Structural and Biochemical Analysis of 3-Dehydroquinate Dehydratase from Corynebacterium glutamicum.

Dehydroquinate dehydratase (DHQD) catalyzes the conversion of 3-dehydroquinic acid (DHQ) into 3-dehydroshikimic acid in the mid stage of the shikimate pathway, which is essential for the biosynthesis of aromatic amino acids and folates. Here, we report two the crystal structures of type II DHQD (CgDHQD) derived from Corynebacterium glutamicum, which is a widely used industrial platform organism. We determined the structures for CgDHQDWT with the citrate at a resolution of 1.80Å and CgDHQDR19A with DHQ complexed forms at a resolution of 2.00 Å, respectively. The enzyme forms a homododecamer consisting of four trimers with three interfacial active sites. We identified the DHQ-binding site of CgDHQD and observed an unusual binding mode of citrate inhibitor in the site with a half-opened lid loop. A structural comparison of CgDHQD with a homolog derived from Streptomyces coelicolor revealed differences in the terminal regions, lid loop, and active site. Particularly, CgDHQD, including some Corynebacterium species, possesses a distinctive residue P105, which is not conserved in other DHQDs at the position near the 5-hydroxyl group of DHQ. Replacements of P105 with isoleucine and valine, conserved in other DHQDs, caused an approximately 70% decrease in the activity, but replacement of S103 with threonine (CgDHQDS103T) caused a 10% increase in the activity. Our biochemical studies revealed the importance of key residues and enzyme kinetics for wild type and CgDHQDS103T, explaining the effect of the variation. This structural and biochemical study provides valuable information for understanding the reaction efficiency that varies due to structural differences caused by the unique sequences of CgDHQD.

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.

Production of various phenolic aldehyde compounds using the 4CL-FCHL biosynthesis platform.

Vanillin (3-methoxy-4-hydroxybenzaldehyde) is one of the most important flavoring substances used in the cosmetic and food industries. Feruloyl-CoA hydratase/lyase (FCHL) is an enzyme that catalyzes the production of vanillin from feruloyl-CoA. In this study, we report kinetic parameters and biochemical properties of FCHL from Sphingomonas paucimobilis SYK-6 (SpFCHL). Also, the crystal structures of an apo-form of SpFCHL and two complexed forms with acetyl-CoA and vanillin/CoA was present. Comparing the apo structure to its complexed forms of SpFCHL, a gate loop with an "open and closed" role was observed at the entrance of the substrate-binding site. With vanillin and CoA complexed to SpFCHL, we captured a conformational change in the feruloyl moiety-binding pocket that repositions the catalytic SpFCHLE146 and other key residues. This binding pocket does not tightly fit the vanillin structure, suggesting substrate promiscuity of this enzyme. This observation is in good agreement with assay results for phenylpropanoid-CoAs and indicates important physicochemical properties of the substrate for the hydratase/lyase reaction mechanism. In addition, we showed that various phenolic aldehydes could be produced using the 4CL-FCHL biosynthesis platform.

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 insight into a molecular mechanism of methenyltetrahydrofolate cyclohydrolase from Methylobacterium extorquens AM1.

Bioconversion of the C1 compounds into value-added products is one of the CO2-reducing strategies. In particular, because CO2 can be easily converted into formate, the efficient and direct bioconversion of CO2 through formate assimilation is attracting attention. The tetrahydrofolate (THF) cycle is the highly efficient reconstructed formate assimilation pathway, and 5,10-methenyltetrahydrofolate cyclohydrolase (FchA) is an essential enzyme involved in the THF cycle. In this study, a kinetic analysis of FchA from Methylobacterium extorquens AM1 (MeFchA) was performed and revealed that the enzyme has much higher cyclization than hydrolyzation activity, making it an optimal enzyme for formate assimilation. The crystal structure of MeFchA in the apo- and the THF-complexed forms was also determined, revealing that the substrate-binding site of the enzyme has three differently charged regions to stabilize the three differently charged moieties of the formyl-THF substrate. The residues involved in the substrate binding were also verified through site-directed mutagenesis. This study provides a biochemical and structural basis for the molecular mechanism underlying formate assimilation.

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.

Extra disulfide and ionic salt bridge improves the thermostability of lignin peroxidase H8 under acidic condition.

The development of a lignin peroxidase (LiP) that is thermostable even under acidic pH conditions is a main issue for efficient enzymatic lignin degradation due to reduced repolymerization of free phenolic products at acidic pH (< 3). Native LiP under mild conditions (half-life (t1/2) of 8.2 days at pH 6) exhibits a marked decline in thermostability under acidic conditions (t1/2 of only 14 min at pH 2.5). Thus, improving the thermostability of LiP in acidic environments is required for effective lignin depolymerization in practical applications. Here, we show the improved thermostability of a synthetic LiPH8 variant (S49C/A67C/H239E, PDB: 6ISS) capable of strengthening the helix-loop interactions under acidic conditions. This variant retained excellent thermostability at pH 2.5 with a 10-fold increase in t1/2 (2.52 h at 25 °C) compared with that of the native enzyme. X-ray crystallography analysis showed that the recombinant LiPH8 variant is the only unique lignin peroxidase containing five disulfide bridges, and the helix-loop interactions of the synthetic disulfide bridge and ionic salt bridge in its structure are responsible for stabilizing the Ca2+-binding region and heme environment, resulting in an increase in overall structural resistance against acidic conditions. Our work will allow the design of biocatalysts for ligninolytic enzyme engineering and for efficient biocatalytic degradation of plant biomass in lignocellulose biorefineries.

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.

Crystal structure of an acetyl-CoA acetyltransferase from PHB producing bacterium Bacillus cereus ATCC 14579.

Bacillus cereus ATCC 14579 is a known polyhydroxybutyrate (PHB)-producing microorganism that possesses genes associated with PHB synthesis such as PhaA, PhaB, and PHA synthases. PhaA (i.e., thiolase) is the first enzyme in the PHA biosynthetic pathway, which catalyze the condensation of two acetyl-CoA molecules to acetoacetyl-CoA. Our study elucidated the crystal structure of PhaA in Bacillus cereus ATCC 14579 (BcTHL) in its apo- and CoA-bound forms. BcTHL adopts a type II biosynthetic thiolase structure by forming a tetramer. The crystal structure of CoA-complexed BcTHL revealed that the substrate binding site of BcTHL is constituted by different residues compared with other known thiolases. Our study also revealed that Arg221, a residue involved in ADP binding, undergoes a positional conformational change upon the binding of the CoA molecule.

Biochemical properties and crystal structure of formate-tetrahydrofolate ligase from Methylobacterium extorquens CM4.

Methylobacterium extorquens is a methylotroph model organism that has the ability to assimilate formate using the tetrahydrofolate (THF) pathway. The formate-tetrahydrofolate ligase from M. extorquens (MeFtfL) is an enzyme involved in the THF pathway that catalyzes the conversion of formate, THF, and ATP into formyltetrahydrofolate and ADP. To investigate the biochemical properties of MeFtfL, we evaluated the metal usage and enzyme kinetics of the enzyme. MeFtfL uses the Mg ion for catalytic activity, but also has activity for Mn and Ca ions. The enzyme kinetics analysis revealed that Km value of farmate was much higher than THF and ATP, which shows that the ligation activity of MeFtfL is highly dependent on formation concentration. We also determined the crystal structure of MeFtfL at 2.8 Å resolution. MeFtfL functions as a tetramer, and each monomer consists of three domains. The structural superposition of MeFtfL with FtfL from Moorella thermoacetica allowed us to predict the substrate binding site of the enzyme.

Enhanced succinic acid production by Mannheimia employing optimal malate dehydrogenase.

Succinic acid (SA), a dicarboxylic acid of industrial importance, can be efficiently produced by metabolically engineered Mannheimia succiniciproducens. Malate dehydrogenase (MDH) is one of the key enzymes for SA production, but has not been well characterized. Here we report biochemical and structural analyses of various MDHs and development of hyper-SA producing M. succiniciproducens by introducing the best MDH. Corynebacterium glutamicum MDH (CgMDH) shows the highest specific activity and least substrate inhibition, whereas M. succiniciproducens MDH (MsMDH) shows low specific activity at physiological pH and strong uncompetitive inhibition toward oxaloacetate (ki of 67.4 and 588.9 µM for MsMDH and CgMDH, respectively). Structural comparison of the two MDHs reveals a key residue influencing the specific activity and susceptibility to substrate inhibition. A high-inoculum fed-batch fermentation of the final strain expressing cgmdh produces 134.25 g L-1 of SA with the maximum productivity of 21.3 g L-1 h-1, demonstrating the importance of enzyme optimization in strain development.

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.

Structural insight into bi-functional malonyl-CoA reductase.

The bi-functional malonyl-CoA reductase is a key enzyme of the 3-hydroxypropionate bi-cycle for bacterial CO2 fixation, catalysing the reduction of malonyl-CoA to malonate semialdehyde and further reduction to 3-hydroxypropionate. Here, we report the crystal structure and the full-length architecture of malonyl-CoA reductase from Porphyrobacter dokdonensis. The malonyl-CoA reductase monomer of 1230 amino acids consists of four tandemly arranged short-chain dehydrogenases/reductases, with two catalytic and two non-catalytic short-chain dehydrogenases/reductases, and forms a homodimer through paring contact of two malonyl-CoA reductase monomers. The complex structures with its cofactors and substrates revealed that the malonyl-CoA substrate site is formed by the cooperation of two short-chain dehydrogenases/reductases and one novel extra domain, while only one catalytic short-chain dehydrogenase/reductase contributes to the formation of the malonic semialdehyde-binding site. The phylogenetic and structural analyses also suggest that the bacterial bi-functional malonyl-CoA has a structural origin that is completely different from the archaeal mono-functional malonyl-CoA and malonic semialdehyde reductase, and thereby constitute an efficient enzyme.

Effects of Synergistic Inhibition on α-glucosidase by Phytoalexins in Soybeans.

To determine the mechanism of action of the effects of phytoalexins in soybeans, we analyzed α-glucosidase inhibition kinetics using Michaelis-Menten plots and Lineweaver-Burk plots. The results showed that the type of inhibition with glyceollin was competitive, that of genistein was noncompetitive, that of daidzein was uncompetitive, and luteolin showed a mixed mode of action. The Ki values were determined using a Dixon plot as glyceollin, 18.99 µM; genistein, 15.42 µM; luteolin, 16.81 µM; and daidzein, 9.99 µM. Furthermore, potential synergistic effects between glyceollin and the three polyphenols were investigated. A combination of glyceollin and luteolin at a ratio of 3:7 exhibited synergistic effects on α-glucosidase inhibition, having a combination index (CI) of 0.64244, according to the CI-isobologram equation. Collectively, these results showed that a combination of glyceollin and luteolin has the potential to inhibit α-glucosidase activity via a synergistic mode of inhibition.