Hermann Pape

Identification, cloning, expression, and characterization of the extracellular acarbose-modifying glycosyltransferase, AcbD, from Actinoplanes sp. strain SE50.

An extracellular enzyme activity in the culture supernatant of the acarbose producer Actinoplanes sp. strain SE50 catalyzes the transfer of the acarviosyl moiety of acarbose to malto-oligosaccharides. This acarviosyl transferase (ATase) is encoded by a gene, acbD, in the putative biosynthetic gene cluster for the alpha-glucosidase inhibitor acarbose. The acbD gene was cloned and heterologously produced in Streptomyces lividans TK23. The recombinant protein was analyzed by enzyme assays. The AcbD protein (724 amino acids) displays all of the features of extracellular alpha-glucosidases and/or transglycosylases of the alpha-amylase family and exhibits the highest similarities to several cyclodextrin glucanotransferases (CGTases). However, AcbD had neither alpha-amylase nor CGTase activity. The AcbD protein was purified to homogeneity, and it was identified by partial protein sequencing of tryptic peptides. AcbD had an apparent molecular mass of 76 kDa and an isoelectric point of 5.0 and required Ca(2+) ions for activity. The enzyme displayed maximal activity at 30 degrees C and between pH 6.2 and 6.9. The K(m) values of the ATase for acarbose (donor substrate) and maltose (acceptor substrate) are 0.65 and 0.96 mM, respectively. A wide range of additional donor and acceptor substrates were determined for the enzyme. Acceptors revealed a structural requirement for glucose-analogous structures conserving only the overall stereochemistry, except for the anomeric C atom, and the hydroxyl groups at positions 2, 3, and 4 of D-glucose. We discuss here the function of the enzyme in the extracellular formation of the series of acarbose-homologous compounds produced by Actinoplanes sp. strain SE50.

Maltokinase (ATP: maltose 1-phosphotransferase) from Actinoplanes sp.: demonstration of enzyme activity and characterization of the reaction product

Cell free extract of the acarbose producer Actinoplanes sp. catalyzes ATP‐dependent phosphorylation of maltose. This was shown by two different assays. The product was purified and its structure determined to be α‐maltose‐1‐phosphate by chemical analysis and NMR spectroscopy.

The Genome of φAsp2, an Actinoplanes Infecting Phage

The first genome of a virus infecting a representative of the eubacterial genus Actinoplanes is presented. Phage φAsp2 has a circularly permutated chromosome that consists of 58,638 bp; its G/C-bias of 70.39% resembles the hosts G + C-content (71–73% within the genus). A total of 76 open reading frames (orfs) were identified, the majority of which (63) displaying equal transcriptional orientations. Functional gene clustering is obvious as orfs coding for head and tail proteins are located close to the center in the first half of the genome and putative DNA-modifying enzymes are encoded by centrally located genes; DNA repair and recombination functions are situated in the remaining part of the genome, adjacent to a small gene cluster, the predicted proteins of which are involved in DNA packaging. Close to the left terminus there are two small regions (approximately 4.5 kb each, separated by 2.8 kb) which are homologous to the recently sequenced mycobacteriophage rosebush, however, the unique overall structure of the φAsp2-genome does not bear resemblance to any other known viral genome. The nucleotide sequence was deposited in GenBank with the accession no. AY576796.