Screening for improved activity of a transglutaminase from Streptomyces mobaraensis created by a novel rational mutagenesis and random mutagenesis

Microbial transglutaminase (MTG) has been used extensively in academic research and the food industries through its cross-linking or posttranslational modification of proteins. To improve MTGT, a novel method of rational mutagenesis, called WASH-ROM (Water Accessible Surface Hot-space Region Oriented Mutagenesis), was first attempted. Based on the three-dimensional structure of MTG, 151 point mutations were selected at 40 different residues bearing high solvent accessibility surface area, within a 15 Å of the active center site nucleophile, Cys64. Among them, 32 mutants showed higher specific activity than the wild type enzyme. We found that beneficial mutations are distributed in two regions and with distinctive amino acid substitutions. Next, random mutagenesis was applied to the entire MTG region by developing a new plate assay-based screening system, using Corynebacterium glutamicum as the secretion host strain. This in vivo screening system allowed us to readily distinguish the change in enzymatic activity upon mutation by monitoring the intensity of enzymatic reaction-derived color zones which appeared around the recombinant cell colonies on the plate. From the library of 24,000 clones, 10 mutants were finally selected as beneficial enzymes exhibiting higher specific activity than wild type. Notably, most of the mutations differed from those obtained by WASH-ROM, except for H289Y. Beneficial mutations were distributed in two other regions as well. Furthermore, we found that the FRAP-S199A mutant (FRAP: N-terminal four amino acid residues extension) showed the highest specific activity (45 U/mg: 1.7 times higher than the wild type enzyme). Through these different mutation approaches, various beneficial positions leading to increased specific activity of MTG were surveyed

Herpesvirus Protease Inhibition by Dimer Disruption

Kaposi's sarcoma-associated herpesvirus (KSHV), like all herpesviruses, encodes a protease (KSHV Pr), which is necessary for the viral lytic cycle. Herpesvirus proteases function as obligate dimers; however, each monomer has an intact, complete active site which does not interact directly with the other monomer across the dimer interface. Protein grafting of an interfacial KSHV Pr α-helix onto a small stable protein, avian pancreatic polypeptide, generated a helical 30-amino-acid peptide designed to disrupt the dimerization of KSHV Pr. The chimeric peptide was optimized through protein modeling of the KSHV Pr-peptide complex. Circular dichroism analysis and gel filtration chromatography revealed that the rationally designed peptide adopts a helical conformation and is capable of disrupting KSHV Pr dimerization, respectively. Additionally, the optimized peptide inhibits KSHV Pr activity by 50% at a ∼200-fold molar excess of peptide to KSHV Pr, and the dissociation constant was estimated to be 300 μM. Mutagenesis of the interfacial residue M197 to a leucine resulted in an inhibitory concentration which was twofold higher for KSHV Pr M197L than for KSHV Pr, in agreement with the model that the dimer interface is involved in peptide binding. These results indicate that the dimer interface, as well as the active sites, of herpesvirus proteases is a viable target for inhibiting enzyme activity