Impact of Drug Conjugation on Thermal and Metabolic Stabilities of Aglycosylated and -Glycosylated Antibodies.

N-linked glycosylation is one of the most common and complex posttranslational modifications that govern the biological functions and physicochemical properties of therapeutic antibodies. We evaluated thermal and metabolic stabilities of antibody-drug conjugates (ADCs) with payloads attached to the C'E loop in the immunoglobulin G (IgG) Fc CH2 domain, comparing the glycosylated and aglycosylated Fc ADC variants. Our study revealed that introduction of small-molecule drugs into an aglycosylated antibody can compensate for thermal destabilization originating from structural distortions caused by elimination of N-linked glycans.

An efficient light-driven P450 BM3 biocatalyst.

P450s are heme thiolate enzymes that catalyze the regio- and stereoselective functionalization of unactivated C-H bonds using molecular dioxygen and two electrons delivered by the reductase. We have developed hybrid P450 BM3 heme domains containing a covalently attached Ru(II) photosensitizer in order to circumvent the dependency on the reductase and perform P450 reactions upon visible light irradiation. A highly active hybrid enzyme with improved stability and a modified Ru(II) photosensitizer is able to catalyze the light-driven hydroxylation of lauric acid with total turnover numbers of 935 and initial reaction rate of 125 mol product/(mol enzyme/min).

A series of hybrid P450 BM3 enzymes with different catalytic activity in the light-initiated hydroxylation of lauric acid.

We have developed a series of hybrid P450 BM3 enzymes to perform the light-activated hydroxylation of lauric acid. These enzymes contain a Ru(II)-diimine photosensitizer covalently attached to single cysteine residues of mutant P450 BM3 heme domains. The library of hybrid enzymes includes four non-native single cysteine mutants (K97C, Q397C, Q109C and L407C).