Single amino acid mutations interchange the reaction specificities of cyclodextrin glycosyltransferase and the acarbose-modifying enzyme acarviosyl transferase

Acarviosyl transferase (ATase) from Actinoplanes sp. SE50/110 is a bacterial enzyme that transfers the acarviosyl moiety of the diabetic drug acarbose to sugar acceptors. The enzyme exhibits 42% sequence identity with cyclodextrin glycosyltransferases (CGTase), and both enzymes are members of the α-amylase family, a large clan of enzymes acting on starch and related compounds. ATase is virtually inactive on starch, however. In contrast, ATase is the only known enzyme to efficiently use acarbose as substrate (2 µmol min-1 mg-1); acarbose is a strong inhibitor of CGTase and of most other α-amylase family enzymes. This distinct reaction specificity makes ATase an interesting enzyme to investigate the variation in reaction specificity of α-amylase family enzymes. Here we show that a G140H mutation in ATase, introducing the typical His of the conserved sequence region I of the α-amylase family, changed ATase into an enzyme with 4-α-glucanotransferase activity (3.4 µmol min-1 mg-1). Moreover, this mutation introduced cyclodextrin-forming activity into ATase, converting 2% of starch into cyclodextrins. The opposite experiment, removing this typical His side chain in CGTase (H140A), introduced acarviosyl transferase activity in CGTase (0.25 µmol min-1 mg-1).

Stabilitaet und Verbleib von rekombinanten Organismen bzw. Nucleinsaeuren in Forschungslabors und unter industriellen Produktionsbedingungen Abschlussbericht

As a contribution to unanswered safety questions when employing genetic modified organisms the following developments are presented: (i) specific oligonucleotide probes and PCR samples for the detection of genetically engineered streptomyces, (ii) isolation and characterization of dimethyl sulfoxide degradating oxidase/reductase from Hyphomicrobium sp. and specific probes/PCR primers for Hyphomicrobium, (iii) new shuttle vectors for E. coli and Streptomyces containing polylinkers and lacZ casettes for blue/white selection on x-gal plates in E. coli, (iv) cloning and sequence analysis of the mel operons for melanin biosynthesis from Streptomyces. Research is continued on the expression of human kallikrein protein as a model system for expression and spreading in actinomycetes and on the development of gene probes for dimethyl sulfoxide degradating genes. (WEN)SIGLEAvailable from TIB Hannover: F96B688+a / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekBundesministerium fuer Bildung, Wissenschaft, Forschung und Technologie, Bonn (Germany)DEGerman

Crystal structures of the bacterial solute receptor AcbH displaying an exclusive substrate preference for beta D galactopyranose

Solute receptors or binding proteins are indispensable components of canonical ATP-binding cassette importers in prokaryotes. Here, we report on the characterization and crystal structures in the closed and open conformations of AcbH, the solute receptor of the putative carbohydrate transporter AcbFG which is encoded in the acarbose (acarviosyl-1,4-maltose) biosynthetic gene cluster from Actinoplanes sp. SE50/110. Binding assays identified AcbH as a high affinity monosaccharide-binding protein with a dissociation constant (K(d)) for beta-D-galactopyranose of 9.8 ± 1.0 nM. Neither galactose-containing di- and trisaccharides, such as lactose and raffinose, nor monosaccharides including D-galacturonic acid, L-arabinose, D-xylose and L-rhamnose competed with (14)C-galactose for binding to AcbH. Moreover, AcbH does not bind D-glucose which is a common property of all but one D-galactose-binding proteins characterized to date. Strikingly, determination of the X-ray structure revealed that AcbH is structurally homologous to maltose binding proteins rather than to glucose binding proteins. In the substrate binding pocket, two helices are inserted which reduce the cavity size and allow the exclusive binding of monosaccharides, specifically beta-D-galactopyranose, in the (4)C(1) conformation. Site directed mutagenesis of three residues from the binding pocket (Arg82, Asp361, Arg362) which interact with the axially oriented O4-H hydroxyl of the bound galactopyranose and subsequent functional analyses indicate that these residues are crucial for galactose binding. To our knowledge, this is the first report on the tertiary structure of a solute receptor with exclusive affinity for beta-D-galactopyranose. The putative role of a galactose import system in the context of acarbose metabolism in Actinoplanes sp. is discussed