Kinetics and Optimization of the Lysine–Isopeptide Bond Forming Sortase Enzyme from Corynebacterium diphtheriae

Site-specifically modified protein bioconjugates have important applications in biology, chemistry, and medicine. Functionalizing specific protein side chains with enzymes using mild reaction conditions is of significant interest, but remains challenging. Recently, the lysine-isopeptide bond forming activity of the sortase enzyme that builds surface pili in Corynebacterium diphtheriae (CdSrtA) has been reconstituted in vitro. A mutationally activated form of CdSrtA was shown to be a promising bioconjugating enzyme that can attach Leu-Pro-Leu-Thr-Gly peptide fluorophores to a specific lysine residue within the N-terminal domain of the SpaA protein (NSpaA), enabling the labeling of target proteins that are fused to NSpaA. Here we present a detailed analysis of the CdSrtA catalyzed protein labeling reaction. We show that the first step in catalysis is rate limiting, which is the formation of the CdSrtA-peptide thioacyl intermediate that subsequently reacts with a lysine ε-amine in NSpaA. This intermediate is surprisingly stable, limiting spurious proteolysis of the peptide substrate. We report the discovery of a new enzyme variant (CdSrtAΔ) that has significantly improved transpeptidation activity, because it completely lacks an inhibitory polypeptide appendage ("lid") that normally masks the active site. We show that the presence of the lid primarily impairs formation of the thioacyl intermediate and not the recognition of the NSpaA substrate. Quantitative measurements reveal that CdSrtAΔ generates its cross-linked product with a catalytic turnover number of 1.4 ± 0.004 h-1 and that it has apparent KM values of 0.16 ± 0.04 and 1.6 ± 0.3 mM for its NSpaA and peptide substrates, respectively. CdSrtAΔ is 7-fold more active than previously studied variants, labeling >90% of NSpaA with peptide within 6 h. The results of this study further improve the utility of CdSrtA as a protein labeling tool and provide insight into the enzyme catalyzed reaction that underpins protein labeling and pilus biogenesis

NEAr Transporter (NEAT) Domains: Unique Surface Displayed Heme Chaperones That Enable Gram-Positive Bacteria to Capture Heme-Iron From Hemoglobin.

Iron is an important micronutrient that is required by bacteria to proliferate and to cause disease. Many bacterial pathogens forage iron from human hemoglobin (Hb) during infections, which contains this metal within heme (iron-protoporphyrin IX). Several clinically important pathogenic species within the Firmicutes phylum scavenge heme using surface-displayed or secreted NEAr Transporter (NEAT) domains. In this review, we discuss how these versatile proteins function in the Staphylococcus aureus Iron-regulated surface determinant system that scavenges heme-iron from Hb. S. aureus NEAT domains function as either Hb receptors or as heme-binding chaperones. In vitro studies have shown that heme-binding NEAT domains can rapidly exchange heme amongst one another via transiently forming transfer complexes, leading to the interesting hypothesis that they may form a protein-wire within the peptidoglycan layer through which heme flows from the microbial surface to the membrane. In Hb receptors, recent studies have revealed how dedicated heme- and Hb-binding NEAT domains function synergistically to extract Hbs heme molecules, and how receptor binding to the Hb-haptoglobin complex may block its clearance by macrophages, prolonging microbial access to Hbs iron. The functions of NEAT domains in other Gram-positive bacteria are also reviewed

NMR experiments redefine the hemoglobin binding properties of bacterial NEAr‐iron Transporter domains

Iron is a versatile metal cofactor that is used in a wide range of essential cellular processes. During infections, many bacterial pathogens acquire iron from human hemoglobin (Hb), which contains the majority of the bodys total iron content in the form of heme (iron protoporphyrin IX). Clinically important Gram-positive bacterial pathogens scavenge heme using an array of secreted and cell-wall-associated receptors that contain NEAr-iron Transporter (NEAT) domains. Experimentally defining the Hb binding properties of NEAT domains has been challenging, limiting our understanding of their function in heme uptake. Here we show that solution-state NMR spectroscopy is a powerful tool to define the Hb binding properties of NEAT domains. The utility of this method is demonstrated using the NEAT domains from Bacillus anthracis and Listeria monocytogenes. Our results are compatible with the existence of at least two types of NEAT domains that are capable of interacting with either Hb or heme. These binding properties can be predicted from their primary sequences, with Hb- and heme-binding NEAT domains being distinguished by the presence of (F/Y)YH(Y/F) and S/YXXXY motifs, respectively. The results of this work should enable the functions of a wide range of NEAT domain containing proteins in pathogenic bacteria to be reliably predicted

The Staphylococcus aureus IsdH Receptor Forms a Dynamic Complex with Human Hemoglobin that Triggers Heme Release via Two Distinct Hot Spots.

Iron is an essential nutrient that is actively acquired by bacterial pathogens during infections. Clinically important Staphylococcus aureus obtains iron by extracting heme from hemoglobin (Hb) using the closely related IsdB and IsdH surface receptors. In IsdH, extraction is mediated by a conserved tridomain unit that contains its second (N2) and third (N3) NEAT domains joined by a helical linker, called IsdHN2N3. Leveraging the crystal structure of the IsdHN2N3:Hb complex, we have probed the mechanism of heme capture using NMR, stopped-flow transfer kinetics measurements, and molecular dynamics (MD) simulations. NMR studies of the 220 kDa IsdHN2N3:Hb complex reveal that it is dynamic, with persistent interdomain motions enabling the linker and N3 domains in the receptor to transiently engage Hb to remove its heme. An alanine mutagenesis analysis reveals that two receptor subsites positioned ~20 Å apart trigger heme release by contacting Hb's F-helix. These subsites are located within the N3 and linker domains and appear to play distinct roles in stabilizing the heme transfer transition state. Linker domain contacts primarily function to destabilize Hb-heme interactions, thereby lowering ΔH‡, while contacts from the N3 subsite play a similar destabilizing role, but also form a bridge through which heme moves from Hb to the receptor. Interestingly, MD simulations suggest that within the transiently forming interface, both the F-helix and receptor bridge are in motion, dynamically sampling conformations that are suitable for heme transfer. Thus, IsdH triggers heme release from Hb via a flexible, low-affinity interface that forms fleetingly in solution

Pyrococcus furiosus Prolyl Oligopeptidase: A Dynamic Supramolecular Host for Peptidase and Dirhodium Catalysis

Supramolecular catalysis involves the design and characterization of synthetic macromolecules that catalyze chemical reactions. While enzymes are often cited as the inspiration for such catalysts, enzymes can also serve as hosts for non-native catalytic components. Protein-based hosts can be readily produced in E. coli and rapidly evolved for particular applications. Moreover, inherent properties of these systems, including their conformational dynamics, can be exploited for non-native transformations that occur within their interior. Studies on the peptidase activity of a prolyl oligopeptidase from Pyrococcus furiosus (Pfu POP) suggest that its unique two-domain architecture regulates substrate access and specificity. We have established that Pfu POP also serves as an efficient host for asymmetric cyclopropanation upon active-site modification with a dirhodium cofactor. To understand how Pfu POP controls both peptidase and dirhodium catalysis, we determined the crystal structures of this enzyme and its S477C mutant and used these structures as starting points for MD simulations of both the apo structures and systems containing a covalently linked peptidase inhibitor or a dirhodium catalyst. Pfu POP was crystalized in an open conformation, and MD simulations reveal spontaneous transitions between open and closed states, in addition to a number of smaller scale conformational changes, suggesting facile inter-domain movement. Importantly, key aspects of previously reported peptidase kinetics and cyclopropanation selectivity can be rationalized in the context of this inter-domain opening and closing. This finding constitutes a remarkable example in which the conformational dynamics of a supramolecular host affect two different catalytic activities and suggests that Pfu POP could serve as a host for a wide range of non-native catalysts

Crystal Structure and Conformational Dynamics of Pyrococcus furio- sus Prolyl Oligopeptidase

Enzymes in the prolyl oligopeptidase family possess unique structures and substrate specificities that are important for their biological activity and for potential biocatalytic applications. The crystal structures of Pyrococcus furiosus (Pfu) prolyl oligopeptidases (POP) and the corresponding S477C mutant were solved to 1.9 and 2.2 Å resolution, respectively. The wild type enzyme crystallized in an open con- formation, indicating that this state is readily accessible, and it contained bound chloride ions and a prolylproline ligand. These structures were used as starting points for molecular dynamics simulations of Pfu POP conformational dynamics. The simulations showed that large-scale do- main opening and closing occurred spontaneously, providing facile substrate access to the active site. Movement of the loop containing the catalytically essential histidine into a conformation similar to those found in structures with fully-formed catalytic triads also occurred. This movement was modulated by chloride binding, providing a rationale for experimentally observed activation of POP peptidase catalysis by chlo- ride. Thus, the structures and simulations reported in this study, combined with existing biochemical data, provide a number of insights into POP catalysis

Transition Metal-Catalyzed C-H and C-C Activation: How Hydrocarbons Go Boom, Gracefully

Carbon-hydrogen and carbon-carbon bonds are ubiquitous in organic molecules, yet suffer from limited reactivity. Modern advances in transition metal catalysis have enabled selective transformation of these compounds into functionalized species. Our seminar will focus on the work of Professor William D. Jones, a mechanistic inorganic chemist at the University of Rochester and member of the Center for Enabling New Technologies through Catalysis (CENTC). Using rhodium and nickel complexes, Dr. Jones examines the kinetic and thermodynamic pathways of hydrocarbon activation. He has been able to fine-tune the selectivity of specific reactions by experimental manipulation of these metal-ligand systems. This research has applications in improving the functionality, selectivity, and efficiency of certain industrial synthetic processes

Crystal Structure and Conformational Dynamics of Pyrococcus furiosus Prolyl Oligopeptidase

Enzymes in the prolyl oligopeptidase family possess unique structures and substrate specificities that are important for their biological activity and for potential biocatalytic applications. The crystal structures of Pyrococcus furiosus (Pfu) prolyl oligopeptidase (POP) and the corresponding S477C mutant were determined to 1.9 and 2.2 Å resolution, respectively. The wild type enzyme crystallized in an open conformation, indicating that this state is readily accessible, and it contained bound chloride ions and a prolylproline ligand. These structures were used as starting points for molecular dynamics simulations of Pfu POP conformational dynamics. The simulations showed that large-scale domain opening and closing occurred spontaneously, providing facile substrate access to the active site. Movement of the loop containing the catalytically essential histidine into a conformation similar to those found in structures with fully formed catalytic triads also occurred. This movement was modulated by chloride binding, providing a rationale for experimentally observed activation of POP peptidase catalysis by chloride. Thus, the structures and simulations reported in this study, combined with existing biochemical data, provide a number of insights into POP catalysis