch, Mark Kutschke, Alex Parker, Fangchao Song, Brian Fox, Paul D. Adams, Markus de Raad, Trent R. Northen
Summary: Mass spectrometry combined with microfluidics in the Drop-NIMS platform enables rapid screening of enzymatic reactions and identification of different combinations of substrates and enzymes for various applications.
Article
Plant Sciences
Pradeep Kumar Prabhakar, Jose Henrique Pereira, Rahil Taujale, Wanchen Shao, Vivek S. Bharadwaj, Digantkumar Chapla, Jeong-Yeh Yang, Yannick J. Bomble, Kelley W. Moremen, Natarajan Kannan, Michal Hammel, Paul D. Adams, Henrik V. Scheller, Breeanna R. Urbanowicz
Summary: The researchers describe the structure of GalS1 from Populus trichocarpa, which consists of an N-terminal domain and a C-terminal catalytic domain. They propose a mechanism for GalS1 catalysis and a new model for pectin biosynthesis based on deep evolutionary analysis, molecular simulations, and biochemical studies.
