Naoki Shibata

Nylon hydrolase (NylC), a member of the N-terminal nucleophile (Ntn) hydrolase superfamily, is responsible for the degradation of various aliphatic nylons, including nylon-6 and nylon-66. NylC is initially expressed as an inactive precursor (36 kDa), but the precursor is autocatalytically cleaved at Asn266/Thr267 to generate an active enzyme composed of 27 and 9 kDa subunits. We isolated various mutants with amino acid changes at the catalytic centre. X-ray crystallographic analysis revealed that the NylC precursor forms a doughnut-shaped quaternary structure composed of four monomers (molecules A-D) with D2 symmetry. Catalytic residues in the precursor are covered by loop regions at the A/B interface (equivalent to the C/D interface). However, the catalytic residues are exposed to the solvent environment through autocleavage followed by movements of the loop regions. T267A, D306A and D308A mutations resulted in a complete loss of autocleavage. By contrast, in the T267S mutant, autocleavage proceeded slowly at a constant reaction rate (k = 2.8 × 10-5  s-1 ) until complete conversion, but the reaction was inhibited by K189A and N219A mutations. Based on the crystallographic and molecular dynamic simulation analyses, we concluded that the Asp308-Asp306-Thr267 triad, resembling the Glu-Ser-Ser triad conserved in Ntn-hydrolase family enzymes, is responsible for autocleavage and that hydrogen-bonding networks connecting Thr267 with Lys189 and Asn219 are required for increasing the nucleophilicity of Thr267-OH in both the water accessible and water inaccessible systems. Furthermore, we determined that NylC employs the Asp308-Asp306-Thr267 triad as catalytic residues for substrate hydrolysis, but the reaction requires Lys189 and Tyr146 as additional catalytic/substrate-binding residues specific for nylon hydrolysis.

Adenosylcobalamin (AdoCbl) or coenzyme B12 is a naturally occurring organometallic compound that serves as a cofactor for enzymes that catalyze intramolecular group-transfer reactions and ribonucleotide reduction in a wide variety of organisms from bacteria to animals. AdoCbl-dependent enzymes are radical enzymes and generate an adenosyl radical by homolysis of the coenzyme's cobalt-carbon (Co-C) bond for catalysis. How do the enzymes activate and cleave the Co-C bond to form the adenosyl radical? How do the enzymes utilize the high reactivity of adenosyl radical for catalysis by suppressing undesirable side reactions? Our recent structural studies aimed to solve these problems with diol dehydratase and ethanolamine ammonia-lyase established the crucial importance of steric strain of the Co-C bond and conformational stabilization of adenosyl radical for coenzyme B12 catalysis. We outline here our results obtained with these eliminating isomerases and compare them with those obtained with other radical B12 enzymes.

The X-ray structures of coenzyme B12 (AdoCbl)-dependent eliminating isomerases complexed with adenosylmethylcobalamin (AdoMeCbl) have been determined. As judged from geometries, the Co-C bond in diol dehydratase (DD) is not activated even in the presence of substrate. In ethanolamine ammonia-lyase (EAL), the bond is elongated in the absence of substrate; in the presence of substrate, the complex likely exists in both pre- and post-homolysis states. The impacts of incorporating an extra CH2 group are different in the two enzymes: the DD active site is flexible, and AdoMeCbl binding causes large conformational changes that make DD unable to adopt the catalytic state, whereas the EAL active site is rigid, and AdoMeCbl binding does not induce significant conformational changes. Such flexibility and rigidity of the active sites might reflect the tightness of adenine binding. The structures provide good insights into the basis of the very low activity of AdoMeCbl in these enzymes.

Adenosylcobalamin (AdoCbl) or coenzyme B12-dependent enzymes catalyze intramolecular group-transfer reactions and ribonucleotide reduction in a wide variety of organisms from bacteria to animals. They use a super-reactive primary-carbon radical formed by the homolysis of the coenzyme's Co-C bond for catalysis and thus belong to the larger class of "radical enzymes." For understanding the general mechanisms of radical enzymes, it is of great importance to establish the general mechanism of AdoCbl-dependent catalysis using enzymes that catalyze the simplest reactions-such as diol dehydratase, glycerol dehydratase and ethanolamine ammonia-lyase. These enzymes are often called "eliminases." We have studied AdoCbl and eliminases for more than a half century. Progress has always been driven by the development of new experimental methodologies. In this chapter, we describe our investigations on these enzymes, including their metabolic roles, gene cloning, preparation, characterization, activity assays, and mechanistic studies, that have been conducted using a wide range of biochemical and structural methodologies we have developed.

Adenosylcobalamin (AdoCbl) or coenzyme B12-dependent enzymes tend to undergo mechanism-based inactivation during catalysis or inactivation in the absence of substrate. Such inactivation may be inevitable because they use a highly reactive radical for catalysis, and side reactions of radical intermediates result in the damage of the coenzyme. How do living organisms address such inactivation when enzymes are inactivated by undesirable side reactions? We discovered reactivating factors for radical B12 eliminases. They function as releasing factors for damaged cofactor(s) from enzymes and thus mediate their exchange for intact AdoCbl. Since multiple turnovers and chaperone functions were demonstrated, they were renamed "reactivases" or "reactivating chaperones." They play an essential role in coenzyme recycling as part of the activity-maintaining systems for B12 enzymes. In this chapter, we describe our investigations on reactivating chaperones, including their discovery, gene cloning, preparation, characterization, activity assays, and mechanistic studies, that have been conducted using a wide range of biochemical and structural methods that we have developed.

The metal active site is precisely designed in metalloproteins. Here we applied 3D domain swapping, a phenomenon in which a partial protein structure is exchanged between molecules, to introduce metal sites in proteins. We designed multiple metal-binding sites specific to domain-swapped myoglobin (Mb) with His mutation. Stable dimeric Mbs with metal-binding sites were obtained by shifting the His position and introducing two Ala residues in the hinge region (K78H/G80A/H82A and K79H/G80A/H81A Mbs). The absorption and circular dichroism spectra of the monomer and dimer of K78H/G80A/H82A and K79H/G80A/H81A Mbs were similar to the corresponding spectra, respectively, of wild-type Mb. No negative peak due to dimer-to-monomer dissociation was observed below the denaturation temperature in the differential scanning calorimetry thermograms of K78H/G80A/H82A and K79H/G80A/H81A Mbs, whereas the dimer dissociates into monomers at 68 °C for wild-type Mb. These results show that the two mutants were stable in the dimer state. Metal ions bound to the metal-binding sites containing the introduced His in the domain-swapped Mb dimers. Co2+-bound and Ni2+-bound K78H/G80A/H82A Mb exhibited octahedral metal-coordination structures, where His78, His81, Glu85, and three H2O/OH- molecules coordinated to the metal ion. On the other hand, Co2+-bound and Zn2+-bound K79H/G80A/H81A Mb exhibited tetrahedral metal-coordination structures, where His79, His82, Asp141, and a H2O/OH- molecule coordinated to the metal ion. The Co2+-bound site exists deep inside the protein in the K79H/G80A/H81A Mb dimer, which may allow the unique tetrahedral coordination for the Co2+ ion. These results show that we can utilize domain swapping to construct artificial metalloproteins.

The tight H-bond network enhanced the helices at the hinge region and stabilized the myoglobin dimer, providing a unique example of using H-bonds in the design of a dimeric protein through 3D domain swapping.

Biodegradation of synthetic polymers is recognized as a useful way to reduce their environmental load and pollution, loss of natural resources, extensive energy consumption, and generation of greenhouse gases. The potential use of enzymes responsible for the degradation of the targeted polymers is an effective approach which enables the conversion of the used polymers to original monomers and/or other useful compounds. In addition, the enzymes are expected to be applicable in industrial processes such as improving the surface structures of the polymers. Especially, conversion of the solid polymers to soluble oligomers/monomers is a key step for the biodegradation of the polymers. Regarding the hydrolysis of polyamides, three enzymes, 6-aminohexanoate-cyclic-dimer hydrolase (NylA), 6-aminohexanoate-dimer hydrolase (NylB), and 6-aminohexanoate-oligomer endo-hydrolase (nylon hydrolase, NylC), are found in several bacterial strains. In this chapter, we describe our approach for the screening of microorganisms which degrade nylons and related compounds; preparation of substrates; assay of hydrolytic activity for soluble and insoluble substrates; and X-ray crystallographic and computational approaches for analysis of structure and catalytic mechanisms of the nylon-degrading enzymes.

Domain swapping is an exception to Anfinsen's dogma, and more than one structure can be produced from the same amino acid sequence by domain swapping. We have previously shown that myoglobin (Mb) can form a domain-swapped dimer in which the hinge region is converted to a helical structure. In this study, we showed that domain-swapped dimerization of Mb was achieved by a single Ala mutation of Gly at position 80. Multiple Ala mutations at positions 81 and 82 in addition to position 80 facilitated dimerization of Mb by stabilization of the dimeric states. Domain swapping tendencies correlated well with the helical propensity of the mutated residue in a series of Mb mutants with amino acids introduced to the hinge region. These findings demonstrate that a single mutation in the hinge loop to modify helical propensity can control oligomer formation, providing new ideas to create high-order protein oligomers using domain swapping.

Protein oligomers have gained interest, owing to their increased knowledge in cells and promising utilization for future materials. Various proteins have been shown to 3D domain swap, but there has been no domain swapping report on a blue copper protein. Here, we found that azurin from Alcaligenes xylosoxidans oligomerizes by the procedure of 2,2,2-trifluoroethanol addition to Cu(i)-azurin at pH 5.0, lyophilization, and dissolution at pH 7.0, whereas it slightly oligomerizes when using Cu(ii)-azurin. The amount of high order oligomers increased with the addition of Cu(ii) ions to the dissolution process of a similar procedure for apoazurin, indicating that Cu(ii) ions enhance azurin oligomerization. The ratio of the absorbance at 460 nm to that at ∼620 nm of the azurin dimer (Abs460/Abs618 = 0.113) was higher than that of the monomer (Abs460/Abs622 = 0.067) and the EPR A‖ value of the dimer (5.85 mT) was slightly smaller than that of the monomer (5.95 mT), indicating a slightly more rhombic copper coordination for the dimer. The redox potential of the azurin dimer was 342 ± 5 mV vs. NHE, which was 50 mV higher than that of the monomer. According to X-ray crystal analysis, the azurin dimer exhibited a domain-swapped structure, where the N-terminal region containing three β-strands was exchanged between protomers. The copper coordination structure was tetrahedrally distorted in the azurin dimer, similar to that in the monomer; however, the Cu-O(Gly45) bond length was longer for the dimer (monomer, 2.46-2.59 Å; dimer, 2.98-3.25 Å). These results open the door for designing oligomers of blue copper proteins by domain swapping.

The Wnt-β-catenin signaling pathway regulates embryonic development and tissue homeostasis throughout the animal kingdom. Signaling through this pathway crucially depends on the opposing activities of two cytoplasmic multiprotein complexes: the Axin destruction complex, which destabilizes the downstream effector β-catenin, and the Dishevelled signalosome, which inactivates the Axin complex and thus enables β-catenin to accumulate and operate a transcriptional switch in the nucleus. These complexes are assembled by dynamic head-to-tail polymerization of the DIX domains of Axin or Dishevelled, respectively, which increases their avidity for signaling effectors. Axin also binds to Dishevelled through its DIX domain. Here, we report the crystal structure of the heterodimeric complex between the two DIX domains of Axin and Dishevelled. This heterotypic interface resembles the interfaces observed in the individual homopolymers, albeit exhibiting a slight rearrangement of electrostatic interactions and hydrogen bonds, consistent with the heterotypic interaction being favored over the homotypic Axin DIX interaction. Last, cell-based signaling assays showed that heterologous polymerizing domains functionally substituted for the DIX domain of Dishevelled provided that these Dishevelled chimeras retained a DIX head or tail surface capable of binding to Axin. These findings indicate that the interaction between Dishevelled and Axin through their DIX domains is crucial for signaling to β-catenin.

Many c-type cytochromes (cyts) can form domain-swapped oligomers. The positively charged Hydrogenobacter thermophilus (HT) cytochrome (cyt) c552 forms domain-swapped oligomers during expression in the Escherichia coli (E. coli) expression system, but the factors influencing the oligomerization remain unrevealed. Here, we found that the dimer of the negatively charged Shewanella violacea (SV) cyt c5 exhibits a domain-swapped structure, in which the N-terminal helix is exchanged between protomers, similar to the structures of the HT cyt c552 and Pseudomonas aeruginosa (PA) cyt c551 domain-swapped dimers. Positively charged horse cyt c and HT cyt c552 domain swapped during expression in E. coli, whereas negatively charged PA cyt c551 and SV cyt c5 did not. Oligomers were formed during expression in E. coli for HT cyt c552 attached to either a co- or post-translational signal peptide for transportation through the cytoplasm membrane, but not for PA cyt c551 attached to either signal peptide. HT cyt c552 formed oligomers in E. coli in the presence and absence of rare codons. More oligomers were obtained from the in vitro folding of horse cyt c and HT cyt c552 by the addition of negatively charged liposomes during folding, whereas the amount of oligomers for the in vitro folding of PA cyt c551 and SV cyt c5 did not change significantly by the addition. These results indicate that the protein surface charge affects the oligomerization of c-type cyts in cells; positively charged c-type cyts assemble on a negatively charged membrane, inducing formation of domain-swapped oligomers during folding.

Dishevelled (Dvl) is a positive regulator of the canonical Wnt pathway that downregulates the phosphorylation of β-catenin and its subsequent degradation. Dvl contains an N-terminal DIX domain, which is involved in its homooligomerization and interactions with regulators of the Wnt pathway. The crystal structure of a Y27W mutant of the Dishevelled2 DIX domain (DIX-Y27W) has been determined at 1.64 Å resolution. DIX-Y27W has a compact ubiquitin-like fold and self-associates with neighbouring molecules through β-bridges, resulting in a head-to-tail helical molecular arrangement similar to previously reported structures of DIX domains. Glu23 of DIX-Y27W forms a hydrogen bond to the side chain of Trp27, corresponding to the Glu762...Trp766 hydrogen bond of the rat Axin DIX domain, whereas Glu23 in the Y27D mutant of the Dishevelled2 DIX domain forms a salt bridge to Lys68 of the adjacent molecule. The high-resolution DIX-Y27W structure provides details of the head-to-tail interaction, including solvent molecules, and also the plausibly wild-type-like structure of the self-association surface compared with previously published Dvl DIX-domain mutants.

BACKGROUND: Head-to-tail polymerising domains generating heterogeneous aggregates are generally difficult to crystallise. DIX domains, exclusively found in the Wnt signalling pathway, are polymerising factors following this head-to-tail arrangement; moreover, they are considered to play a key role in the heterotypic interaction between Dishevelled (Dvl) and Axin, which are cytoplasmic proteins also positively and negatively regulating the canonical Wnt/β- catenin signalling pathway, but this interaction mechanism is still unknown. OBJECTIVE: This study mainly aimed to clarify whether the Dvl2 and Axin-DIX domains (Dvl2-DIX and Axin-DIX, respectively) form a helical polymer in a head-to-tail way during complexation. METHODS: Axin-DIX (DAX) and Dvl2-DIX (DIX), carrying polymerisation-blocking mutations, were expressed as a fusion protein by using a flexible peptide linker to fuse the C-terminal of the former to the N-terminal of the latter, enforcing a defined 1:1 stoichiometry between them. RESULTS: The crystal of the DAX-DIX fusion protein diffracted to a resolution of about 0.3 nm and a data set was collected at a 0.309 nm resolution. The structure was solved via the molecular replacement method by using the DIX and DAX structures. A packing analysis of the crystal revealed the formation of a tandem heterodimer in a head-to-tail way, as predicted by the Wntsignalosome model. CONCLUSION: The results demonstrated that the combination of polymerisation-blocking mutations and a fusion protein of two head-to-tail polymerising domains is effective especially for crystallising complexes among heterologous polymerising proteins or domains.

Geometric and electronic structure changes in the copper (Cu) centers in bilirubin oxidase (BOD) upon a four-electron reduction were investigated by quantum mechanics/molecular mechanics (QM/MM) calculations. For the QM region, the unrestricted density functional theory (UDFT) method was adopted for the open-shell system. We found new candidates of the native intermediate (NI, intermediate II) and the resting oxidized (RO) states, i.e., NIH+ and RO₀. Elongations of the Cu-Cu atomic distances for the trinuclear Cu center (TNC) and very small structural changes around the type I Cu (T1Cu) were calculated as the results of a four-electron reduction. The QM/MM optimized structures are in good agreement with recent high-resolution X-ray structures. As the structural change in the TNC upon reduction was revealed to be the change in the size of the triangle spanned by the three Cu atoms of TNC, we introduced a new index (l) to characterize the specific structural change. Not only the wild-type, but also the M467Q, which mutates the amino acid residue coordinating T1Cu, were precisely analyzed in terms of their molecular orbital levels, and the optimized redox potential of T1Cu was theoretically reconfirmed.

Bilirubin oxidase (BOD) belongs to the family of blue multicopper oxidases, and catalyzes the concomitant oxidation of bilirubin to biliverdin and the reduction of molecular oxygen to water via a four-electron reduction system. The active sites of BOD comprise four copper atoms; type I copper (T1Cu) forms a mononuclear site, and a cluster of three copper atoms forms a trinuclear center. In the present study, we determined the high-resolution crystal structures of BOD from the fungus Myrothecium verrucaria. We investigated wild-type (WT) BOD and a BOD mutant called Met467Gln, which is inactive against bilirubin. The structures revealed that a novel post-translational crosslink between Trp396 and His398 is formed in the vicinity of the T1Cu site in WT BOD, whereas it is absent in the Met467Gln mutant. Our structural and computational studies suggest that the His-Trp crosslink adjusts the redox potential of T1Cu to that of bilirubin to efficiently abstract electrons from the substrate.

Nylon hydrolase (NylC) is initially expressed as an inactive precursor (36 kDa). The precursor is cleaved autocatalytically at Asn266/Thr267 to generate an active enzyme composed of an α subunit (27 kDa) and a β subunit (9 kDa). Four αβ heterodimers (molecules A-D) form a doughnut-shaped quaternary structure. In this study, the thermostability of the parental NylC was altered by amino acid substitutions located at the A/D interface (D122G/H130Y/D36A/L137A) or the A/B interface (E263Q) and spanned a range of 47 °C. Considering structural, biophysical, and biochemical analyses, we discuss the structural basis of the stability of nylon hydrolase. From the analytical centrifugation data obtained regarding the various mutant enzymes, we conclude that the assembly of the monomeric units is dynamically altered by the mutations. Finally, we propose a model that can predict whether the fate of the nascent polypeptide will be correct subunit assembly, inappropriate protein-protein interactions causing aggregation, or intracellular degradation of the polypeptide.

Arthrobacter sp. (formerly Flavobacterium sp.) KI72 has enzymes which are responsible for the degradation of nylon-6 industry by-products (nylon-oligomer). NylB encoded on plasmid pOAD2 is one of these enzymes and has a specific activity toward the degradation of 6-aminohexanoate-linear dimer (Ald). The plasmid, pOAD2, has also an analogous protein, NylB', which has 88% homology to NylB but only about 0.5% of the specific activity. We constructed Hyb24 (a hybrid between the NylB and NylB' with NylB'-level activity), Hyb24DN (with double mutation of G181D and H266N with NylB-level activity) and Hyb24DNA (a mutant of Hyb24DN with an additional mutation of S112A at the active site) proteins and solved the three-dimensional structures by x-ray crystallography. In case of Hyb24DNA, the structure of a complex with substrate, Ald, was determined. The overall structures of three proteins are almost identical with a two-domain structure that is categorized in β-lactamase fold. On the basis of the spatial arrangements of amino acid residues at the active site of Hyb24DN and Hyb24DNA-Ald complex, we conclude that the nylon-oligomer hydrolase has evolved from the ester hydrolysis enzymes, of which essential residue is nucleophilic Ser112, with a β-lactamase fold as an ancestral protein by substitution of two residues G181D and H266N at the active site pocket.