SummaryProtein N-glycosylation is a widespread post-translational modification. The first committed step in this process is catalysed by dolichyl-phosphate N-acetylglucosamine-phosphotransferase DPAGT1 (GPT/E.C. 2.7.8.15). Missense DPAGT1 variants cause congenital myasthenic syndrome and disorders of glycosylation. In addition, naturally-occurring bactericidal nucleoside analogues such as tunicamycin are toxic to eukaryotes due to DPAGT1 inhibition, preventing their clinical use. Our structures of DPAGT1 with the substrate UDP-GlcNAc and tunicamycin reveal substrate binding modes, suggest a mechanism of catalysis, provide an understanding of how mutations modulate activity (thus causing disease) and allow design of non-toxic “lipid-altered” tunicamycins. The structure-tuned activity of these analogues against several bacterial targets allowed the design of potent antibiotics for Mycobacterium tuberculosis, enabling treatment in vitro, in cellulo and in vivo, providing a promising new class of antimicrobial drug.

Using directed evolution, a variant N-acetyl amino acid racemase (NAAAR G291D/F323Y) has been developed with up to 6-fold higher activity than the wild-type on a range of N-acetylated amino acids. The variant has been coupled with an enantiospecific acylase to give a preparative scale dynamic kinetic resolution which allows 98% conversion of N-acetyl-DL-allylglycine into D-allylglycine in 18 h at high substrate concentrations (50 g L(-1)). This is the first example of NAAAR operating under conditions which would allow it to be successfully used on an industrial scale for the production of enantiomerically pure α-amino acids. X-ray crystal analysis of the improved NAAAR variant allowed a comparison with the wild-type enzyme. We postulate that a network of novel interactions that result from the introduction of the two side chains is the source of improved catalytic performance.

2-Keto-3-deoxygluconate aldolase from the hyperthermophile Sulfolobus solfataricus is a highly thermostable type I aldolase that can catalyze carbon-carbon bond formation using nonphosphorylated substrates. However, it exhibits poor diastereocontrol in many of its aldol reactions, including the reaction of its natural substrates, pyruvate and D-glyceraldehyde, which afford a 55:45 mixture of D-2-keto-3-deoxygluconate (D-KDGlu) and D-2-keto-3-deoxy-galactonate (D-KDGal). We have employed detailed X-ray crystallographic structural information of this aldolase bound to these diastereoisomeric aldol products to selectively target specific amino acids for mutation for the rapid creation of stereochemically complementary mutants that catalyze either (Re)- or (Si)-facial selective aldol reactions to afford either D-KDGlu or D-KDGal with good levels of diastereocontrol.

Highlights Structures of DPAGT1 with UDP-GlcNAc and tunicamycin reveal mechanisms of catalysis  DPAGT1 mutants in patients with glycosylation disorders modulate DPAGT1 activity  Structures, kinetics and biosynthesis reveal role of lipid in tunicamycin  Lipid-altered, tunicamycin analogues give non-toxic antibiotics against TB by Trp122 holding the Dol-chain into the tunnel observed in the tunicamycin•DPAGT1 complex.Representative residues proposed to bind Dol-P, sugar and pyrophosphate were probed by mutagenesis. Mutation of Mg 2+ -chelating residues to Ala in Asn185Ala and Asp252Ala reduced the DPAGT1 activity to 1.2% and 7%, respectively ( Figure 3A). The more conservative Asn185Asp mutation, which would be expected to retain Mg 2+ -binding activity, also ablated activity (0.7% of WT) suggesting an additional role for Asn185 in catalysis. The amide group of Asn185 lies within 4 Å of the predicted Dol-P phosphate-binding site, forming hydrogen bonds with the nucleophilic oxygen of Dol-P to guide it towards the -phosphate.Mutations of Lys125, which also lies near the Dol-P phosphate binding site, to Lys125Ala, Lys125Glu and Lys125Gln, all reduced the activity to below 2.2%, consistent with a critical guiding role for Lys125. Interestingly, an Asp252Asn mutation increased activity 5-fold ( Figure 3A). This mutation removes a coordinating negative charge from the Mg 2+ , making the Mg 2+ more electropositive and the -phosphorus more electrophilic, thus potentially increasing its susceptibility to nucleophilic attack.Mutation of His302, which hydrogen bonds to the O4 oxygen of GlcNAc in UDP-GlcNAc to hold it in its bent-back conformation, causes 98% loss of activity, again consistent with the guiding role that active site residues play in aligning access of the nucleophile to the -phosphate. Finally, mutation of the Arg301 sidechain that, along with the Mg 2+ ion, completes the 'pincer' of the pyrophosphate caused almost complete loss of activity and this mutation has been found in patients with CDG-Ij (Imtiaz et al., 2012).

Practical syntheses of 2-keto-3-deoxy-D-xylonate (D-KDX) and 2-keto-3-deoxy-L-arabinonate (L-KDA) that rely on reaction of the anion of ethyl 2-[(tert-butyldimethylsilyl)oxy]-2-(dimethoxy phosphoryl) acetate with enantiopure glyceraldehyde acetonide, followed by global deprotection of the resultant O-silyl-enol esters, have been developed. This has enabled us to confirm that a 2-keto-3-deoxy-D-gluconate aldolase from the archaeon Sulfolobus solfataricus demonstrates good activity for catalysis of the retro-aldol cleavage of both these enantiomers to afford pyruvate and glycolaldehyde. The stereochemical promiscuity of this aldolase towards these enantiomeric aldol substrates confirms that this organism employs a metabolically promiscuous pathway to catabolise the C5-sugars D-xylose and L-arabinose.