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Lignin, flavonoids, melatonin, and stilbenes are plant specialized metabolites with diverse physiological and biological functions, supporting plant growth and conferring stress resistance. Their biosynthesis requires O -methylations catalyzed by 5-hydroxyconiferaldehyde O -methyltransferase (CAldOMT; also called caffeic acid O -methyltransferase, COMT). CAldOMT was first known for its roles in syringyl (S) lignin biosynthesis in angiosperm cell walls and later found to be multifunctional. This enzyme also catalyzes O -methylations in flavonoid, melatonin, and stilbene biosynthetic pathways. Phylogenetic analysis indicated the convergent evolution of enzymes with OMT activities towards the monolignol biosynthetic pathway intermediates in some gymnosperm species that lack S-lignin and Selaginella moellendorffii , a lycophyte which produces S-lignin. Furthermore, neofunctionalization of CAldOMTs occurred repeatedly during evolution, generating unique O -methyltransferases (OMTs) with novel catalytic activities and/or accepting novel substrates, including lignans, 1,2,3-trihydroxybenzene, and phenylpropenes. This review summarizes multiple aspects of CAldOMTs and their related proteins in plant metabolism and discusses their evolution, molecular mechanism, and roles in biorefineries, agriculture, and synthetic biology. [ABSTRACT FROM AUTHOR]

Tetrahydrofuran ring formation from dibenzylbutyrolactone lignans is a key step in the biosynthesis of aryltetralin lignans including deoxypodophyllotoxin and podophyllotoxin. Previously, Fe(II)- and 2-oxoglutarate-dependent dioxygenase (2-ODD) from Podophyllum hexandrum (Himalayan mayapple, Berberidaceae) was found to catalyze the cyclization of a dibenzylbutyrolactone lignan, yatein, to give deoxypodophyllotoxin and designated as deoxypodophyllotoxin synthase (DPS). Recently, we reported that the biosynthesis of deoxypodophyllotoxin and podophyllotoxin evolved in a lineage-specific manner in phylogenetically unrelated plant species such as P. hexandrum and Anthriscus sylvestris (cow parsley, Apiaceae). Therefore, a comprehensive understanding of the characteristics of DPSs that catalyze the cyclization of yatein to deoxypodophyllotoxin in various plant species is important. However, for plant species other than P. hexandrum , the isolation of the DPS enzyme gene and the type of the enzyme, e.g. whether it is 2-ODD or another type of enzyme such as cytochrome P-450, have not been reported. In this study, we report the identification and characterization of A. sylvestris DPS (AsDPS). Phylogenetic analysis showed that AsDPS belonged to the 2-ODD superfamily and shared moderate amino acid sequence identity (40.8%) with P. hexandrum deoxypodophyllotoxin synthase (PhDPS). Recombinant protein assay indicated that AsDPS and PhDPS differ in terms of the selectivity of substrate enantiomers. Protein modeling using AlphaFold2 and site-directed mutagenesis indicated that the Tyr305 residue of AsDPS probably contributes to substrate recognition. This study advances our understanding of the podophyllotoxin biosynthetic pathway in A. sylvestris and provides new insight into 2-ODD involved in plant secondary (specialized) metabolism. [ABSTRACT FROM AUTHOR]

The mechanism of thermostabilization of GH10 xylanase, XynR, from Bacillus sp. strain TAR-1 by the mutation of S92 to E was investigated. Thermodynamic analysis revealed that thermostabilization was driven by the decrease in entropy change of activation for thermal inactivation. Crystallographic analysis suggested that this mutation suppressed the fluctuation of the amino acid residues at position 92-95. [ABSTRACT FROM AUTHOR]

The covalent conjugation of a 14-carbon fatty acid (myristic acid) to the N-terminal Gly residue, termed N-myristoylation, occurs in some viral proteins to dictate their pathological function. This protein lipidation reaction, however, is monitored by host cytotoxic T lymphocytes that are capable of recognizing N-terminal lipopeptide fragments in the context of major histocompatibility complex (MHC) class I molecules. In a rhesus model of human AIDS, for example, the classical MHC class I allomorph, Mamu-B*05104, was shown to bind SIV Nef-derived 4-mer lipopeptides (myristic acid-Gly-Gly-Ala-Ile; C14nef4) and present them to the CD8+ T-cell line, SN45. These lipopeptides accommodated in MHC class I molecules expose much shorter peptide chains than conventional MHC class I-presented 8–10-mer peptides, and the molecular mechanisms by which αβ T-cell receptors (TCRs) recognize lipopeptides currently remain unclear. An X-ray crystallographic analysis of the SN45 TCR α and β heterodimer in a form that was co-crystallized with the C14nef4-bound Mamu-B*05104 complex indicated that the amide group of the N-myristoylated glycine residue offered a primary T-cell epitope by establishing a sole hydrogen bond between its nitrogen atom and the side chain of Glu at position 101 of CDR3β. Accordingly, the Glu to Ala mutation at this position resulted in the loss of lipopeptide recognition. On the other hand, TCRs were positioned remotely from the peptide portion of C14nef4, and strong interactions were not observed. Thus, these observations provide novel structural insights into lipopeptide recognition by TCRs, which contrast sharply with the general molecular principle of peptide recognition. [ABSTRACT FROM AUTHOR]

Glycosaminoglycans (GAGs) such as hyaluronan and chondroitin in animal extracellular matrices contain disaccharide-repeating units. In a Gram-negative pathogenic Streptobacillus moniliformis, which belongs to Fusobacteria phylum and resides in rodent oral cavities, the solute-binding protein (Smon0123)-dependent ATP-binding cassette transporter imports unsaturated hyaluronan/chondroitin disaccharides into the cytoplasm after GAG lyase-dependent depolymerization. Here we show substrate recognition of unsaturated hyaluronan disaccharide by Smon0123. Moreover, Smon0123 exhibited no affinity for unsaturated chondroitin disaccharides containing three sulfate groups, distinct from non-sulfated, mono-sulfated, and di-sulfated chondroitin disaccharides previously identified as substrates. Crystal structure of Smon0123 with unsaturated hyaluronan disaccharide demonstrates that several residues, including Trp284 and Glu410, are crucial for binding to unsaturated hyaluronan/chondroitin disaccharides, whereas arrangements of water molecules at binding sites are found to be substrate dependent through comparison with substrate-bound structures determined previously. These residues are well conserved in Smon0123-like proteins of fusobacteria, and probably facilitate the fusobacterial residence in hyaluronan-rich oral cavities. Import of hyaluronan/chondroitin by bacteria. [ABSTRACT FROM AUTHOR]

Aspergillus oryzae RIB40 formate oxidase has Arg87 and Arg554 near the formyl group and O(4) atom of 8-formyl-flavin adenine dinucleotide (FAD), respectively, with Asp396 neighbouring Arg554. Herein, we probed the roles of these three residues in modification of FAD to 8-formyl-FAD. Replacement of Arg87 or Arg554 with Lys or Ala decreased and abolished the modification, respectively. Replacement of Asp396 with Ala or Asn lowered the modification rate. The observation of unusual effects of maintaining pH 7.0 on the modification in R87K, R554K and D396 variants indicates initial and subsequent processes with different pH dependencies. Comparison of the initial process at pH 4.5 and 7.0 suggests that the microenvironment around Arg87 and the protonation state of Asp396 affect the initial process in the native enzyme. Comparison of the crystal structures of native and R554 variants showed that the replacements had minimal effect on catalytic site structure. The positively charged Arg87 might contribute to the formation of an anionic quinone-methide tautomer intermediate, while the positively charged Arg554, in collaboration with the negatively charged Asp396, might stabilize this intermediate and form a hydrogen bonding network with the N(5)/O(4) region, thereby facilitating efficient FAD modification. [ABSTRACT FROM AUTHOR]

The article focuses on the study which describes the structure and oxidation-reduction potential (ORP) of the 8-replaced FAD with molecular mass 0f 799 as cofactor. It says that the hydrogen-nuclear magnetic resonance (H-NMR) spectrum of the cofactor fraction from enzyme showed that the 8-replaced FAD was 8-formyl-FAD. It adds that the open and hemiacetal forms of the oxidation-reduction potentials were estimated by cyclic voltammetry.

The article looks into the structures, sorting determinants, and protein engineering of seed storage proteins and crop development with engineered seed proteins. It discusses a strategy for the development of transgenic crops to utilize seed protein sources that are used to prevent lifestyle-related diseases and improve human health by consumption. It notes the significance of protein engineering in designing seed storage proteins that contain bioactive peptides which improve human health.

The article presents a study concerning soybean basic 7S globulin. It mentions that basic 7S globulin is a cysteine-rich protein from soybean seeds comprised of subunits containing 27 kD and 16 kD chains connected by disulfide bonding. It notes that the basic 7S globulin is characterized as a member of a superfamily of structurally linked but functionally unique proteins descended from certain group of plant aspartic proteinases.

The article presents a study on the structural and functional characteristics of recombinant human serum transferrin produced in the yeast Pichia pastoris. The study showed high-level secretion with the use of signal sequence of the a factor of the yeast Saccharomyces cerevisiae. Moreover, the use of matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy analysis has helped determine carbohydrate modification.

The article presents a study which aims to understand the crystal structure of a cold-active alkaline phosphatase (SCAP) from psychrophile, which elicits high catalytic efficiency at low temperatures. It says that the study used Shewanella sp., and the crystal structure of SCAP was determined by X-ray analysis. Results show that the SCAP molecule consisted of crown and core domains and highly conserved in alkaline phosphatase, and the refined model shows a homodimer with six metal ligand sites.

Aminoacyl-tRNA synthetase forms an enzyme-bound intermediate, aminoacyladenylate in the amino-acid activation reaction. This reaction is monitored by measuring the ATP-PPi exchange reason in which [32P]PPi is incorporated into ATP. We previously reported that l-lysine hydroxamate completely inhibited the l-lysine-dependent ATP-PPi exchange reaction catalysed by lysyl-tRNA synthetase from Bacillus stearothermophilus (BsLysRS). Several experiments suggested that BsLysRS can adenylate l-lysine hydroxamate, but the enzyme-bound lysyladenylate-like compound does not undergo the nucleophilic attack of PPi. This contrasts with the two reports for seryl-tRNA synthetase (SerRS): (i) l-serine hydroxamate was utilized by yeast SerRS as a substrate in the ATP-PPi exchange; and (ii) a seryladenylate-like compound was formed from l-serine hydroxamate in the crystal structure of Thermus thermophilus SerRS. To gain clues about the mechanistic difference, we have determined the crystal structures of two complexes of BsLysRS with the adenylate of l-lysine hydroxamate and with 5′-O-[N-(l-Lysyl)sulphamoyl] adenosine. The comparisons of the two BsLysRS structures and the above SerRS structure revealed the specific side-chain shift of Glu411 of BsLysRS in the complex with the adenylate of l-lysine hydroxamate. In support of other structural comparisons, the result suggested that Glu411 plays a key role in the arrangement of PPi for the nucleophilic attack. [ABSTRACT FROM PUBLISHER]

The article reports on Sphingomonas species A1, a pit-forming bacterium that directly incorporates a macro-molecule into its cytoplasm through a pit-dependent transport system, termed a superchannel. It describes the superchannel and argues for its biological significance, including genome sequence, cell surface flagellin, alginate lyases and alginate-binding proteins.

Examines the structure and function of the bacterial biosystem responsible for import and depolymerization of macromolecules. Importance of cell surface in the acceptance and transport of molecules, energy and signals; Establishment of a novel concept regarding the import of macromolecules into organisms; Details on the structural and functional aspects of the superchannel and polysaccharide depolymerization system.

The cold-active protein-tyrosine phosphatase (CAPTPase) of a psychrophile, Shewanella sp., shows high catalytic activity below 20°C. The catalytic residue of CAPTPase is histidine, as opposed to the cysteine of known protein-tyrosine phosphatases (PTPases), and the enzyme protein has three amino acid sequences, Asp-Xaa-His, Gly-Asp-Xaa-Xaa-Asp-Arg and Gly-Asn-His-Glu, that are observed in many protein-serine/threonine phosphatases (PS/TPases). We have determined the crystal structures of CAPTPase at 1.82 Å and the enzyme bound with a phosphate ion at 1.90 Å resolution using X-ray crystallography and the multiple isomorphous replacement method. The final refined models are comprised of 331 amino acid residues, two metal ions, 447 water molecules, and an acetate or phosphate ion in an asymmetric unit. The enzyme protein consists of three β-sheets, termed Sheet I, Sheet I′, and Sheet II, and 14 α-helices. The CAPTPase has a different overall structure from known protein-tyrosine phosphatases. The arrangement of two metal ions, a phosphate ion and the adjacent amino acid residues in the catalytic site of CAPTPase is identical to that of PS/TPases. Thus, it was confirmed that the CAPTPase was a novel PTPase with a conformation similar to the catalytic site of PS/TPase. We speculate that the hydrophobic moiety around the catalytic residue of CAPTPase might play an important role in eliciting high activity at low temperature. [ABSTRACT FROM PUBLISHER]

A novel enzyme, pheophorbidase, which catalyzes the conversion of pheophorbide a to C-132-carboxylpyropheophorbide a, was purified from Chenopodium album leaves. The purified enzyme showed two bands of 28 kDa and 29 kDa on SDS-PAGE. The molecular mass of the native pheophorbidase was 105 kDa. The N-terminal amino acid sequence for the 28-kDa protein could be determined, whereas the N-terminus of the 29-kDa protein was blocked. Immunochemical and enzyme activity analyses revealed that pheophorbidase is located in an extra-plastidic part of the cell. [ABSTRACT FROM AUTHOR]

Alanine racemase of Bacillus stearothermophilus has been proposed to catalyze alanine racemization by means of two catalytic bases: lysine 39 (K39) abstracting specifically the α-hydrogen of D-alanine and tyrosine 265 (Y265) playing the corresponding role for the antipode L-alanine. The role of K39 as indicated has already been verified Watanabe, A., Kurokawa, Y., Yoshimura, T., Kurihara, T., Soda, K., and Esaki, N. (1999) J. Biol. Chenu 274, 4189–4194. We here present evidence for the functioning of Y265 as the base catalyst specific to L-alanine. The Y265↑Ala mutant enzyme (Y265A), like Y265S and Y265F, was a poor catalyst for alanine racemization. However, Y265A and Y265S catalyzed trans-amination with D-alanine much more rapidly than the wild-type enzyme, and the bound coenzyme, pyridoxal 5'-phosphate (PLP), was converted to pyridoxamine 5' -phosphate (PMP). The rate of transamination catalyzed by Y265F was about 9% of that by the wild-type enzyme. However, Y265A, Y265S, and Y265F were similar in that L-alanine was inert as a substrate in transamination. The apo-form of the wild-type enzyme catalyzes the abstraction of tritium non-specifically from both (4'S)- and (4'R)-[4'-3H]PMP in the presence of pyruvate. In contrast, apo-Y265A abstracts tritium virtually from only the R-isomer. This indicates that the side-chain of Y265 abstracts the α-hydrogen of L-alanine and transfers it supra-facially to the pro-S position at C-4' of PMP. Y265 is the counterpart residue to K39 that transfers the α-hydrogen of D-alanine to the pro-R position of PMP. [ABSTRACT FROM AUTHOR]

Chlorophyllase (Chlase) was purified from mature leaves of Chenopodium album, and its enzymatic properties were investigated. Chlase was extracted from acetone powder of C. album and purified by the following chroma-tographic procedures: hydrophobic chromatography, Con A Sepharose, Heparin affinity chromatography, Mono Q ion-exchange chromatography, and gel-filtration. Con A Sepharose affinity chromatography and gel-filtration were the most effective steps on the purification. On Mono Q chromatography, the Chlase preparation separated into two major and one minor fractions that exhibited Chlase activity. The two major Chlases were purified to homogeneity. Their molecular masses were estimated as 41.3 kDa and 40.2 kDa by SDS-PAGE. The optimum pH and Km values of these two Chlases were similar. Their N-terminal amino acid sequences were almost identical except for a deletion in the tenth amino acid residue in one of the Chlase; there was no homologous protein detected by database search. [ABSTRACT FROM AUTHOR]

The crystal and molecular structure of Lipase II from Rhizopus niveus was analyzed using X-ray single crystal diffraction data at a resolution of 2.2 Å. The structure was refined than R-factor of 0.19 for all available data. This lipase was purified and crystallized as Lipase I, which contains two polypeptide chains combined through non-covalent interaction. However, during crystal growth, Lipase I was converted to Lipase II, which consists of a single polypeptide chain of 269 amino acid residues, by limited proteolysis. The structure of Lipase II shows a typical α/β hydrolase fold containing the so-called nucleophilic elbow. The catalytic center of this enzyme is analogous to those of other neutral lipases and serine proteases. This catalytic center is sheltered by an α-helix lid, which appears in neutral lipases, opening the active site at the oil-water interface. [ABSTRACT FROM AUTHOR]

The article discusses a study which examines the crystal structure of pyridoxal 4-dehydrogenase (PLDH) of Mesorhizobium loti. It explores the crystal parameters and diffraction data statistics of PLDH which include detector, wavelength, and redundancy. It also highlights the molecular replacement of PLDH through a software program.

N-Acyl-D-glucosamine 2-epimerase from porcine kidney, which was cloned and expressed in Escherichia coli, was crystallized by the vapor-diffusion method, using polyethylene glycol and ammonium acetate as precipitants. The crystals were resistant to X-ray radiation damage and diffracted to more than 2.0 Å resolution. The diffraction pattern indicated that the crystals belong to the orthorhombic system, space group P21,21 21 with unit-cell dimensions of a=78.1, b=97.2, and c=100.7 Å. It is supposed that the asymmetric unit consists of two N-acyl-D-glucosamine 2-epimerase molecules. Collection of data on the native crystals indicated that they are suitable for X-ray structural analysis. [ABSTRACT FROM AUTHOR]