Highly efficient site-specific protein modification using tyrosinase from Streptomyces avermitilis: Structural insight.

Tyrosinase-mediated protein conjugation has recently drawn attention as a site-specific protein modification tool under mild conditions. However, the tyrosinases reported to date act only on extremely exposed tyrosine residues, which limits where the target tyrosine can be located. Herein, we report a tyrosinase from Streptomyces avermitilis (SaTYR), that exhibits a much higher activity against tyrosine residues on the protein surface than other tyrosinases. We determined the crystal structure of SaTYR and revealed that the enzyme has a relatively flat and shallow substrate-binding pocket to accommodate a protein substrate. We demonstrated SaTYR-mediated fluorescence dye tagging and PEGylation of a surface tyrosine residue that was unreacted by other tyrosinases with an approximately 95.2 % conjugation yield in 1 h. We also present a structural rationale that considers the steric hindrance from adjacent residues and surrounding structures along with the extent of solvent exposure of residues, as necessary when determining the optimal positions for introducing target tyrosine residues in SaTYR-mediated protein modification. The study demonstrated that the novel tyrosinase, SaTYR, extends the scope of tyrosinase-mediated protein modification, and we propose that site-specific tyrosine conjugation using SaTYR is a promising strategy for protein bioconjugation in various applications.

Three-directional engineering of IsPETase with enhanced protein yield, activity, and durability.

The mesophilic PETase from Ideonella sakaiensis (IsPETase) has been shown to exhibit high PET hydrolysis activity, but its low stability limits its industrial applications. Here, we developed a variant, Z1-PETase, with enhanced soluble protein yield and durability while maintaining or improving activity at lower temperatures. The selected Z1-PETase not only exhibited a 20-fold improvement in soluble protein yield compared to the previously engineered IsPETaseS121E/D186H/S242T/N246D (4p) variant, but also demonstrated a 30% increase in low-temperature activity at 40 °C, along with an 11 °C increase in its TmD value. The PET depolymerization test across a temperature range low to high (30-70 °C) confirmed that Z1-PETase exhibits high accessibility of mesophilic PET hydrolase and rapid depolymerizing rate at higher temperature in accordance with the thermal behaviors of polymer and enzyme. Additionally, structural interpretation indicated that the stabilization of specific active site loops in Z1-PETase contributes to enhanced thermostability without adversely impacting enzymatic activity. In a pH-stat bioreactor, Z1-PETase depolymerized > 90% of both transparent and colored post-consumer PET powders within 24 and 8 h at 40 °C and 55 °C, respectively, demonstrating that the utility of this IsPETase variant in the bio-recycling of PET.

Discovery and rational engineering of PET hydrolase with both mesophilic and thermophilic PET hydrolase properties.

Excessive polyethylene terephthalate (PET) waste causes a variety of problems. Extensive research focused on the development of superior PET hydrolases for PET biorecycling has been conducted. However, template enzymes employed in enzyme engineering mainly focused on IsPETase and leaf-branch compost cutinase, which exhibit mesophilic and thermophilic hydrolytic properties, respectively. Herein, we report a PET hydrolase from Cryptosporangium aurantiacum (CaPETase) that exhibits high thermostability and remarkable PET degradation activity at ambient temperatures. We uncover the crystal structure of CaPETase, which displays a distinct backbone conformation at the active site and residues forming the substrate binding cleft, compared with other PET hydrolases. We further develop a CaPETaseM9 variant that exhibits robust thermostability with a Tm of 83.2 °C and 41.7-fold enhanced PET hydrolytic activity at 60 °C compared with CaPETaseWT. CaPETaseM9 almost completely decompose both transparent and colored post-consumer PET powder at 55 °C within half a day in a pH-stat bioreactor.

Molecular mechanism underlying high-affinity terephthalate binding and conformational change of TBP from Ideonella sakaiensis.

Ideonella sakaiensis is the bacterium that can survive by degrading polyethylene terephthalate (PET) plastic, and terephthalic acid (TPA) binding protein (IsTBP) is an essential periplasmic protein for uptake of TPA into the cytosol for complete degradation of PET. Here, we demonstrated that IsTBP has remarkably high specificity for TPA among 33 monophenolic compounds and two 1,6-dicarboxylic acids tested. Structural comparisons with 6-carboxylic acid binding protein (RpAdpC) and TBP from Comamonas sp. E6 (CsTphC) revealed the key structural features that contribute to high TPA specificity and affinity of IsTBP. We also elucidated the molecular mechanism underlying the conformational change upon TPA binding. In addition, we developed the IsTBP variant with enhanced TPA sensitivity, which can be expanded for the use of TBP as a biosensor for PET degradation.

Crystal Structure and Functional Characterization of Acetylornithine Aminotransferase from Corynebacterium glutamicum.

The amino acids l-arginine and l-ornithine are widely used in animal feed and as health supplements and pharmaceutical compounds. In arginine biosynthesis, acetylornithine aminotransferase (AcOAT) uses pyridoxal-5'-phosphate (PLP) as a cofactor for amino group transfer. Here, we determined the crystal structures of the apo and PLP complex forms of AcOAT from Corynebacterium glutamicum (CgAcOAT). Our structural observations revealed that CgAcOAT undergoes an order-to-disorder conformational change upon binding with PLP. Additionally, we observed that unlike other AcOATs, CgAcOAT exists as a tetramer. Subsequently, we identified the key residues involved in PLP and substrate binding based on structural analysis and site-directed mutagenesis. This study might provide structural insights on CgAcOAT, which can be utilized for the development of improved l-arginine production enzymes.

Structural studies of a novel auxiliary-domain-containing phenylalanine hydroxylase from Bacillus cereus ATCC 14579.

Phenylalanine hydroxylase (PAH), which belongs to the aromatic amino-acid hydroxylase family, is involved in protein synthesis and pyomelanine production through the hydroxylation of phenylalanine to tyrosine. In this study, the crystal structure of PAH from Bacillus cereus ATCC 14579 (BcPAH) with an additional 280 amino acids in the C-terminal region was determined. The structure of BcPAH consists of three distinct domains: a core domain with two additional inserted α-helices and two novel auxiliary domains: BcPAH-AD1 and BcPAH-AD2. Structural homologues of BcPAH-AD1 and BcPAH-AD2 are known to be involved in mRNA regulation and protein-protein interactions, and thus it was speculated that BcPAH might utilize the auxiliary domains for interaction with its partner proteins. Furthermore, phylogenetic tree analysis revealed that the three-domain PAHs, including BcPAH, are completely distinctive from both conventional prokaryotic PAHs and eukaryotic PAHs. Finally, biochemical studies of BcPAH showed that BcPAH-AD1 might be important for the structural integrity of the enzyme and that BcPAH-AD2 is related to enzyme stability and/or activity. Investigations into the intracellular functions of the two auxiliary domains and the relationship between these functions and the activity of PAH are required.

Structural and functional characterization of an auxiliary domain-containing PET hydrolase from Burkholderiales bacterium.

Biodegradation of polyethylene terephthalate (PET) is one of fundamental ways to solve plastic pollution. As various microbial hydrolases have an extra domain unlike PETase from Ideonella sakaiensis (IsPETase), research on the role of these extra domain in PET hydrolysis is crucial for the identification and selection of a novel PET hydrolase. Here, we report that a PET hydrolase from Burkholderiales bacterium RIFCSPLOWO2_02_FULL_57_36 (BbPETase) with an additional N-terminal domain (BbPETaseAND) shows a similar hydrolysis activity toward microcrystalline PET and a higher thermal stability than IsPETase. Based on detailed structural comparisons between BbPETase and IsPETase, we generated the BbPETaseS335N/T338I/M363I/N365G variant with an enhanced PET-degrading activity and thermal stability. We further revealed that BbPETaseAND contributes to the thermal stability of the enzyme through close contact with the core domain, but the domain might hinder the adhesion of enzyme to PET substrate. We suggest that BbPETase is an enzyme in the evolution of efficient PET degradation and molecular insight into a novel PET hydrolase provides a novel strategy for the development of biodegradation of PET.

Implications for the PET decomposition mechanism through similarity and dissimilarity between PETases from Rhizobacter gummiphilus and Ideonella sakaiensis.

The development of a superb polyethylene terephthalate (PET) hydrolyzing enzyme requires an accurate understanding of the PET decomposition mechanism. However, studies on PET degrading enzymes, including the PET hydrolase from Ideonella sakaiensis (IsPETase), have not provided sufficient knowledge of the molecular mechanisms for the hardly accessible substrate. Here, we report a novel PET hydrolase from Rhizobacter gummiphilus (RgPETase), which has a hydrolyzing activity similar to IsPETase toward microcrystalline PET but distinct behavior toward low crystallinity PET film. Structural analysis of RgPETase reveals that the enzyme shares the key structural features of IsPETase for high PET hydrolysis activity but has distinguished structures at the surface-exposed regions. RgPETase shows a unique conformation of the wobbling tryptophan containing loop (WW-loop) and change of the electrostatic surface charge on the loop dramatically affects the PET-degrading activity. We further show that effect of the electrostatic surface charge to the activity varies depending on locations. This work provides valuable information underlying the uncovered PET decomposition mechanism.

Dual α-1,4- and ß-1,4-Glycosidase Activities by the Novel Carbohydrate-Binding Module in α-l-Fucosidase from Vibrio sp. Strain EJY3.

Carbohydrates are structurally and functionally diverse materials including polysaccharides, and marine organisms are known to have many enzymes for the breakdown of complex polysaccharides. Here, we identified an α-l-fucosidase enzyme from the marine bacterium Vibrio sp. strain EJY3 (VejFCD) that has dual α-1,4-glucosidic and ß-1,4-galactosidic specificities. We determined the crystal structure of VejFCD and provided the structural basis underlying the dual α- and ß-glycosidase activities of the enzyme. Unlike other three-domain FCDs, in VejFCD, carbohydrate-binding module-B (CBM-B) with a novel ß-sandwich fold tightly contacts with the CatD/CBM-B main body and provides key residues for the ß-1,4-glycosidase activity of the enzyme. The phylogenetic tree analysis suggests that only a few FCDs from marine microorganisms have the key structural features for dual α-1,4- and ß-1,4-glycosidase activities. This study provides the structural insights into the mechanism underlying the novel glycoside hydrolase activities and could be applied for more efficient utilization in the hydrolysis of complex carbohydrates in biotechnological applications.

High Galacto-Oligosaccharide Production and a Structural Model for Transgalactosylation of ß-Galactosidase II from Bacillus circulans.

The transgalactosylase activity of ß-galactosidase produces galacto-oligosaccharides (GOSs) with prebiotic effects similar to those of major oligosaccharides in human milk. ß-Galactosidases from Bacillus circulans ATCC 31382 are important enzymes in industrial-scale GOS production. Here, we show the high GOS yield of ß-galactosidase II from B. circulans (ß-Gal-II, Lactazyme-B), compared to other commercial enzymes. We also determine the crystal structure of the five conserved domains of ß-Gal-II in an apo-form and complexed with galactose and an acceptor sugar, showing the heterogeneous mode of transgalactosylation by the enzyme. Truncation studies of the five conserved domains reveal that all five domains are essential for enzyme catalysis, while some truncated constructs were still expressed as soluble proteins. Structural comparison of ß-Gal-II with other ß-galactosidase homologues suggests that the GOS linkage preference of the enzyme might be quite different from other enzymes. The structural information on ß-Gal-II might provide molecular insights into the transgalactosylation process of the ß-galactosidases in GOS production.

Structural bioinformatics-based protein engineering of thermo-stable PETase from Ideonella sakaiensis.

Poly(ethylene terephthalate) (PET), a widely used plastic around the world, causes various environmental and health problems. Several groups have been extensively conducting research to solve these problems through enzymatic degradation of PET at high temperatures around 70 °C. Recently, Ideonella sakaiensis, a bacterium that degrades PET at mild temperatures, has been newly identified, and further protein engineering studies on the PET degrading enzyme from the organism (IsPETase) have also been conducted to overcome the low thermal stability of the enzyme. In this study, we performed structural bioinformatics-based protein engineering of IsPETase to optimize the substrate binding site of the enzyme and developed two variants, IsPETaseS242T and IsPETaseN246D, with higher enzymatic activity at both 25 and 37 °C compared with IsPETaseWT. We also developed the IsPETaseS121E/D186H/S242T/N246D variant by integrating the S242 T and N246D mutations into the previously reported IsPETaseS121E/D186H/R208A variant. At the 37 °C incubation, the quadruple variant maintained the PET degradation activity for 20 days, unlike IsPETaseWT that lost its activity within a day. Consequently, this study exhibited 58-fold increase in the activity compared with IsPETaseWT.

Sequence, structure and function-based classification of the broadly conserved FAH superfamily reveals two distinct fumarylpyruvate hydrolase subfamilies.

Fumarylacetoacetate hydrolase (FAH) superfamily proteins are found ubiquitously in microbial pathways involved in the catabolism of aromatic substances. Although extensive bioinformatic data on these proteins have been acquired, confusion caused by problems with the annotation of these proteins hinders research into determining their physiological functions. Here we classify 606 FAH superfamily proteins using a maximum likelihood (ML) phylogenetic tree, comparative gene-neighbourhood patterns and in vitro enzyme assays. The FAH superfamily proteins used for the analyses are divided into five distinct subfamilies, and two of them, FPH-A and FPH-B, contain the majority of the proteins of undefined function. These subfamilies include clusters designated FPH-I and FPH-II, respectively, which include two distinct types of fumarylpyruvate hydrolase (FPH), an enzyme involved in the final step of the gentisate pathway. We determined the crystal structures of these FPH enzymes at 2.0 Å resolutions and investigate the substrate binding mode by which these types of enzymes can accommodate fumarylpyruvate as a substrate. Consequentially, we identify the molecular signatures of the two types of FPH enzymes among the broadly conserved FAH superfamily proteins. Our studies allowed us to predict the relationship of unknown FAH superfamily proteins using their sequence information.

Structure and biochemical studies of a pseudomonad maleylpyruvate isomerase from Pseudomonas aeruginosa PAO1.

Pseudomonas aeruginosa PAO1 can utilize various aromatic hydrocarbons as a carbon source. Among the three genes involved in the gentisate pathway of P. aeruginosa, the gene product of PA2473 belongs to the ζ-class glutathione S-transferase and is predicted to be a maleylpyruvate isomerase. In this study, we determined the crystal structure of maleylpyruvate isomerase from Pseudomonas aeruginosa PAO1 (PaMPI) at a resolution of 1.8 Å. PaMPI functions as a dimer and shows the glutathione S-transferase fold. The structure comparison with other glutathione S-transferase structures enabled us to predict the glutathione cofactor binding site and suggests that PaMPI has differences in residues that make up the putative substrate binding site. Biochemical study of PaMPI showed that the protein has an MPI activity. Interestingly, unlike the reported glutathione S-transferases so far, the purified PaMPI showed isomerase activity without the addition of the reduced glutathione, although the protein showed much higher activity when the glutathione cofactor was added to the reaction mixture. Taken together, our studies reveal that the gene product of PA2473 functions as a maleylpyruvate isomerase and might be involved in the gentisate pathway.

Structural insights into a maleylpyruvate hydrolase from sphingobium sp. SYK-6, a bacterium degrading lignin-derived aryls.

Sphingobium sp. strain SYK-6, an aerobic gram-negative bacillus found in soil, is known for utilizing lignin-derived monoaryls and biaryls as carbon sources and degrading aromatic compounds. The Sphingobium sp. strain SYK-6 genome contains three genes involved in salicylate catabolism: SLG_11260, SLG_11270, and SLG_11280. Here, we report that the gene product of SLG_11280 functions as a maleylpyruvate hydrolase (SsMPH) with Km and Kcat values of 166.2 µM and 3.76 min-1, respectively. This study also reveals the crystal structures of both the apo and pyruvate-manganese ion-bound SsMPH, which revealed that like other fumarylacetoacetate hydrolases, SsMPH dimerizes and has nine unique 310-helices. Molecular docking studies of maleylpyruvate also revealed the likely binding mode of SsMPH and its substrate.