Engineering topics

Wakatsuki, Soichi

Publications and source records attributed to Wakatsuki, Soichi.

5 records

DOE OSTI2025

Deciphering the altered conformational states of bifunctional thaumarchaeal crotonyl-CoA hydratase and 3-hydroxypropionyl-CoA dehydratase from Nitrosopumilus maritimus

Abstract The thaumarchaeal 3-hydroxypropionate/4-hydroxybutyrate (3HP/4HB) cycle represents one of the most efficient mechanisms for CO2 fixation discovered to date. Within this cycle, the enzyme encoded by Nmar_1308 from Nitrosopumilus maritimus SCM1 plays a crucial role due to its dual functionality as both a crotonyl-CoA hydratase (CCAH) and a 3-hydroxypropionyl-CoA dehydratase (3HPD). Although the importance of a bifunctional enzyme for lowering the cost of biosynthesis, the details of structural dynamics are still missing. Here, in addition to our cryogenic temperature structures, we determined the first ambient temperature structures of the Nmar_1308 protein by Serial Femtosecond X-ray Crystallography (SFX). The determined structures capture previously unobserved conformational dynamics of the Nmar_1308 protein, providing invaluable information for future synthetic biology applications.

Destan, Ebru (ORCID:0000000231290827)↗

DOE OSTI2024

Crystal structure of the 4-hydroxybutyryl-CoA synthetase (ADP-forming) from nitrosopumilus maritimus

The 3-hydroxypropionate/4-hydroxybutyrate (3HP/4HB) cycle from ammonia-oxidizing Thaumarchaeota is currently considered the most energy-efficient aerobic carbon fixation pathway. The Nitrosopumilus maritimus 4-hydroxybutyryl-CoA synthetase (ADP-forming; Nmar_0206) represents one of several enzymes from this cycle that exhibit increased efficiency over crenarchaeal counterparts. This enzyme reduces energy requirements on the cell, reflecting thaumarchaeal success in adapting to low-nutrient environments. Here we show the structure of Nmar_0206 from Nitrosopumilus maritimus SCM1, which reveals a highly conserved interdomain linker loop between the CoA-binding and ATP-grasp domains. Phylogenetic analysis suggests the widespread prevalence of this loop and highlights both its underrepresentation within the PDB and structural importance within the (ATP-forming) acyl-CoA synthetase (ACD) superfamily. This linker is shown to have a possible influence on conserved interface interactions between domains, thereby influencing homodimer stability. These results provide a structural basis for the energy efficiency of this key enzyme in the modified 3HP/4HB cycle of Thaumarchaeota.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Time-resolved cryogenic electron tomography for the study of transient cellular processes

Cryogenic electron tomography (cryo-ET) is the highest resolution imaging technique applicable to the life sciences, enabling sub-nanometer visualization of specimens preserved in their near native states. The rapid plunge freezing process used to prepare samples lends itself to time-resolved studies, which researchers have pursued for in vitro samples for decades. Here, we focus on developing a freezing apparatus for time-resolved studies in situ. The device mixes cellular samples with solution-phase stimulants before spraying them directly onto an electron microscopy grid that is transiting into cryogenic liquid ethane. By varying the flow rates of cell and stimulant solutions within the device, we can control the reaction time from tens of milliseconds to over a second prior to freezing. In a proof-of-principle demonstration, the freezing method is applied to a model bacterium, Caulobacter crescentus, mixed with an acidic buffer. Through cryo-ET we resolved structural changes throughout the cell, including surface-layer protein dissolution, outer membrane deformation, and cytosolic rearrangement, all within 1.5 seconds of reaction time. This new approach, Time-Resolved cryo-ET (TR-cryo-ET), enhances the capabilities of cryo-ET by incorporating a sub-second temporal axis and enables the visualization of induced structural changes at the molecular, organelle, or cellular level.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Structural and biophysical analysis of a Haemophilus influenzae tripartite ATP-independent periplasmic (TRAP) transporter

Tripartite ATP-independent periplasmic (TRAP) transporters are secondary-active transporters that receive their substrates via a soluble-binding protein to move bioorganic acids across bacterial or archaeal cell membranes. Recent cryo-electron microscopy (cryo-EM) structures of TRAP transporters provide a broad framework to understand how they work, but the mechanistic details of transport are not yet defined. Here we report the cryo-EM structure of the Haemophilus influenzae N-acetylneuraminate TRAP transporter (HiSiaQM) at 2.99 Å resolution (extending to 2.2 Å at the core), revealing new features. The improved resolution (the previous HiSiaQM structure is 4.7 Å resolution) permits accurate assignment of two Na + sites and the architecture of the substrate-binding site, consistent with mutagenic and functional data. Moreover, rather than a monomer, the HiSiaQM structure is a homodimer. We observe lipids at the dimer interface, as well as a lipid trapped within the fusion that links the SiaQ and SiaM subunits. We show that the affinity (K D ) for the complex between the soluble HiSiaP protein and HiSiaQM is in the micromolar range and that a related SiaP can bind HiSiaQM. This work provides key data that enhances our understanding of the ‘elevator-with-an-operator’ mechanism of TRAP transporters.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2023

Genomes to Structure and Function Workshop Report 2022

The goal of the U.S. Department of Energy (DOE) Biological and Environmental Research (BER) Program is to achieve a predictive understanding of complex biological, earth, and environmental systems with the aim of advancing the nation’s energy and infrastructure security. (https://www.energy.gov/science/ ber/biological-and-environmental-research). To pursue this goal, collaborations among experts in diverse research areas that lead to multidisciplinary projects are indispensable. The roles of DOE’s User Facilities, which offer unique and powerful resources for such research projects, are evolving, and expectations for the facilities are increasing. To respond to Users’ needs, the Joint Genome Institute (JGI) and Environmental Molecular Sciences Laboratory (EMSL) initiated the Facilities Integrating Collaborations for User Science (FICUS) program in 2014. This collaboration has grown into a popular and successful program, advancing more than 100 multidisciplinary projects to date. Similarly, the new interFacility collaborations among the JGI, EMSL, and User resources for BER structural biology and imaging at the Basic Energy Science (BES) Program’s synchrotron and neutron facilities are becoming essential for cutting-edge transdisciplinary science. To further explore the need for the BER research community to combine genomic, functional, and structural approaches to advance their research, an organizing committee was formed to develop and jointly host a 3-part workshop. The committee’s members represented seven DOE National Laboratory User Facilities (Appendix 1 lists the members). The “Genomes to Structure and Function” virtual workshop (see Appendices 2–5) was composed of three sessions. The first session, titled “Molecular Structures” (October 27– 28, 2021), highlighted diverse integrative experimental and computational approaches correlating structural data with sequencing and functional information, as well as predicting protein structures to model complex biological systems. The second session, “Intracellular Organization, and Material Synthesis and Decomposition” (December 15–16, 2021), covered imaging methods for observing, quantifying, and manipulating biosystems. The third session, “Imaging the Rhizosphere and Cellular Organization” (January 26–27, 2022) emphasized advanced and non-invasive imaging techniques applied to plant root-microbe-soil interactions.

59 BASIC BIOLOGICAL SCIENCES↗