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Purification of the coenzyme B12-dependent isobutyryl-CoA mutase (ICM) from Streptomyces cinnamonensis gave a protein of approximately 65 kDa by SDS-polyacrylamide gel electrophoresis, whose gene icmA was cloned using sequences derived from tryptic peptide fragments. The gene encodes a protein of 566 residues (62, 487 Da), with 43-44% sequence identity to the large subunit of methylmalonyl-CoA mutase (MCM) from S. cinnamonensis and Propionibacterium shermanii. Targeted disruption of the icmA gene yielded an S. cinnamonensis mutant devoid of ICM activity. The IcmA protein is approximately 160 residues shorter than the large subunit of the bacterial MCMs, corresponding to a loss of the entire C-terminal coenzyme B12 binding domain. The sequence of the (beta/alpha)8-barrel comprising residues A1-A400 in P. shermanii MCM is highly conserved in IcmA. The protein was produced in Streptomyces lividans and Escherichia coli with an N-terminal His6 tag (His6-IcmA), but after purification His6-IcmA showed no ICM activity. In the presence of coenzyme B12, protein from S. lividans and S. cinnamonensis of approximately 17 kDa by SDS-polyacrylamide gel electrophoresis could be selectively eluted with His6-IcmA from a Ni2+ affinity column. After purification, this small subunit showed no ICM activity but gave active enzyme when recombined with coenzyme B12 and IcmA or His6-IcmA.

The extracellular interferon gamma receptor alpha-chain comprises two immunoglobulin-like domains, each with fibronectin type-III topology, which are responsible for binding interferon gamma at the cell surface. The epitopes on the human receptor recognized by three neutralizing antibodies, A6, gammaR38 and gammaR99, have been mapped by homolog scanning mutagenesis. In this way, a loop connecting beta-strands C and C' in the N-terminal domain was identified as a key component of the epitopes bound by A6 and gammaR38, whereas gammaR99 binds to the C-terminal domain in a region including strands A and B and part of the large C'E loop. The epitope for A6 was confirmed in a crystal structure of a complex between a recombinant N-terminal receptor domain and the Fab fragment from A6, determined by X-ray diffraction to 2.8 A resolution. The antibody-antigen interface buries 1662 A2 of protein surface, including 22 antibody residues from five complementarity determining regions, primarily through interactions with the CC' surface loop of the receptor. The floor of the antigen binding cavity is formed mainly by residues from CDR L3 and CDR H3 while a surrounding ridge is formed by residues from all other CDRs except L2. Many potential polar interactions, as well as 13 aromatic side-chains, four in VL, six in VH and three in the receptor, are situated at the interface. The surface of the receptor contacted by A6 overlaps to a large extent with that contacted by interferon-gamma, in the ligand-receptor complex. However, the conformation of this epitope is very different in the two complexes, demonstrating that conformational mobility in a surface loop on this cytokine receptor permits steric and electrostatic complementarity to two quite differently shaped binding sites.

The catabolism of branched chain amino acids, especially valine, appears to play an important role in furnishing building blocks for macrolide antibiotic biosynthesis. To determine for the first time the importance of valine dehydrogenase (vdh) in polyether antibiotic biosynthesis, the vdh gene from Streptomyces cinnamonensis has been cloned and sequenced. The enzyme (M(r)37,718 Da) has been produced in large amounts in an active form in the E. coli cytoplasm using a T7 RNA-polymerase expression system. Upon inactivation of the gene in S. cinnamonensis by a double-crossover mechanism, a hyg::vdh mutant was isolated that was devoid of vdh activity. Upon growth in chemically defined media, as well as a complex medium optimised for monensin production, the mutant and wild-type grew equally well and reached the same levels of monensin production. In both strains a valine transaminase activity could be detected that provides an alternative route for converting valine into 2-oxoisovaleric acid. The results show that vdh is not essential for normal growth of S. cinnamonensis, and its inactivation does not significantly affect normal levels of monensin production in this strain.