A novel milk-clotting enzyme from Aspergillus oryzae and A. luchuensis is an aspartic endopeptidase PepE presumed to be a vacuolar enzyme

Aspergillus oryzae RIB40 has 11 aspartic endopeptidase genes. We searched for milk-clotting enzymes based on the homology of the deduced amino acid sequence with chymosins. As a result, we identified a milk-clotting enzyme in A. oryzae. We expected other Aspergillus species to have a homologous enzyme with milk-clotting activity, and we found the most homologous aspartic endopeptidase from A. luchuensis had milk-clotting activity. Surprisingly, two enzymes were considered as vacuole enzymes according to a study on A. niger proteases. The two enzymes from A. oryzae and A. luchuensis cleaved a peptide between the 105Phe-106Met bond in κ-casein, similar to chymosin. Although both enzymes showed proteolytic activity using casein as a substrate, the optimum pH values for milk-clotting and proteolytic activities were different. Furthermore, the substrate specificities were highly restricted. Therefore, we expected that the Japanese traditional fermentation agent, koji, could be used as an enzyme source for cheese production.

Machine learning discovery of missing links that mediate alternative branches to plant alkaloids

Engineering the microbial production of secondary metabolites is limited by the known reactions of correctly annotated enzymes in sequence databases. To expand the range of biosynthesis pathways, machine learning is herein demonstrated for the discovery of missing link enzymes, using benzylisoquinoline alkaloid production as a model application with potential to revolutionize the paradigm of sustainable biomanufacturing. Bacterial studies utilize a tetrahydropapaveroline pathway, whereas plants are reported to contain a more stable norcoclaurine pathway, which is exploited in yeast. However, committed aromatic precursors are currently produced by microbial enzymes that remain elusive in plants. Accordingly, the machine learning enzyme selection algorithm is first applied to clarify the early missing links in plant alkaloid pathways. Characterization of predicted sequences via metabolomics reveals distinct oxidases and carboxy-lyases, which complete a plant gene-only benzylisoquinoline alkaloid pathway from tyrosine. Synergistic application of aryl acetaldehyde producing enzymes results in enhanced production through hybrid norcoclaurine and tetrahydropapaveroline pathways. Transplantation of features into homologous enzyme templates leads to the highest levels of bacterial norcoclaurine and N-methylcoclaurine. Mechanism-directed isotope tracing patterns confirm alternative flux branches from aromatic precursors to alkaloids. This machine learning-driven workflow can be adapted to numerous pathways.

Structure of an unusualS-adenosylmethionine synthetase fromCampylobacter jejuni

S-Adenosylmethionine (AdoMet) participates in a wide range of methylation and other group-transfer reactions and also serves as the precursor for two groups of quorum-sensing molecules that function as regulators of the production of virulence factors in Gram-negative bacteria. The synthesis of AdoMet is catalyzed by AdoMet synthetases (MATs), a ubiquitous family of enzymes found in species ranging from microorganisms to mammals. The AdoMet synthetase from the bacteriumCampylobacter jejuni(cjMAT) is an outlier among this homologous enzyme family, with lower sequence identity, numerous insertions and substitutions, and higher catalytic activity compared with other bacterial MATs. Alterations in the structure of this enzyme provide an explanation for its unusual dimeric quaternary structure relative to the other MATs. Taken together with several active-site substitutions, this new structure provides insights into its improved kinetic properties with alternative substrates.

A Novel Mutation in the Transglutaminase-1 Gene in an Autosomal Recessive Congenital Ichthyosis Patient

Structure-function implication on a novel homozygous Trp250/Gly mutation of transglutaminase-1 (TGM1) observed in a patient of autosomal recessive congenital ichthyosis is invoked from a bioinformatics analysis. Structural consequences of this mutation are hypothesized in comparison to homologous enzyme human factor XIIIA accepted as valid in similar structural analysis and are projected as guidelines for future studies at an experimental level on TGM1 thus mutated.

Biosynthesis of Galactofuranose in Kinetoplastids: Novel Therapeutic Targets for Treating Leishmaniasis and Chagas' Disease

Cell surface proteins of parasites play a role in pathogenesis by modulating mammalian cell recognition and cell adhesion during infection. β-Galactofuranose (Galf) is an important component of glycoproteins and glycolipids found on the cell surface of Leishmania spp. and Trypanosoma cruzi. β-Galf-containing glycans have been shown to be important in parasite-cell interaction and protection against oxidative stress. Here, we discuss the role of β-Galf in pathogenesis and recent studies on the Galf-biosynthetic enzymes: UDP-galactose 4′ epimerase (GalE), UDP-galactopyranose mutase (UGM), and UDP-galactofuranosyl transferase (GalfT). The central role in Galf formation, its unique chemical mechanism, and the absence of a homologous enzyme in humans identify UGM as the most attractive drug target in the β-Galf-biosynthetic pathway in protozoan parasites.