Unnaturally Distorted Hexagonal Protein Ring Alternatingly Reorganized from Two Distinct Chemically Modified Proteins.

In this study, we constructed a semiartificial protein assembly of alternating ring type, which was modified from the natural assembly state via incorporation of a synthetic component at the protein interface. For the redesign of a natural protein assembly, a scrap-and-build approach employing chemical modification was used. Two different protein dimer units were designed based on peroxiredoxin from Thermococcus kodakaraensis, which originally forms a dodecameric hexagonal ring with six homodimers. The two dimeric mutants were reorganized into a ring by reconstructing the protein-protein interactions via synthetic naphthalene moieties introduced by chemical modification. Cryo-electron microscopy revealed the formation of a uniquely shaped dodecameric hexagonal protein ring with broken symmetry, distorted from the regular hexagon of the wild-type protein. The artificially installed naphthalene moieties were arranged at the interfaces of dimer units, forming two distinct protein-protein interactions, one of which is highly unnatural. This study deciphered the potential of the chemical modification technique that constructs semiartificial protein structures and assembly hardly accessible by conventional amino acid mutations.

Reducing substrate inhibition of malate dehydrogenase from Geobacillus stearothermophilus by C-terminal truncation.

Malate dehydrogenase (MDH) catalyzes the reduction of oxaloacetate to L-malate. Geobacillus stearothermophilus MDH (gs-MDH) is used as a diagnostic reagent; however, gs-MDH is robustly inhibited at high substrate concentrations, which limits its reaction rate. Here, we reduced substrate inhibition of gs-MDH by deleting its C-terminal residues. Computational analysis showed that C-terminal residues regulate the position of the active site loop. C-terminal deletions of gs-MDH successfully increased Ki values by 5- to 8-fold with maintained thermal stability (>90% of the wild-type enzyme), although kcat/Km values were decreased by <2-fold. The structure of the mutant showed a shift in the location of the active site loop and a decrease in its volume, suggesting that substrate inhibition was reduced by eliminating the putative substrate binding site causing inhibition. Our results provide an effective method to reduce substrate inhibition of the enzyme without loss of other parameters, including binding and stability constants.

Functional analysis of the N-terminal region of acetylxylan esterase from Caldanaerobacter subterraneus subsp. tengcongensis.

Acetylxylan esterase from Caldanaerobacter subterraneus subsp. tengcongensis (TTE0866) has an N-terminal region (NTR; residues 23-135) between the signal sequence (residues 1-22) and the catalytic domain (residues 136-324), which is of unknown function. Our previous study revealed the crystal structure of the wild-type (WT) enzyme containing the NTR and the catalytic domain. Although the structure of the catalytic domain was successfully determined, that of the NTR was undetermined, as its electron density was unclear. In this study, we investigated the role of the NTR through functional and structural analyses of NTR truncation mutants. Based on sequence and secondary structure analyses, NTR was confirmed to be an intrinsically disordered region. The truncation of NTR significantly decreased the solubility of the proteins at low salt concentrations compared with that of the WT. The NTR-truncated mutant easily crystallized in a conventional buffer solution. The crystal exhibited crystallographic properties comparable with those of the WT crystals suitable for structural determination. These results suggest that NTR plays a role in maintaining the solubility and inhibiting the crystallization of the catalytic domain.

Increasing loop flexibility affords low-temperature adaptation of a moderate thermophilic malate dehydrogenase from Geobacillus stearothermophilus.

Malate dehydrogenase (MDH) catalyzes the reversible reduction of nicotinamide adenine dinucleotide from oxaloacetate to L-malate. MDH from moderate thermophilic Geobacillus stearothermophilus (gs-MDH) has high thermal stability and substrate specificity and is used as a diagnostic reagent. In this study, gs-MDH was engineered to increase its catalytic activity at low temperatures. Based on sequential and structural comparison with lactate dehydrogenase from G. stearothermophilus, we selected G218 as a mutation site to increase the loop flexibility pivotal for MDH catalysis. The G218 mutants showed significantly higher specific activities than the wild type at low temperatures and maintained thermal stability. The crystal structure of the G218Y mutant, which had the highest catalytic efficiency among all the G218 mutants, suggested that the flexibility of the mobile loop was successfully increased by the bulky side chain. Therefore, this study demonstrated the low-temperature adaptation of MDH by facilitating conformational changes during catalysis.

Crystal structure of acetylxylan esterase from Caldanaerobacter subterraneus subsp. tengcongensis.

The acetylxylan esterases (AXEs) classified into carbohydrate esterase family 4 (CE4) are metalloenzymes that catalyze the deacetylation of acetylated carbohydrates. AXE from Caldanaerobacter subterraneus subsp. tengcongensis (TTE0866), which belongs to CE4, is composed of three parts: a signal sequence (residues 1-22), an N-terminal region (NTR; residues 23-135) and a catalytic domain (residues 136-324). TTE0866 catalyzes the deacetylation of highly substituted cellulose acetate and is expected to be useful for industrial applications in the reuse of resources. In this study, the crystal structure of TTE0866 (residues 23-324) was successfully determined. The crystal diffracted to 1.9 Šresolution and belonged to space group I212121. The catalytic domain (residues 136-321) exhibited a (ß/α)7-barrel topology. However, electron density was not observed for the NTR (residues 23-135). The crystal packing revealed the presence of an intermolecular space without observable electron density, indicating that the NTR occupies this space without a defined conformation or was truncated during the crystallization process. Although the active-site conformation of TTE0866 was found to be highly similar to those of other CE4 enzymes, the orientation of its Trp264 side chain near the active site was clearly distinct. The unique orientation of the Trp264 side chain formed a different-shaped cavity within TTE0866, which may contribute to its reactivity towards highly substituted cellulose acetate.

Structural analysis and reaction mechanism of malate dehydrogenase from Geobacillus stearothermophilus.

Malate dehydrogenase (MDH) catalyzes the reversible reduction of oxaloacetate (OAA) to L-malate using nicotinamide adenine dinucleotide hydrogen. MDH has two characteristic loops, the mobile loop and the catalytic loop, in the active site. On binding to the substrate, the enzyme undergoes a structural change from the open-form, with an open conformation of the mobile loop, to the closed-form, with the loop in a closed conformation. In this study, three crystals of MDH from a moderate thermophile, Geobacillus stearothermophilus (gs-MDH) were used to determine four different enzyme structures (resolutions, 1.95-2.20 Å), each of which was correspondingly assigned to its four catalytic states. Two OAA-unbound structures exhibited the open-form, while the other two OAA-bound structures exhibited both the open- and closed-form. The structural analysis suggested that the binding of OAA to the open-form gs-MDH promotes conformational change in the mobile loop and simultaneously activates the catalytic loop. The mutations on the key amino acid residues involving the proposed catalytic mechanism significantly affected the gs-MDH activity, supporting our hypothesis. These findings contribute to the elucidation of the detailed molecular mechanism underlying the substrate recognition and structural switching during the MDH catalytic cycle.

Rebuilding Ring-Type Assembly of Peroxiredoxin by Chemical Modification.

Direct control of the protein quaternary structure (QS) is challenging owing to the complexity of the protein structure. As a protein with a characteristic QS, peroxiredoxin from Aeropyrum pernix K1 (ApPrx) forms a decamer, wherein five dimers associate to form a ring. Here, we disrupted and reconstituted ApPrx QS via amino acid mutations and chemical modifications targeting hot spots for protein assembly. The decameric QS of an ApPrx* mutant, wherein all cysteine residues in wild-type ApPrx were mutated to serine, was destructed to dimers via an F80C mutation. The dimeric ApPrx*F80C mutant was then modified with a small molecule and successfully assembled as a decamer. Structural analysis confirmed that an artificially installed chemical moiety potentially facilitates suitable protein-protein interactions to rebuild a native structure. Rebuilding of dodecamer was also achieved through an additional amino acid mutation. This study describes a facile method to regulate the protein assembly state.

Artificial Metalloenzymes: From Selective Chemical Transformations to Biochemical Applications.

Artificial metalloenzymes (ArMs) comprise a synthetic metal complex in a protein scaffold. ArMs display performances combining those of both homogeneous catalysts and biocatalysts. Specifically, ArMs selectively catalyze non-natural reactions and reactions inspired by nature in water under mild conditions. In the past few years, the construction of ArMs that possess a genetically incorporated unnatural amino acid and the directed evolution of ArMs have become of great interest in the field. Additionally, biochemical applications of ArMs have steadily increased, owing to the fact that compartmentalization within a protein scaffold allows the synthetic metal complex to remain functional in a sea of inactivating biomolecules. In this review, we present updates on: 1) the newly reported ArMs, according to their type of reaction, and 2) the unique biochemical applications of ArMs, including chemoenzymatic cascades and intracellular/in vivo catalysis. We believe that ArMs have great potential as catalysts for organic synthesis and as chemical biology tools for pharmaceutical applications.

Disassembly of the ring-type decameric structure of peroxiredoxin from Aeropyrum pernix K1 by amino acid mutation.

The quaternary structure of peroxiredoxin from Aeropyrum pernix K1 (ApPrx) is a decamer, in which five homodimers are assembled in a pentagonal ring through hydrophobic interactions. In this study, we determined the amino acid (AA) residues of ApPrx crucial for forming the decamer using AA mutations. The ApPrx0Cys mutant, wherein all cysteine residues were mutated to serine, was prepared as a model protein to remove the influence of the redox states of the cysteines on its assembling behavior. The boundary between each homodimer of ApPrx0Cys contains characteristic aromatic AA residues forming hydrophobic interactions: F46, F80, W88, W210, and W211. We found that a single mutation of F46, F80, or W210 to alanine completely disassembled the ApPrx0Cys decamer to homodimers, which was clarified by gel-filtration chromatography and dynamic light scattering measurements. F46A, F80A, and W210A mutants lacked only one aromatic ring compared with ApPrx0Cys, indicating that the assembly is very sensitive to the surface structure of the protein. X-ray structures revealed two mechanisms of disassembly of the ApPrx decamer: loss of hydrophobicity between homodimers and flip of the arm domain. The AA residues targeted in this study are well conserved in ring-type Prx proteins, suggesting the importance of these residues in the assembly. This study demonstrates the sensitivity and modifiability of peroxiredoxin assembly by a simple AA mutation.

Cooperative Catalysis of an Alcohol Dehydrogenase and Rhodium-Modified Periodic Mesoporous Organosilica.

The combined use of a metal-complex catalyst and an enzyme is attractive, but typically results in mutual inactivation. A rhodium (Rh) complex immobilized in a bipyridine-based periodic mesoporous organosilica (BPy-PMO) shows high catalytic activity during transfer hydrogenation, even in the presence of bovine serum albumin (BSA), while a homogeneous Rh complex exhibits reduced activity due to direct interaction with BSA. The use of a smaller protein or an amino acid revealed a clear size-sieving effect of the BPy-PMO that protected the Rh catalyst from direct interactions. A combination of Rh-immobilized BPy-PMO and an enzyme (horse liver alcohol dehydrogenase; HLADH) promoted sequential reactions involving the transfer hydrogenation of NAD+ to give NADH followed by the asymmetric hydrogenation of 4-phenyl-2-butanone with high enantioselectivity. The use of BPy-PMO as a support for metal complexes could be applied to other systems consisting of a metal-complex catalyst and an enzyme.

Distinct molecular assembly of homologous peroxiredoxins from Pyrococcus horikoshii and Thermococcus kodakaraensis.

Peroxiredoxins from Pyrococcus horikoshii (PhPrx) and Thermococcus kodakaraensis (TkPrx) are highly homologous proteins sharing 196 of the 216 residues. We previously reported a pentagonal ring-type decameric structure of PhPrx. Here, we present the crystal structure of TkPrx. Despite their homology, unlike PhPrx, the quaternary structure of TkPrx was found to be a dodecamer comprised of six homodimers arranged in a hexagonal ring-type assembly. The possibility of the redox-dependent conversion of the molecular assembly, which had been observed in PhPrx, was excluded for TkPrx based on the crystal structure of a mutant in which all of the cysteine residues were substituted with serine. The monomer structures of the dodecameric TkPrx and decameric PhPrx coincided well, but there was a slight difference in the relative orientation of the two domains. Molecular assembly of PhPrx and TkPrx in solution evaluated by gel-filtration chromatography was consistent with the crystallographic results. For both PhPrx and TkPrx, the gel-filtration elution volume slightly increased with a decrease in the protein concentration, suggesting the existence of an equilibrium state between the decameric/dodecameric ring and lower-order assembly. This structural assembly difference between highly homologous Prxs suggests a significant influence of quaternary structure on function, worthy of further exploration.

A Pyrene-Linked Cavity within a ß-Barrel Protein Promotes an Asymmetric Diels-Alder Reaction.

A unique π-expanded reaction cavity tethering a polycyclic moiety which provides a platform for substrate binding was constructed within the robust ß-barrel structure of nitrobindin (NB). NB variants with cavities of different sizes and shapes are coupled with N-(1-pyrenyl)maleimide (Pyr) to prepare a series of NB-Pyr conjugates. The orientation of the pyrene moiety is fixed within the cavity by the coupling reaction. The fluorescent quenching analysis of NB-Pyr indicates that azachalcone (aza), which is a dienophile for a Diels-Alder (DA) reaction, is efficiently incorporated within the pyrene-linked reaction cavity by the aromatic interaction. The DA reaction between aza and cyclopentadiene proceeds within the reaction cavity of NB-Pyr in the presence of CuII ion in high yield and high enantio- and regioselectivity.

Artificial Diels-Alderase based on the transmembrane protein FhuA.

Copper(I) and copper(II) complexes were covalently linked to an engineered variant of the transmembrane protein Ferric hydroxamate uptake protein component A (FhuA ΔCVF(tev)). Copper(I) was incorporated using an N-heterocyclic carbene (NHC) ligand equipped with a maleimide group on the side arm at the imidazole nitrogen. Copper(II) was attached by coordination to a terpyridyl ligand. The spacer length was varied in the back of the ligand framework. These biohybrid catalysts were shown to be active in the Diels-Alder reaction of a chalcone derivative with cyclopentadiene to preferentially give the endo product.

Construction of a hybrid biocatalyst containing a covalently-linked terpyridine metal complex within a cavity of aponitrobindin.

A hybrid biocatalyst containing a metal terpyridine (tpy) complex within a rigid ß-barrel protein nitrobindin (NB) is constructed. A tpy ligand with a maleimide group, N-[2-([2,2':6',2''-terpyridin]-4'-yloxy)ethyl]maleimide (1), was covalently linked to Cys96 inside the cavity of NB to prepare a conjugate NB-1. Binding of Cu(2+), Zn(2+), or Co(2+) ion to the tpy ligand in NB-1 was confirmed by UV-vis spectroscopy and ESI-TOF MS measurements. Cu(2+)-bound NB-1 is found to catalyze a Diels-Alder reaction between azachalcone and cyclopentadiene in 22% yield, which is higher than that of the Cu(2+)-tpy complex without the NB matrix. The results suggest that the hydrophobic cavity close to the copper active site within the NB scaffold supports the binding of the two substrates, dienophile and diene, to promote the reaction.

Photochemical properties of a myoglobin-CdTe quantum dot conjugate.

A myoglobin-cadmium telluride quantum dot conjugate was constructed using an artificial heme modified with a thiol moiety as a linker. Irradiation of the myoglobin-cadmium telluride conjugate generated the photoreduced ferrous myoglobin via an electron transfer from the photoexcited quantum dot, leading to the formation of CO-bound myoglobin under a CO atmosphere.