Klaus Sauber

Identification of the bile acid-binding site of the ileal lipid-binding protein by photoaffinity labeling, matrix-assisted laser desorption ionization-mass spectrometry, and NMR structure.

The ileal lipid-binding protein (ILBP) is the only physiologically relevant bile acid-binding protein in the cytosol of ileocytes. To identify the bile acid-binding site(s) of ILBP, recombinant rabbit ILBP photolabeled with 3-azi- and 7-azi-derivatives of cholyltaurine was analyzed by a combination of enzymatic fragmentation, gel electrophoresis, and matrix-assisted laser desorption ionization (MALDI)-mass spectrometry. The attachment site of the 3-position of cholyltaurine was localized to the amino acid triplet His100-Thr101-Ser102using the photoreactive 3,3-azo-derivative of cholyltaurine. With the corresponding 7,7-azo-derivative, the attachment point of the 7-position could be localized to the C-terminal part (position 112–128) as well as to the N-terminal part suggesting more than one binding site for bile acids. By chemical modification and NMR structure of ILBP, arginine residue 122 was identified as the probable contact point for the negatively charged side chain of cholyltaurine. Consequently, bile acids bind to ILBP with the steroid nucleus deep inside the protein cavity and the negatively charged side chain near the entry portal. The combination of photoaffinity labeling, enzymatic fragmentation, MALDI-mass spectrometry, and NMR structure was successfully used to determine the topology of bile acid binding to ILBP.

Structure-based prediction of modifications in glutarylamidase to allow single-step enzymatic production of 7-aminocephalosporanic acid from cephalosporin C

Glutarylamidase is an important enzyme employed in the commercial production of 7‐aminocephalosporanic acid, a starting compound in the synthesis of cephalosporin antibiotics. 7‐aminocephalosporanic acid is obtained from cephalosporin C, a natural antibiotic, either chemically or by a two‐step enzymatic process utilizing the enzymes D‐amino acid oxidase and glutarylamidase. We have investigated possibilities for redesigning glutarylamidase for the production of 7‐aminocephalosporanic acid from cephalosporin C in a single enzymatic step. These studies are based on the structures of glutarylamidase, which we have solved with bound phosphate and ethylene glycol to 2.5 Å resolution and with bound glycerol to 2.4 Å. The phosphate binds near the catalytic serine in a way that mimics the hemiacetal that develops during catalysis, while the glycerol occupies the side‐chain binding pocket. Our structures show that the enzyme is not only structurally similar to penicillin G acylase but also employs essentially the same mechanism in which the α‐amino group of the catalytic serine acts as a base. A subtle difference is the presence of two catalytic dyads, His B23/Glu B455 and His B23/Ser B1, that are not seen in penicillin G acylase. In contrast to classical serine proteases, the central histidine of these dyads interacts indirectly with the Oγ through a hydrogen bond relay network involving the α‐amino group of the serine and a bound water molecule. A plausible model of the enzyme–substrate complex is proposed that leads to the prediction of mutants of glutarylamidase that should enable the enzyme to deacylate cephalosporin C into 7‐aminocephalosporanic acid.

The Tendamistat Expression-Secretion System: Synthesis of Proinsulin Fusion Proteins with Streptomyces Lividans

Tendamistat (HOE 467), a potent inhibitor of the human pancreatic α-amylase, is an acidic protein of 74 amino acids (Vertesy et al., 1984). The inhibitor gene was cloned from an amplified genomic sequence of an over-producing strain of Streptomyces tendae and further characterized. By expression of the gene in the heterologous host Streptomyces lividans we demonstrated that secretion of this protein was mediated by a signal peptide dependent mechanism (Koller & Ries, 1989). We have also evaluated the tendamistat-based secretion system for a number of foreign proteins, for example interleukin II and proinsulin (Bender et al., 1990, Koller et al., 1989). Our main interest focussed on the formation of disulphide bonds, stability and activity of secreted proteins.