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Despite the advent of many new therapies, therapeutic monoclonal antibodies remain a prominent biologics product, with a market value of billions of dollars annually. A variety of downstream processing technological advances have led to a paradigm shift in how therapeutic antibodies are developed and manufactured. A key driver of change has been the increased adoption of single-use technologies for process development and manufacturing. An early-stage developability assessment of potential lead antibodies, using both in silico and high-throughput experimental approaches, is critical to de-risk development and identify molecules amenable to manufacturing. Both statistical and mechanistic modelling approaches are being increasingly applied to downstream process development, allowing for deeper process understanding of chromatographic unit operations. Given the greater adoption of perfusion processes for antibody production, continuous and semi-continuous downstream processes are being increasingly explored as alternatives to batch processes. As part of the Quality by Design (QbD) paradigm, ever more sophisticated process analytical technologies play a key role in understanding antibody product quality in real-time. We should expect that computational prediction and modelling approaches will continue to be advanced and exploited, given the increasing sophistication and robustness of predictive methods compared to the costs, time, and resources required for experimental studies.

The cysteine desulfurase IscS is a highly conserved master enzyme initiating sulfur transfer via persulfide to a range of acceptor proteins involved in Fe-S cluster assembly, tRNA modifications, and sulfur-containing cofactor biosynthesis. Several IscS-interacting partners including IscU, a scaffold for Fe-S cluster assembly; TusA, the first member of a sulfur relay leading to sulfur incorporation into the wobble uridine of several tRNAs; ThiI, involved in tRNA modification and thiamine biosynthesis; and rhodanese RhdA are sulfur acceptors. Other proteins, such as CyaY/frataxin and IscX, also bind to IscS, but their functional roles are not directly related to sulfur transfer. We have determined the crystal structures of IscS-IscU and IscS-TusA complexes providing the first insight into their different modes of binding and the mechanism of sulfur transfer. Exhaustive mutational analysis of the IscS surface allowed us to map the binding sites of various partner proteins and to determine the functional and biochemical role of selected IscS and TusA residues. IscS interacts with its partners through an extensive surface area centered on the active site Cys328. The structures indicate that the acceptor proteins approach Cys328 from different directions and suggest that the conformational plasticity of a long loop containing this cysteine is essential for the ability of IscS to transfer sulfur to multiple acceptor proteins. The sulfur acceptors can only bind to IscS one at a time, while frataxin and IscX can form a ternary complex with IscU and IscS. Our data support the role of frataxin as an iron donor for IscU to form the Fe-S clusters.

The chain length distribution of complex polysaccharides present on the bacterial surface is determined by polysaccharide co-polymerases (PCPs) anchored in the inner membrane. We report crystal structures of the periplasmic domains of three PCPs that impart substantially different chain length distributions to surface polysaccharides. Despite very low sequence similarities, they have a common protomer structure with a long central α-helix extending 100 Å into the periplasm. The protomers self-assemble into bell-shaped oligomers of variable sizes, with a large internal cavity. Electron microscopy shows that one of the full-length PCPs has a similar organization as that observed in the crystal for its periplasmic domain alone. Functional studies suggest that the top of the PCP oligomers is an important region for determining polysaccharide modal length. These structures provide a detailed view of components of the bacterial polysaccharide assembly machinery.

The Escherichia coli dihydroxyacetone (Dha) kinase is an unusual kinase because (i) it uses the phosphoenolpyruvate carbohydrate: phosphotransferase system (PTS) as the source of high-energy phosphate, (ii) the active site is formed by two subunits, and (iii) the substrate is covalently bound to His218 K * of the DhaK subunit. The PTS transfers phosphate to DhaM, which in turn phosphorylates the permanently bound ADP coenzyme of DhaL This phosphoryl group is subsequently transferred to the Dha substrate bound to DhaK. Here we report the crystal structure of the E. coli Dha kinase complex, DhaK-DhaL The structure of the complex reveals that DhaK undergoes significant conformational changes to accommodate binding of DhaL. Combined mutagenesis and enzymatic activity studies of kinase mutants allow us to propose a catalytic mechanism for covalent Dha binding, phosphorylation, and release of the Dha-phosphate product. Our results show that His56 K is involved in formation of the covalent hemiaminal bond with Dha. The structure of H56N K with noncovalently bound substrate reveals a somewhat different positioning of Dha in the binding pocket as compared to covalently bound Dha, showing that the covalent attachment to His218 K orients the substrate optimally for phosphoryl transfer. Asp109 K is critical for activity, likely acting as a general base activating the γ-OH of Dha. Our results provide a comprehensive picture of the roles of the highly conserved active site residues of dihydroxyacetone kinases.

Heme oxygenases (HOs) catalyze the oxidation of heme to biliverdin, carbon monoxide (CO), and free iron. Iron acquisition is critical for invading microorganisms to enable survival and growth. Here we report the crystal structure of ChuS, which displays a previously uncharacterized fold and is unique compared with other characterized HOs. Despite only 19% sequence identity between the N- and C-terminal halves, these segments of ChuS represent a structural duplication, with a root-mean-square deviation of 2.1 Å between the two repeats. ChuS is capable of using ascorbic acid or cytochrome P450 reductase-NADPH as electron sources for heme oxygenation. CO detection confirmed that ChuS is a HO, and we have identified it in pathogenic Escherichia coli O157:H7. Based on sequence analysis, this HO is present in many bacteria, although not in the E. coli K-12 strain. The N- and C-terminal halves of ChuS are each a functional HO.

The histidine biosynthetic pathway is an ancient one found in bacteria, archaebacteria, fungi, and plants that converts 5-phosphoribosyl 1-pyrophosphate to L-histidine in 10 enzymatic reactions. This pathway provided a paradigm for the operon, transcriptional regulation of gene expression, and feedback inhibition of a pathway. L-histidinol dehydrogenase (HisD, EC 1.1.1.23) catalyzes the last two steps in the biosynthesis of L-histidine: sequential NAD-dependent oxidations of L-histidinol to L-histidinaldehyde and then to L-histidine. HisD functions as a homodimer and requires the presence of one Zn2+cation per monomer. We have determined the three-dimensional structure of Escherichia coli HisD in the apo state as well as complexes with substrate, Zn2+, and NAD+(best resolution is 1.7 Å). Each monomer is made of four domains, whereas the intertwined dimer possibly results from domain swapping. Two domains display a very similar incomplete Rossmann fold that suggests an ancient event of gene duplication. Residues from both monomers form the active site. Zn2+plays a crucial role in substrate binding but is not directly involved in catalysis. The active site residue His-327 participates in acid-base catalysis, whereas Glu-326 activates a water molecule. NAD+binds weakly to one of the Rossmann fold domains in a manner different from that previously observed for other proteins having a Rossmann fold.