uPIC-M: Efficient and Scalable Preparation of Clonal Single Mutant Libraries for High-Throughput Protein Biochemistry.

New high-throughput biochemistry techniques complement selection-based approaches and provide quantitative kinetic and thermodynamic data for thousands of protein variants in parallel. With these advances, library generation rather than data collection has become rate-limiting. Unlike pooled selection approaches, high-throughput biochemistry requires mutant libraries in which individual sequences are rationally designed, efficiently recovered, sequence-validated, and separated from one another, but current strategies are unable to produce these libraries at the needed scale and specificity at reasonable cost.

Formylglycine-generating enzyme binds substrate directly at a mononuclear Cu(I) center to initiate O activation.

The formylglycine-generating enzyme (FGE) is required for the posttranslational activation of type I sulfatases by oxidation of an active-site cysteine to C-formylglycine. FGE has emerged as an enabling biotechnology tool due to the robust utility of the aldehyde product as a bioconjugation handle in recombinant proteins. Here, we show that Cu(I)-FGE is functional in O activation and reveal a high-resolution X-ray crystal structure of FGE in complex with its catalytic copper cofactor.

Piperidine-based glycodendrons as protein N-glycan prosthetics.

The generation of homogeneously glycosylated proteins is essential for defining glycoform-specific activity and improving protein-based therapeutics. We present a novel glycodendron prosthetic which can be site-selectively appended to recombinant proteins to create 'N-glycosylated' glycoprotein mimics. Using computational modeling, we designed the dendrimer scaffold and protein attachment point to resemble the native N-glycan architecture.

Formylglycine, a post-translationally generated residue with unique catalytic capabilities and biotechnology applications.

Formylglycine (fGly) is a catalytically essential residue found almost exclusively in the active sites of type I sulfatases. Formed by post-translational oxidation of cysteine or serine side chains, this aldehyde-functionalized residue participates in a unique and highly efficient catalytic mechanism for sulfate ester hydrolysis. The enzymes that produce fGly, formylglycine-generating enzyme (FGE) and anaerobic sulfatase-maturating enzyme (anSME), are as unique and specialized as fGly itself.

Mycobacterium tuberculosis Rv3406 is a type II alkyl sulfatase capable of sulfate scavenging.

The genome of Mycobacterium tuberculosis (Mtb) encodes nine putative sulfatases, none of which have a known function or substrate. Here, we characterize Mtb's single putative type II sulfatase, Rv3406, as a non-heme iron (II) and α-ketoglutarate-dependent dioxygenase that catalyzes the oxidation and subsequent cleavage of alkyl sulfate esters. Rv3406 was identified based on its homology to the alkyl sulfatase AtsK from Pseudomonas putida.