Shedding light on silica biomineralization by comparative analysis of the silica-associated proteomes from three diatom species.

Morphogenesis of the intricate patterns of diatom silica cell walls is a protein-guided process, yet to date only very few such silica biomineralization proteins have been identified. Therefore, it is currently unknown whether all diatoms share conserved proteins of a basal silica forming machinery, and whether unique proteins are responsible for the morphogenesis of species-specific silica patterns. To answer these questions, we extracted proteins from the silica of three diatom species (Thalassiosira pseudonana, Thalassiosira oceanica, and Cyclotella cryptica) by complete demineralization of the cell walls. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis of the extracts identified 92 proteins that we name 'soluble silicome proteins' (SSPs). Surprisingly, no SSPs are common to all three species, and most SSPs showed very low similarity to one another in sequence alignments. In-depth bioinformatics analyses revealed that SSPs could be grouped into distinct classes based on short unconventional sequence motifs whose functions are yet unknown. The results from the in vivo localization of selected SSPs indicates that proteins, which lack sequence homology but share unconventional sequence motifs may exert similar functions in the morphogenesis of the diatom silica cell wall.

Natural hybrid silica/protein superstructure at atomic resolution.

Formation of highly symmetric skeletal elements in demosponges, called spicules, follows a unique biomineralization mechanism in which polycondensation of an inherently disordered amorphous silica is guided by a highly ordered proteinaceous scaffold, the axial filament. The enzymatically active proteins, silicateins, are assembled into a slender hybrid silica/protein crystalline superstructure that directs the morphogenesis of the spicules. Furthermore, silicateins are known to catalyze the formation of a large variety of other technologically relevant organic and inorganic materials. However, despite the biological and biotechnological importance of this macromolecule, its tertiary structure was never determined. Here we report the atomic structure of silicatein and the entire mineral/organic hybrid assembly with a resolution of 2.4 Å. In this work, the serial X-ray crystallography method was successfully adopted to probe the 2-µm-thick filaments in situ, being embedded inside the skeletal elements. In combination with imaging and chemical analysis using high-resolution transmission electron microscopy, we provide detailed information on the enzymatic activity of silicatein, its crystallization, and the emergence of a functional three-dimensional silica/protein superstructure in vivo. Ultimately, we describe a naturally occurring mineral/protein crystalline assembly at atomic resolution.

Control of biosilica morphology and mechanical performance by the conserved diatom gene Silicanin-1.

The species-specifically patterned biosilica cell walls of diatoms are paradigms for biological mineral morphogenesis and the evolution of lightweight materials with exceptional mechanical performance. Biosilica formation is a membrane-mediated process that occurs in intracellular compartments, termed silica deposition vesicles (SDVs). Silicanin-1 (Sin1) is a highly conserved protein of the SDV membrane, but its role in biosilica formation has remained elusive. Here we generate Sin1 knockout mutants of the diatom Thalassiosira pseudonana. Although the mutants grow normally, they exhibit reduced biosilica content and morphological aberrations, which drastically compromise the strength and stiffness of their cell walls. These results identify Sin1 as essential for the biogenesis of mechanically robust diatom cell walls, thus providing an explanation for the conservation of this gene throughout the diatom realm. This insight paves the way for genetic engineering of silica architectures with desired structures and mechanical performance.

Convergence of theory and experiment on the role of preorganization, quantum tunneling and enzyme motions into flavoenzyme-catalyzed hydride transfer.

Hydride transfer is one of the most common reactions catalyzed by enzymatic systems and it has become an object of study due to possible significant quantum tunneling effects. In the present work, we provide a combination of theoretical QM/MM simulations and experimental measurements of the rate constants and kinetic isotopic effects (KIEs) for the hydride transfer reaction catalyzed by morphinone reductase, MR. Quantum mechanical tunneling coefficients, computed in the framework of variational transition-state theory, play a significant role in this reaction, reaching values of 23.8 ± 5.5 for the lightest isotopologue; one of the largest values reported for enzymatic systems. This prediction is supported by the agreement between the theoretically predicted rate constants and the corresponding experimental values. Simulations indicate that the role of protein motions can be satisfactorily described as equilibrium fluctuations along the reaction coordinate, in line with a high degree of preorganization displayed by this enzyme.

Untangling Heavy Protein and Cofactor Isotope Effects on Enzyme-Catalyzed Hydride Transfer.

"Heavy" (isotopically labeled) enzyme isotope effects offer a direct experimental probe of the role of protein vibrations on enzyme-catalyzed reactions. Here we have developed a strategy to generate isotopologues of the flavoenzyme pentaerythritol tetranitrate reductase (PETNR) where the protein and/or intrinsic flavin mononucleotide (FMN) cofactor are isotopically labeled with 2H, 15N, and 13C. Both the protein and cofactor contribute to the enzyme isotope effect on the reductive hydride transfer reaction, but their contributions are not additive and may partially cancel each other out. However, the isotope effect specifically arising from the FMN suggests that vibrations local to the active site play a role in the hydride transfer chemistry, while the protein-only "heavy enzyme" effect demonstrates that protein vibrations contribute to catalysis in PETNR. In all cases, enthalpy-entropy compensation plays a major role in minimizing the magnitude of "heavy enzyme" isotope effects. Fluorescence lifetime measurements of the intrinsic flavin mononucleotide show marked differences between "light" and "heavy" enzymes on the nanosecond-picosecond time scale, suggesting relevant time scale(s) for those vibrations implicated in the "heavy enzyme" isotope effect on the PETNR reaction.