Biology subjects

Lo, Y. K.

Publications and source records attributed to Lo, Y. K..

5 recordsLinked to original sources

bioRxiv2026

Structural Basis of Redox-Coupled CO2 hydration by the Cyanobacterial NDH-1MS' complex

The NAD(P)H dehydrogenase-like complex, NDH-1MS, is a constitutively expressed CO2-concentrating mechanism critical for sustaining CO2 fixation in cyanobacteria. This complex has been proposed to function as a vectorial carbonic anhydrase and accumulates intracellular bicarbonate against chemical equilibrium by coupling CO2 hydration to the photosynthetic cyclic electron flow. Using cryo-electron microscopy, we determined the structure of NDH-1MS from Thermosynechococcus vestitus. Contrary to its inducible counterpart NDH-1MS, our data suggest that a candidate CO2 hydration site may reside in the carbon-concentrating subunit CupB instead of the CupB-NdhF4 interface previously proposed. The active-site region partially resembles metal ion-independent iota-carbonic anhydrases and may bind CO2 or bicarbonate. We further observe features consistent with a Grotthuss-type proton transfer network connecting the active-site region to the antiporter-like subunits NdhF4 and NdhD4; however, unlike complex I, the proton export channel in NdhF4 appears to be blocked by bulky hydrophobic amino acids. Structural comparison between the oxygenic photosynthesis-specific subunit NdhV-bound state and dissociated state further reveals a correlation between plastoquinone stability and NdhV association. Taken together, we propose a revised working model in which NDH-1MS may function as a metal ion-independent vectorial carbonic anhydrase, with CO2 hydration coupled to proton transfer events in the antiporter-like subunits across the thylakoid membrane. SignificancePhotosynthetic complex I-like CO2-uptake systems are central to cyanobacterial carbon fixation, yet the molecular basis for coupling CO2 hydration to electron transfer has remained unresolved. Here, structures of NDH-1MS in NdhV-bound and NdhV-free states revise the architectural framework for catalysis and coupling in the constitutive CO2-uptake module. The data identify a CupB-centered candidate active-site region, reveal a non-canonical terminal antiporter-like subunit with an apparently blocked P-side exit, and link NdhV association to altered plastoquinone stability. Together, these findings refine current models for vectorial carbonic anhydrase activity in cyanobacterial NDH-1 and provide a structural basis for understanding how photosynthetic redox chemistry may be coupled to inorganic carbon concentration.

plant biology↗

bioRxiv2026

Structural Basis of Membrane Potential Coupled Vectorial CO2 Hydration by the DAB2 Complex in Chemolithoautotrophs

The fixation of dissolved inorganic carbon (DIC) such as CO2 and bicarbonate by autotrophic microorganisms is fundamental to the global primary production. Many autotrophs depend on a diversity of CO2-concentrating mechanisms (CCMs) to overcome the inefficiency of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and the limited supply of DIC. While cyanobacterial CCMs are well characterized, analogous systems in chemolithoautotrophs, specifically active DIC uptake systems have long been overlooked. Here, we present the first cryo-EM structural analysis of DAB2, an essential membrane-associated protein complex for CO2 uptake in Halothiobacillus neapolitanus. The cytoplasmic subunit DabA2 displays a {beta}-carbonic anhydrase-like fold including a zinc ion, while the transmembrane subunit DabB2 resembles the proton-conducting subunits of respiratory Complex I. Purified DAB2 binds CO2 independent of proton motive force (PMF) however, did not spontaneously hydrate CO2. This suggests that CO2 hydration is PMF-dependent and may involve a gating mechanism. Structural analysis reveals an unconventional deeply buried active site only accessible via gated substrate tunnels, implying that substrate access, product release, and catalytic activation are tightly regulated. A unique transmembrane helix of DabA2 constitutes part of the proton conduction pathway and potentially couples proton translocation to enzymatic turnover. These features define a vectorial CO2 hydration mechanism that prohibits reverse bicarbonate dehydration and requires a proton gradient to initiate catalytic turnover. Our findings establish DAB2 as a prototype of a previously unrecognized family of PMF-driven carbonic anhydrases, elucidating a novel strategy for CO2 capture in non-photosynthetic autotrophs and expanding the mechanistic landscape of bacterial CCMs.

biochemistry↗

bioRxiv2025

Structural insights into late-stage photosystem II assembly by Psb32

Photosystem II (PSII) assembly is a stepwise process in which intermediate complexes with auxiliary proteins are transiently formed to allow efficient de novo biogenesis or repair of damaged PSII. In particular, the role of extrinsic PSII subunits (PsbO, PsbU, PsbV) and auxiliary proteins such as Psb27 for the formation and photoactivation of the Mn4O5Ca cluster, which catalyzes the unique water splitting reaction in mature PSII, remains unclear. Using cryo-electron microscopy, we have determined the structure of two novel late-stage PSII assembly intermediates from Thermosynechococcus vestitus BP-1. In contrast to previous studies, the resulting monomeric PSII complexes contain both PsbJ and Psb27 and exhibit a fully mature acceptor side, while the oxygen evolving complex (OEC) is still in an immature state. The second complex additionally associates with the late-acting assembly factor Psb32 and the extrinsic subunit PsbV. While Psb32 has received little attention, its proposed role in the complex challenges the previous assumption that all extrinsic subunits associate spontaneously, as well as the notion that PsbO initiates binding and solely drives OEC formation. Our structures of the Psb27-PSII and Psb32-PSII intermediates provide novel insights, how structural changes of C-termini of the D1 and D2 core proteins regulate maturation of the OEC and how the catalytic side is prepared for binding of the Mn4O5Ca cluster. The Psb32-PSII complex potentially represents the final PSII assembly intermediate that precedes the incorporation and photoactivation of the Mn4O5Ca cluster, allowing us to explain the final steps in the PSII biogenesis and assembly pipeline in great detail, as only the two extrinsic subunits PsbO and PsbU are missing.

plant biology↗

bioRxiv2024

Frequent transitions in self-assembly across the evolution of a central metabolic enzyme

Many enzymes assemble into homomeric protein complexes comprising multiple copies of one protein. Because structural form is usually assumed to follow function in biochemistry, these assemblies are thought to evolve because they provide some functional advantage. In many cases, however, no specific advantage is known and, in some cases, quaternary structure varies among orthologs. This has led to the proposition that self-assembly may instead vary neutrally within protein families. The extent of such variation has been difficult to ascertain because quaternary structure has until recently been difficult to measure on large scales. Here, we employ mass photometry, phylogenetics, and structural biology to interrogate the evolution of homo-oligomeric assembly across the entire phylogeny of prokaryotic citrate synthases - an enzyme with a highly conserved function. We discover a menagerie of different assembly types that come and go over the course of evolution, including cases of parallel evolution and reversions from complex to simple assemblies. Functional experiments in vitro and in vivo indicate that evolutionary transitions between different assemblies do not strongly influence enzyme catalysis. Our work suggests that enzymes can wander relatively freely through a large space of possible assemblies and demonstrates the power of characterizing structure-function relationships across entire phylogenies.

evolutionary biology↗

bioRxiv2018

A multidrug resistant clinical P. aeruginosa isolate in the MLST550 clonal complex: uncoupled quorum sensing modulates the interplay of virulence and resistance

Pseudomonas aeruginosa is a prevalent and pernicious pathogen equipped with both extraordinary capabilities to infect the host and to develop antimicrobials resistance (AMR). Monitoring the emergence of AMR high risk clones and understanding the interplay of their pathogenicity and antibiotic resistance is of paramount importance to avoid resistance dissemination and to control P. aeruginosa infections. In this study, we report the identification of a multidrug resistant (MDR) P. aeruginosa strain PA154197 isolated from a blood stream infection in Hong Kong. PA154197 belongs to a distinctive MLST550 clonal complex shared by two international P. aeruginosa isolates VW0289 and AUS544. Comparative genome and transcriptome analysis with the reference strain PAO1 led to the identification of a variety of genetic variations in antibiotic resistance genes and the hyper-expression of three multidrug efflux pumps MexAB-OprM, MexEF-OprN, and MexGHI-OpmD in PA154197. Unlike many resistant isolates displaying an attenuated virulence, PA154197 produces a significantly high level of the P. aeruginosa major virulence factor pyocyanin (PYO) and displays an uncompromised virulence compared to PAO1. Further analysis revealed that the secondary quorum sensing system Pqs which primarily controls the PYO production is hyper-active in PA154197 independent of the master QS systems Las and Rhl. Together, these investigations disclose a unique, uncoupled QS mediated pathoadaptation mechanism in clinical P. aeruginosa which may account for the high pathogenic potentials and antibiotics resistance in the MDR isolate PA154197.

microbiology↗