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 ␣-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 ␣-glucosidases and/or transglycosylases of the ␣-amylase family and exhibits the highest similarities to several cyclodextrin glucanotransferases (CGTases). However, AcbD had neither ␣-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°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.
Weak D is recently reported to be encoded by 14 different weak D genotypes. The amount of RHD mRNA in donors expressing weak D was measured in previous studies. By semiquantitative RT‐PCR analysis Rouillac et al. (Blood 1996) found lower RHD mRNA levels in donors expressing weak D compared to normal RhD donors, whereas Beckers et al. (Transfusion 1997) described equal mRNA levels. The aim of the present study is to explore whether the pointmutations found in donors carrying the weak D phenotype can be responsible for weak D expression. Real‐time quantitative PCR analysis showed that levels of RHD mRNA from donors expressing different weak D genotypes (type 1, 2 and 3) did not differ from RHD mRNA levels from normal heterozygous RhD donors. This indicates that the mutations found in weak D genotypes do not affect RhD expression on transcription level. To investigate whether the described mutations influence translation of mRNA into RhD protein or the configuration of the RhD protein, weak D type 1 cDNA (T809G) and weak D type 3 cDNA (C8G) was transfected in K562 cells. Preliminary results indicate that the ratios between RhD expression and RHD mRNA level (RhD expression/RHD mRNA level) do not differ between weak D type 1 and normal D K562 cells, but are reduced in weak D type 3 transfectants compared to normal D K562 cells. This suggests that in K562 neither the translation nor the configuration of RhD is influenced by the mutation T809G. There might be a missing or limited factor in the K562 model involved in the expression of RhD, hampering the interpretation of the weak D type 1. However, the lower expression of RhD with the mutation C8G might be caused by changed configuration or changed intracellular transport to the membrane.
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