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SummaryCytosolic innate immune sensing is critical for protecting barrier tissues. NOD1 and NOD2 are cytosolic sensors of small peptidoglycan fragments (muropeptides) derived from the bacterial cell wall. These muropeptides enter cells, especially epithelial cells, through unclear mechanisms. We previously implicated SLC46 transporters in muropeptide transport in Drosophila immunity. Here we focus on Slc46a2, which is highly expressed in mammalian epidermal keratinocytes, and show that it is critical for delivery of DAP-muropeptides and activation of NOD1 in keratinocytes, while the related transporter Slc46a3 is critical for responding to MDP, the NOD2 ligand. In a mouse model, Slc46a2 and Nod1 deficiency strongly suppressed psoriatic inflammation, while methotrexate, a commonly used psoriasis therapeutic, inhibited Slc46a2-dependent transport of DAP-muropeptides. Collectively these studies define SLC46A2 as a transporter of NOD1 activating muropeptides, with critical roles in the skin barrier, and identify this transporter as an important target for anti-inflammatory intervention.

Glycan-protein interactions facilitate some of the most important biomolecular processes in and between cells. They are involved in different cellular pathways, cell-cell interactions and associated with many diseases, making these interactions of great interest. However, their structural and functional diversity poses great challenges in studying them at the molecular level. Surface plasmon resonance (SPR) technology presents great advantages to study glycan-protein interactions due to its superior sensitivity, ability to monitor real-time interactions, relatively simple data interpretation, and most importantly, direct measurement of binding without a need for fluorescent labeling. Here, another dimensionality of SPR in studying glycan-protein interactions is demonstrated via examples of binding between human innate immune receptors and their bacterial peptidoglycan ligands. In order to best resemble interactions in solution, a novel strategy of tethering the carbohydrate at different positions to the biosensor surface is applied to represent the potential displays of the carbohydrate ligand to the receptor. Subsequent kinetic analysis provides insights into the optimized configuration of peptidoglycan fragments for binding with its receptors. The manuscript contains a "how-to guide" to help with the implementation of these methods in other glycan-protein binding systems.

Uridine diphosphate (UDP) sugars are essential precursors for glycosylation reactions in all forms of life. Reactions that transfer the carbohydrate from the UDP donor are catalyzed by glycosyltransferases (Gtfs). While the stereochemistry and negative physiological charge of UDP-sugars are essential for their biochemical function in the cell, these characteristics make them challenging molecules to synthesize and purify on scale in the laboratory. This chapter focuses on the utilization of a chemoenzymatic synthesis of muramyl UDP-sugars, key building blocks in the bacterial cell peptidoglycan. A scalable strategy to obtain UDP-N-acetyl muramic acid derivatives (UDP-NAM), the first committed intermediate used solely in peptidoglycan biosynthesis, is described herein. This methodology utilizes two enzymes involving the cell wall recycling enzymes MurNAc/GlcNAc anomeric kinase (AmgK) and NAM α-1-phosphate uridylyl transferase (MurU), respectively. The promiscuity of these enzymes allows for the unique chemical functionality to be embedded in bacterial peptidoglycan both in vitro and in whole bacterial cells for subsequent structural and functional studies of this important biopolymer.

There are barriers to adoption of research-based teaching methods. Professional development workshops may inform faculty of these methods, but effective adoption often does not follow. In addition, newly-minted research-active faculty are often overwhelmed by the many new responsibilities (grant writing, group management, laboratory setup, teaching) that accompany the position and normally do not have the time to consider novel teaching approaches. This case study documents how over a three-year period, the responsibility for teaching a nontraditional "Introduction to Biochemistry" course in a problem-based learning format was successfully transferred from a senior faculty member nearing retirement (HBW) to a newly-hired research-active assistant professor (CLG). We describe our apprenticeship project involving modeling, scaffolding, fading, and coaching. We suggest that involving faculty in active-learning pedagogy early in their career with mentoring by senior faculty overcomes barriers to adopting these methods. This case describes a specific example from which potentially useful elements can be adopted and adapted wherever biochemistry is taught.

Glycosyltransferases involved in the biosynthesis of bacterial secondary metabolites may be useful for the generation of sugar-modified analogues of bioactive natural products. Some glycosyltransferases have relaxed substrate specificity, and it has been assumed that promiscuity is a feature of the class. As part of a program to explore the synthetic utility of these enzymes, we have analyzed the substrate selectivity of glycosyltransferases that attach similar 2-deoxy-L-sugars to glycopeptide aglycons of the vancomycin-type, using purified enzymes and chemically synthesized TDP beta-2-deoxy-L-sugar analogues. We show that while some of these glycopeptide glycosyltransferases are promiscuous, others tolerate only minor modifications in the substrates they will handle. For example, the glycosyltransferases GtfC and GtfD, which transfer 4-epi-L-vancosamine and L-vancosamine to C-2 of the glucose unit of vancomycin pseudoaglycon and chloroorienticin B, respectively, show moderately relaxed donor substrate specificities for the glycosylation of their natural aglycons. In contrast, GtfA, a transferase attaching 4-epi-L-vancosamine to a benzylic position, only utilizes donors that are closely related to its natural TDP sugar substrate. Our data also show that the spectrum of donors utilized by a given enzyme can depend on whether the natural acceptor or an analogue is used, and that GtfD is the most versatile enzyme for the synthesis of vancomycin analogues.

Glycosylating complex molecules remains a major synthetic challenge, making it hard to explore carbohydrate diversity in biologically active glycosylated natural products and their derivatives. In this paper we compare the efficiency of chemical and enzymatic methods for glycosylation of glycopeptides related to vancomycin and describe the parameters that should be considered in designing synthetic approaches to glycosylated natural product derivatives.

In the maturation of the glycopeptide antibiotic teicoplanin, two glycosyltransferases (tGtfA and tGtfB) and one acyltransferase (tAtf) act on the nascent scaffold to produce the final natural product. In this report, we present detailed kinetic characterization of each of these enzymes, demonstrating the ordered nature of these tailoring transformations. tGtfB acts first on the crosslinked peptide aglycone, adding either an N-acetylglucosamine or 2-aminoglucose moiety to the 4-position of the scaffold. The second glycosyltransferase, tGtfA, preferentially acts on this glycosylated scaffold to condense it with another molecule of N-acetylglucosamine. Although the acyltransferase tAtf is capable of N-acylating the free UDP sugar, UDP-2-acetylglucosamine, its catalytic efficiency is 100000-fold higher when acting on the 4-(2-aminoglucosyl)-teicoplanin scaffold, showing that it prefers to act on the aglycone-tethered sugar. This work expands on our previous studies of ordered tailoring of the vancomycin and ...

Moenomycin A is the only known natural product that inhibits peptidoglycan biosynthesis by binding the bacterial transglycosylases. We describe a degradation/reconstruction route to manipulate the reducing end of moenomycin A. A comparison of the biological and enzyme inhibitory activity of moenomycin A and an analogue containing a nerol lipid in place of the natural C25 lipid chain provides insight into the role of the moenocinol unit. Our results show that a lipid chain having ten carbons in moenocinol is sufficient for enzyme inhibition, but a longer chain is required for biological acitivity, apparently because the molecule must partition into biological membranes to reach its target in bacterial cells.

We report the heterologous overexpression and purification of Staphylococcus aureus PBP2 and demonstrate efficient glycan polymerization from lipid II in vitro. S. aureus PBP2 is the first purified gram-positive class A penicillin-binding protein to show good transglycosylase activity. This enables further studies on this important class of enzymes.

Beta-2-Deoxy sugar nucleotides are substrates used by a variety of glycosyltransferases (Gtfs). We have developed a chemical route to synthesize beta-2-deoxy sugar phosphates that starts from alpha-glycosyl chlorides. Our approach reliably provides access to a range of NDP beta-2-deoxy sugars essential for studying glycosyltransferases involved in the synthesis of biologically active natural products.

The antitumor drug aclacinomycin A is a representative member of the anthracycline subgroup that contains a C(7)-O-trisaccharide chain composed of L-2-deoxysugars. The sugar portion of the molecule, which greatly affects its biological activity, is assembled by dedicated glycosyltransferases; however, these enzymes have not been well-studied. Here we report the heterologous expression and purification of one of these enzymes, AknK, as well as the preparation of dTDP-L-2-deoxysugar donors, dTDP-L-2-deoxyfucose and dTDP-L-daunosamine, and the monoglycosyl aglycone, rhodosaminyl aklavinone. Our experiments show that AknK catalyzes the addition of the second sugar to the chain, using dTDP-L-2-deoxyfucose and rhodosaminyl aklavinone, to create the L-2-deoxyfucosyl-L-rhodosaminyl aklavinone. AknK also accepts an alternate dTDP-L-sugar, dTDP-L-daunosamine, and other monoglycosylated anthracyclines, including daunomycin, adriamycin, and idarubicin, to build alternate disaccharides on variant anthracycline backbones. Remarkably, AknK also catalyzes a tandem addition of a second L-2-deoxyfucosyl moiety, albeit with reduced activity, to the natural disaccharide chain to produce L-deoxyfucosyl-L-deoxyfucosyl-L-rhodosaminyl aklavinone, a variant of the natural aclacinomycin A. These results demonstrate that AknK may be a useful enzyme for the chemoenzymatic synthesis of anthracycline variants.

Chloroeremomycin, a vancomycin family glycopeptide antibiotic has three sugars, one d -glucose and two l -4- epi -vancosamines, attached to the crosslinked heptapeptide backbone by three glycosyltransferases, GtfA, -B, and -C. Prior efforts have revealed that GtfB and -C in tandem build an epivancosaminyl-1,2-glucosyldisaccharide chain on residue 4 of the aglycone; however, the characterization of GtfA and glycosylation sequence of chloroeremomycin have been lacking. Here, we report the expression and purification of GtfA, as well as synthesis of its sugar donor, 2′-deoxy-thymidine 5′-diphosphate (dTDP)-β- l -4- epi -vancosamine. GtfA transfers 4- epi -vancosamine from the chemically synthesized dTDP-4- epi -vancosamine to the β-OH-Tyr 6 residue of the aglycone, preferentially after GtfB action, to generate chloroorienticin B. With the preferred kinetic order of GtfB, then GtfA, then GtfC established, we have succeeded in reconstitution of chloroeremomycin from the heptapeptide aglycone by the sequential actions of the three enzymes.

Macrolides and anthracyclines are clinically important drugs produced by streptomycetes. Both classes of natural products contain aminosugar moieties that are attached by glycosyltransferases (Gtfs) at a late stage in the biosynthetic pathway and are essential for biological activity (Scheme 1).1 These Gtfs are potentially useful for the combinatorial biosynthesis or chemoenzymatic synthesis of structurally novel compounds with new biological activities.2 For many years, however, all attempts to reconstitute the activity of these Gtfs in vitro failed. Then, in 2004, Liu and coworkers reported that DesVII, a TDP-D-desosaminyl tranferase involved in pikromycin biosynthesis, requires the presence of an “auxiliary protein”, DesVIII, encoded in the same gene cluster, to catalyze glycosyltransfer in vitro, although the role of DesVIII was not clear.3 Walsh and coworkers subsequently reported that AknS, a Gtf involved in the glycosylation of aklavinone, requires the presence of the DesVIII homolog, AknT, to effect glycosyltransfer in vitro.4 Walsh and coworkers proposed that AknT functions as a regulatory subunit that increases the catalytic activity of AknS by influencing substrate affinity.4 There is precedent for this hypothesis because a well-characterized eukaryotic glycosyltransferase, β-1,4-galactosyltransferase, contains a regulatory subunit that alters substrate affinity and selectivity.5 Scheme 1 It is important to understand the role of Gtf auxiliary proteins in order to facilitate the use of aminosugar transferases in the chemoenzymatic synthesis of macrolide and anthracycline analogs. Therefore, we have been studying another aminosugar transferase, EryCIII, which is involved in erythromycin biosynthesis (Scheme 1B).6, 7 EryCIII catalyzes the transfer of D-desosamine to 3-α-mycarosylerthronolide B (αMEB), making erythromycin D (EryD, 4). We previously reported that EryCIII can be expressed in active form in E. coli provided that the GroEL/ES chaperonin complex is coexpressed.6 However, the purified protein was unstable and lost activity within hours of isolation6, 8 and attempts to stabilize the activity failed. Because the erythromycin gene cluster encodes a DesVIII homolog, EryCII, we sought to determine whether EryCII influenced the stability or activity of EryCIII. Studies described below show that EryCII does activate EryCIII. Surprisingly, however, we found that EryCIII remains active when EryCII is removed, which rules out models for EryCIII activation that involve a stable EryCII:EryCIII complex during glycosyltransfer. We also found that the non-cognate auxiliary protein AknT stably activates EryCIII. Moreover, activated EryCIII is capable of utilizing an unnatural sugar donor, enabling the preparation of erythromycin analogs. Thus, this work shows how to reconstitute the activity of EryCIII in vitro for the synthesis of unnatural macrolides. We PCR amplified eryCII from a mutant strain of Saccharopolyspora erythraea NRRL 2338, ΔeryCI-609 and cloned the gene into a pET22b expression vector to obtain the plasmid pERY2, which was then transformed into E. coli BL21(DE3). Expressed as a C-terminal His6 fusion protein, EryCII was partially purified via Ni-NTA affinity chromatography and then added to a reaction mixture containing inactive EryCIII expressed as previously described.6 EryCIII treated with EryCII mixture was found to be catalytically active, effecting transfer of desosamine to αMEB. The untreated EryCIII sample showed no activity. We then examined whether AknT, shown to be required for AknS activity in the aclarubicin pathway, can activate purified, inactive EryCIII. AknT is 26% homologous to EryCII and AknS is 48% homologous to EryCIII. To our surprise, we found that the addition of purified AknT to inactive EryCIII restores the activity of EryCIII in vitro (Figure 2C).10 Thus, both EryCII and AknT can function as glycosyltransferase activator proteins for EryCIII, although we note that larger amounts of AknT are required to achieve the same level of activation (Figure 2C). Given the structural differences in the substrates of EryCIII and AknS, this result rules out models for activation that involve binding of substrates by the auxiliary proteins. Figure 2 TIC traces for glycosylation reactions catalyzed by different batches of EryCIII (for detailed reaction conditions, see supporting information). (A) 1.2 μM EryCIII; (B) 1.2 μM EryCIII, 0.4 μM EryCII; (C) 1.2 μM EryCIII, ... To evaluate whether EryCII forms a stable complex with EryCIII during the glycosyltransfer reaction, we prepared a plasmid encoding both native EryCII and C-terminal His6-tagged EryCIII. Protein expression was induced with IPTG and EryCIII was purified away from EryCII by Ni-NTA affinity chromatography. (We designate the coexpressed, purified enzyme as EryCIII*.) No EryCII was visible in a SDS-PAGE gel of the purified EryCIII* sample, and mass spectral analysis showed that it contained less than 2% EryCII. Nevertheless, EryCIII* was not only fully active (Figure 2D), but it retained activity for at least two months under a range of different storage conditions. The above experiments suggest that once EryCIII is activated by EryCII, EryCII is no longer required. To assess the generality of this observation, we treated inactive EryCIII with native AknT for 3 hours, and then purified EryCIII by Ni-NTA affinity chromatography to remove AknT. This EryCIII sample, like EryCIII*, proved to have stable enzymatic activity. Although we cannot rule out the possibility that minute amounts of auxiliary protein influence enzymatic activity, we note that relatively high concentrations of other auxiliary proteins (0.25−3 equivalents) seem to be required to effect glycosyltransfer.3, 4 Having identified conditions to obtain pure, active EryCIII, we were able to characterize the kinetic behavior of the enzyme. The apparent Km of EryCIII* for αMEB (at 1 mM TDP-D-desosamine) was found to be 50 μM and the apparent kcat was 1 min−1 (Supporting Information), which is comparable to the turnover numbers observed for many other antibiotic Gtfs. We also investigated the substrate selectivity of the purified sample and found that D-mycaminose can be transferred to the αMEB aglycone (3) after an overnight incubation to produce the corresponding mycaminosyl erythromycin D derivative, erythromycin M (EryM, 5) (Figure 3).6 Thus, the activated enzyme is capable of transferring an alternative sugar donor. This is the first report of EryCIII having some substrate flexibility for alternative sugar donors.11 Because the desosamine sugar contacts the 50S subunit of ribosome,12 the discovery that an unnatural sugar can be transferred has implications for the synthesis of new erythromycin derivatives with improved properties. Figure 3 EryCIII* catalyzed glycosylation of αMEB 3 with TDP-D-mycaminose. Reaction conditions are as in Figure 2 except the sugar donor and the reactions are quenched at the times indicated in the figure ([M+H]+ calculated 720.45; observed 720.5). EB: ... In conclusion, we have shown that EryCIII can be activated by both EryCII and the non-cognate Gtf auxiliary protein AknT. We can remove >98% of the auxiliary proteins without any apparent loss of activity, which suggest that these proteins do not function as a regulatory subunit during glycosyltransfer reaction. Given that EryCIII can be transiently recovered in active form when expressed with GroEL/ES,6 and that including high concentrations of glycerol (>15%), known to facilitate folding, restores partial activity to the enzyme,13 we propose that EryCII or AknT facilitates a conformational change of EryCIII from an inactive to an active form. We are currently trying to understand in greater detail the mechanism by which pre-exposure to EryCII or AknT activates EryCIII for subsequent glycosyltransfer. In the meantime, this report sets the stage for more detailed investigations of the substrate selectivity of EryCIII, which could lead to new antibiotics with activity against resistant microorganisms.

The glycopeptide antibiotics prevent maturation of the bacterial cell wall by binding to the terminal d-alanyl-d-alanine moiety of peptidoglycan precursors, thereby inhibiting the enzymes involved in the final stages of peptidoglycan synthesis. However, there are significant differences in the biological activity of particular glycopeptide derivatives that are not related to their affinity for d-Ala-d-Ala. We compare the ability of vancomycin and a set of clinically relevant glycopeptides to inhibit Staphylococcus aureus PBP2 (penicillin binding protein), the major transglycosylase in a clinically relevant pathogen, S. aureus. We report experiments suggesting that activity differences between glycopeptides against this organism reflect a combination of substrate binding and secondary interactions with key enzymes involved in peptidoglycan synthesis.