David H. Kwan

Prediction and Manipulation of the Stereochemistry of Enoylreduction in Modular Polyketide Synthases

When an enoylreductase enzyme of a modular polyketide synthase reduces a propionate extender unit that has been newly added to the growing polyketide chain, the resulting methyl branch may have either S or R configuration. We have uncovered a correlation between the presence or absence of a unique tyrosine residue in the ER active site and the chirality of the methyl branch that is introduced. When this position in the active site is occupied by a tyrosine residue, the methyl branch has S configuration, otherwise it has R configuration. In a model PKS in vivo, a mutation (Tyr to Val) in an erythromycin PKS-derived ER caused a switch in the methyl branch configuration in the product from S to R. In contrast, alteration (Val to Tyr) at this position in a rapamycin-derived PKS ER was insufficient to achieve a switch from R to S, showing that additional residues also participate in stereocontrol of enoylreduction.

Self‐Immobilizing Fluorogenic Imaging Agents of Enzyme Activity

Chromogenic and fluorogenic substrates are valuable tools for locating endogenousand reporter-enzyme activities, and thus for visualizing gene expression within cells and tissues. They therefore find use in applications ranging from histological analyses to fluorescence-activated cell sorting (FACS). A limiting factor in such studies is the tendency of the colored dye product to diffuse from the site of cleavage, whereby resolution and utility are decreased. This problem was addressed many years ago for visible dyes with the introduction of bromochloroindolyl conjugates, such as 5-bromo-4-chloro-3-indolyl b-d-galactopyranoside (X-Gal), which, when cleaved, dimerize to form an intensely blue product and precipitate; thus, diffusion is minimized. Few such systems have been developed for fluorescent reagents, largely because their precipitation results in fluorescence quenching. An alternative strategy for the reduction of product diffusion would be for the enzyme to release a highly reactive fluorescent species that could covalently derivatize nucleophilic sites in the nearby medium. Sitespecific immobilization of the fluorophore would result, without precipitation, and quenching would therefore be minimized. For example, tyramide signal amplification (TSA) takes advantage of this strategy. Herein, we report the development of simply modified derivatives of coumarin glycosides that are not innately fluorescent, but when cleaved release a fluorescent aglycone that readily forms a quinone methide, which rapidly reacts with a nearby nucleophile. Their use in histological studies and in FACS sorting of cell types is demonstrated. Enzyme substrates that generate latent quinone methides have been developed in the past for a range of enzymes, including proteases, esterases, phosphatases, sulfatases, and glycosidases. The most commonly employed are those containing orthoor para-(di)halomethyl phenols, which, when liberated, generate reactive quinone methides (Scheme 1). These compounds were originally developed as mechanism-based inactivators for the selective inhibition of specific activities or the labeling and identification of activesite residues. However, the time taken for the (di)halomethyl phenol to decompose, and for the quinone methide thereby generated to react, is often sufficiently long for the reagent to leave the active site and react with other nearby nucleophiles, including water. This problem is further aggravated when, as in most cases, the aryl moiety has no specific affinity for the active site. This behavior has significantly reduced the utility of such compounds and, despite numerous reports, has rendered them essentially useless as probes for activity-based proteomics studies, since, although generated by the specific enzyme activity, the quinone methides react indiscriminately. However, this behavior is ideal for imaging agents of the type proposed. Indeed, the perfect reagent would never react at the active site, since that may inactivate the enzyme, but rather would react on the exterior of the protein, or with cellular components in the immediate vicinity. To test this approach, we synthesized glycosides 1, 2, and 3 (Scheme 1). The choice of the difluoromethyl over the monofluoromethyl derivative was based upon the greater stability of the parent compound towards solvolysis and upon the longer anticipated “lifetime” of the dihalomethyl phenol; the longer lifetime improves its chances of diffusing out of the active site. The simple synthetic route employed (see the Experimental Section and the Supporting Information) could be Scheme 1. A) Generation of and nucleophilic addition to a quinone methide. B) Fluorogenic quinone methide generating glycoside substrates synthesized in this study.

The stereochemistry of complex polyketide biosynthesis by modular polyketide synthases.

Polyketides are a diverse class of medically important natural products whose biosynthesis is catalysed by polyketide synthases (PKSs), in a fashion highly analogous to fatty acid biosynthesis. In modular PKSs, the polyketide chain is assembled by the successive condensation of activated carboxylic acid-derived units, where chain extension occurs with the intermediates remaining covalently bound to the enzyme, with the growing polyketide tethered to an acyl carrier domain (ACP). Carboxylated acyl-CoA precursors serve as activated donors that are selected by the acyltransferase domain (AT) providing extender units that are added to the growing chain by condensation catalysed by the ketosynthase domain (KS). The action of ketoreductase (KR), dehydratase (DH), and enoylreductase (ER) activities can result in unreduced, partially reduced, or fully reduced centres within the polyketide chain depending on which of these enzymes are present and active. The PKS-catalysed assembly process generates stereochemical diversity, because carbon–carbon double bonds may have either cis- or trans- geometry, and because of the chirality of centres bearing hydroxyl groups (where they are retained) and branching methyl groups (the latter arising from use of propionate extender units). This review shall cover the studies that have determined the stereochemistry in many of the reactions involved in polyketide biosynthesis by modular PKSs.

Mutagenesis of a Modular Polyketide Synthase Enoylreductase Domain Reveals Insights into Catalysis and Stereospecificity

Modular type I polyketide synthases (PKSs) such as the 6-deoxyerythronolide B synthase (DEBS) or the rapamycin synthase (RAPS) biosynthesize their polyketide products in a fashion similar to fatty acid biosynthesis. Each module of these enzymes consists of multiple catalytic domains. The constituent enoylreductase (ER) domain of a given module stereospecifically reduces an enzyme-bound 2-enoyl intermediate. In a recombinant model PKS containing an ER domain derived from module 13 of RAPS, we have previously used site-specific mutagenesis to identify a key active site residue that influences the stereochemistry of enoylreduction. In this study we have identified further residues involved in stereospecificity. We show here that several other residues, previously considered as catalytically important in the medium-chain dehydrogenase/reductase family of enzymes to which PKS ERs belong, are not essential for enoylreduction in polyketide biosynthesis. However, our results suggest that a lysine residue, also modeled to lie at the active site, might serve as a proton donor to the C-2 position during enoylreduction, as previously proposed for an analogously placed lysine in mammalian fatty acid synthase. These findings further highlight the close mechanistic link between fatty acid and polyketide synthases and provide useful guidance for future biosynthetic engineering of complex polyketide biosynthesis.

Insights into the stereospecificity of ketoreduction in a modular polyketide synthase

Ketoreductase enzymes are responsible for the generation of hydroxyl stereocentres during the biosynthesis of complex polyketide natural products. Previous studies of isolated polyketide ketoreductases have shown that the stereospecificity of ketoreduction can be switched by mutagenesis of selected active site amino acids. We show here that in the context of the intact polyketide synthase multienzyme the same changes do not alter the stereochemical outcome in the same way. These findings point towards additional factors that govern ketoreductase stereospecificity on intact multienzymes in vivo.

Structure of human ST8SiaIII sialyltransferase provides insight into cell-surface polysialylation

Sialyltransferases of the mammalian ST8Sia family catalyze oligo- and polysialylation of surface-localized glycoproteins and glycolipids through transfer of sialic acids from CMP–sialic acid to the nonreducing ends of sialic acid acceptors. The crystal structure of human ST8SiaIII at 1.85-Å resolution presented here is, to our knowledge, the first solved structure of a polysialyltransferase from any species, and it reveals a cluster of polysialyltransferase-specific structural motifs that collectively provide an extended electropositive surface groove for binding of oligo–polysialic acid chain products. The ternary complex of ST8SiaIII with a donor sugar analog and a sulfated glycan acceptor identified with a sialyltransferase glycan array provides insight into the residues involved in substrate binding, specificity and sialyl transfer.

Toward Efficient Enzymes for the Generation of Universal Blood through Structure-Guided Directed Evolution

Blood transfusions are critically important in many medical procedures, but the presence of antigens on red blood cells (RBCs, erythrocytes) means that careful blood-typing must be carried out prior to transfusion to avoid adverse and sometimes fatal reactions following transfusion. Enzymatic removal of the terminal N-acetylgalactosamine or galactose of A- or B-antigens, respectively, yields universal O-type blood, but is inefficient. Starting with the family 98 glycoside hydrolase from Streptococcus pneumoniae SP3-BS71 (Sp3GH98), which cleaves the entire terminal trisaccharide antigenic determinants of both A- and B-antigens from some of the linkages on RBC surface glycans, through several rounds of evolution, we developed variants with vastly improved activity toward some of the linkages that are resistant to cleavage by the wild-type enzyme. The resulting enzyme effects more complete removal of blood group antigens from cell surfaces, demonstrating the potential for engineering enzymes to generate antigen-null blood from donors of various types.

Enhancement of Biological Reactions on Cell Surfaces via Macromolecular Crowding

The reaction of macromolecules such as enzymes and antibodies with cell surfaces is often an inefficient process, requiring large amounts of expensive reagent. Here we report a general method based on macromolecular crowding with a range of neutral polymers to enhance such reactions, using red blood cells (RBCs) as a model system. Rates of conversion of Type A and B red blood cells to universal O type by removal of antigenic carbohydrates with selective glycosidases are increased up to 400-fold in the presence of crowders. Similar enhancements are seen for antibody binding. We further explore the factors underlying these enhancements using confocal microscopy and fluorescent recovery after bleaching (FRAP) techniques with various fluorescent protein fusion partners. Increased cell-surface concentration due to volume exclusion, along with two-dimensionally confined diffusion of enzymes close to the cell surface, appear to be the major contributing factors.

Chemoenzymatic Synthesis of a Type 2 Blood Group A Tetrasaccharide and Development of High-throughput Assays Enables a Platform for Screening Blood Group Antigen-cleaving Enzymes.

A facile enzymatic synthesis of the methylumbelliferyl β-glycoside of the type 2 A blood group tetrasaccharide in good yields is reported. Using this compound, we developed highly sensitive fluorescence-based high-throughput assays for both endo-β-galactosidase and α-N-acetylgalactosaminidase activity specific for the oligosaccharide structure of the blood group A antigen. We further demonstrate the potential to use this assay to screen the expressed gene products of metagenomic libraries in the search for efficient blood group antigen-cleaving enzymes.

Toward Efficient Enzymatic Glycan Synthesis: Directed Evolution and Enzyme Engineering

Enzymatic synthesis of oligosaccharides has traditionally employed glycosyltransferases, or glycosidases run in transglycosylation mode. Glycosynthases, mutant glycosidases in which the catalytic nucleophile has been removed, function as efficient transferases with glycosyl fluoride donors, frequently giving stoicheometric yields. Glycoligases, in which the acid/base catalyst has been mutated, synthesise sulfur-linked oligosaccharides when an activated donor is used in conjunction with a thiosugar acceptor, or in some cases O-glycosides. Recent results in the engineering and evolution of these two classes of mutant enzymes along with “classical” glycosyltransferases using a variety of screening methodologies, including robot-assisted ELISA assays and FACS cell sorting, are discussed.