Yoshihiro Asada

Cloning and Expression of 1,2-α-Mannosidase Gene (fmanIB) from Filamentous Fungus Aspergillus oryzae: in Vivo Visualization of the FmanIBp-GFP Fusion Protein

1,2-α-Mannosidase catalyzes the specific cleavage of 1,2-α-mannose residues from protein-linked N-glycan. In this study, a novel DNA sequence homologous to the authentic 1,2-α-mannosidase was cloned from a cDNA library prepared from solid-state cultured Aspergillus oryzae. The fmanIB cDNA consisted of 1530 nucleotides and encoded a protein of 510 amino acids in which all consensus motifs of the class I α-mannosidase were conserved. Expression of the full length of 1,2-α-mannosidase cDNA by the Aspergillus host, though it has rarely been done with other filamentous-fungal mannosidase, was successful with fmanIB and caused an increase in both intracellular and extracellular mannosidase activity. The expressed protein (FmanIBp) specifically hydrolyzed 1,2-α-mannobiose with maximal activity at a pH of 5.5 and a temperature of 45 °C. With Man9GlcNAc2 as the substrate, Man5GlcNAc2 finally accumulated while hydrolysis of the 1,2-α-mannose residue of the middle branch was rate-limiting. To examine the intracellular localization of the enzyme, a chimeric protein of FmanIBp with green fluorescent protein was constructed. It showed a dotted fluorescence pattern in the mycelia of Aspergillus, indicative of the localization in intracellular vesicles. Based on these enzymatic and microscopic results, we estimated that FmanIBp is a fungal substitute for the mammalian Golgi 1,2-α-mannosidase isozyme IB. This and our previous report on the presence of another ER-type mannosidase in A. oryzae (Yoshida et al., 2000) support the notion that the filamentous fungus has similar steps of N-linked glycochain trimming to those in mammalian cells.

N-Carbamoyl-L-Cysteine as an Intermediate in the Bioconversion from D,L-2-Amino-Δ 2-Thiazoline-4-Carboxylic Acid to L-Cysteine by Pseudomonas sp. ON-4a

We investigated the conversion of D,L-2-amino-Δ (2)-thiazoline-4-carboxylic acid (D,L-ATC) to L-cysteine with Pseudomonas sp. ON-4a, an ATC-assimilating bacterium. Cysteine and N-carbamoylcysteine (NCC), but not S-carbamoylcysteine (SCC), were produced from D,L-ATC by a cell-free extract from the strain. These products were isolated from the reaction mixture and then identified as the L-form. Similar results were obtained with P. putida AJ3865 and unidentified strain TG-3, an ATC-assimilating bacteria. It became clear that L-NCC is an intermediate in the conversion of D,L-ATC to L-cysteine in these Pseudomonas strains. Furthermore, it was suggested that these bacteria have L-ATC hydrolase and L-NCC amidohydrolase.

Identification, Cloning, and Sequencing of the Genes Involved in the Conversion of D,L-2-Amino-Δ2-Thiazoline-4-Carboxylic Acid to …

The newly isolated strain Pseudomonas sp. ON-4a converts D,L-2-amino-Δ2-thiazoline-4-carboxylic acid to L-cysteine viaN-carbamoyl-L-cysteine. A genomic DNA fragment from this strain containing the gene(s) encoding enzymes that convert D,L-2-amino-Δ2-thiazoline-4-carboxylic acid into L-cysteine was cloned in Escherichia coli. Transformants expressing cysteine-forming activity were selected by growth of an E. coli mutant defective in the cysB gene. A positive clone, denoted CM1, carrying the plasmid pCM1 with an insert DNA of approximately 3.4 kb was obtained, and the nucleotide sequence of a complementing region was analyzed. Analysis of the sequence found two open reading frames, ORF1 and ORF2, which encoded proteins of 183 and 435 amino acid residues, respectively. E. coli DH5α harboring pTrCM1, which was constructed by inserting the subcloned sequence into an expression vector, expressed two proteins of 25 kDa and 45 kDa. From the analyses of crude extracts of E. coli DH5α carrying deletion derivatives of pTrCM1 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by enzymatic activity, it was found that the 25-kDa protein encoded by ORF1 was the enzyme L-2-amino-Δ2-thiazoline-4- carboxylic acid hydrolase, which catalyzes the conversion of L-2-amino-Δ2-thiazoline-4-carboxylic acid to N-carbamoyl-L-cysteine, and that the 45-kDa protein encoded by ORF2 was the enzyme N-carbamoyl-L-cysteine amidohydrolase, which catalyzes the conversion of N-carbamoyl-L-cysteine to L-cysteine.

Filamentous fungus Aspergillus oryzae has two types of α-1,2-mannosidases, one of which is a microsomal enzyme that removes a single mannose residue from Man9GlcNAc2

Abstractα-Mannosidase activities towards high-mannose oligosaccharides were examined with a detergent-solubilized microsomal preparation from a filamentous fungus, Aspergillus oryzae. In the enzymatic reaction, the pyridylaminated substrate Man9GlcNAc2-PA was trimmed to Man8GlcNAc2-PA which lacked one α-1,2-mannose residue at the nonreducing terminus of the middle branch (Man8B isomer), and this mannooligosaccharide remained predominant through the overall reaction. Trimming was optimal at pH 7.0 in PIPES buffer in the presence of calcium ion and kifunensine was inhibitory with IC50 below 0.1[emsp4 ]μM. These results suggest that the activity is the same type as was previously observed with human and yeast endoplasmic reticulum (ER) α-mannosidases. Considering these results together with previous data on a fungal α-1,2-mannosidase that trimmed Man9GlcNAc2 to Man5GlcNAc2 (Ichishima, E., et al. (1999) bit>Biochem J, 339: 589–597), the filamentous fungi appear to have two types of α-1,2-mannosidases, each of which acts differently on N-linked mannooligosaccharides.