Highly regioselective C-H functionalization/halogenation of acetanilides to produce ortho-haloacetanilides was catalyzed by Pd(OAc)2 and Cu(OAc) 2 with CuX2 as the halogen source.

SUMMARY The adenylation (A) domains of nonribosomal peptide synthetases (NRPSs) activate aryl acids or amino acids to launch their transfer through the NRPS assembly line for the biosynthesis of many medicinally important natural products. In order to expand the substrate pool of NRPSs, we developed a method based on yeast cell surface display to engineer the substrate specificities of the A-domains. We acquired A-domain mutants of DhbE that have 11- and 6-fold increases in kcat/Km with nonnative substrates 3-hydroxybenzoic acid and 2-aminobenzoic acid, respectively and corresponding 3- and 33-fold decreases in kcat/Km values with the native substrate 2,3-dihydroxybenzoic acid, resulting in a dramatic switch in substrate specificity of up to 200-fold. Our study demonstrates that yeast display can be used as a high throughput selection platform to reprogram the “nonribosomal code” of A-domains.

No abstract

SUMMARY Protein modification by ubiquitin (UB) controls diverse cellular processes. UB is conjugated to cellular proteins by sequential transfer through an E1-E2-E3 enzymatic cascade. The cross-activities of 2 E1s, 50 E2s and thousands of E3s encoded by the human genome make it difficult to identify the substrate proteins of a specific E3 enzyme in the cell. One way to solve this problem is to engineer an orthogonal UB transfer (OUT) cascade in which the engineered UB (xUB) is relayed by engineered E1, E2 and E3 enzymes (xE1, xE2, xE3) to modify the substrate proteins of a specific E3. Here, we use phage display and mutagenesis to construct xUB-xE1 and xE1-xE2 pairs that are orthogonal to the native E1 and E2 enzymes. Our work on engineering the UB transfer cascades will enable us to use OUT to map the signal transduction networks mediated by protein ubiquitination.

Ubiquitin (UB) is a protein modifier that regulates many essential cellular processes. To initiate protein modification by UB, the E1 enzyme activates the C-terminal carboxylate of UB to launch its transfer through the E1-E2-E3 cascade onto target proteins. In this study, we used phage display to profile the specificity of the two human E1 enzymes, Ube1 and Uba6, towards the C-terminal sequence of UB ending with 71LRLRGG76. Phage selection revealed that while Arg72 of UB is absolutely required for E1 recognition, UB residues at positions 71, 73 and 74 can be replaced with bulky aromatic side chains, and Gly75 of UB can be changed to Ser, Asp and Asn for efficient E1 activation. We have thus found that the E1 enzymes have substantial promiscuity regarding the UB C-terminal sequence. The UB variants from phage selection can also be transferred from E1 to E2 enzymes, however, they are blocked from further transfer to the E3 enzymes. This suggests that the C-terminal sequence of UB is important for its discharge from E2 and subsequent transfer to E3. In addition, we observed that the Leu73Phe and Leu73Tyr single mutants of UB are resistant to cleavage by deubiquitinating enzymes (DUBs), although they can be assembled by the E1-E2-E3 cascade into poly-UB chains, thus indicating differences in UB C-terminal specificities between the E1 and DUBs. Consequently these UB mutants may provide stability to UB polymers attached to cellular proteins and facilitate the elucidation of the biological signals encoded in the UB chains.