Human Mat2A Uses an Ordered Kinetic Mechanism and Is Stabilized but Not Regulated by Mat2B.

Methionine adenosyltransferase (MAT) catalyzes the adenosine 5'-triphosphate (ATP) and l-methionine (l-Met) dependent formation of S-adenosyl-l-methionine (SAM), the principal methyl donor of most biological transmethylation reactions. We carried out in-depth kinetic studies to further understand its mechanism and interaction with a potential regulator, Mat2B. The initial velocity pattern and results of product inhibition by SAM, phosphate, and pyrophosphate, and dead-end inhibition by the l-Met analog cycloleucine (l-cLeu) suggest that Mat2A follows a strictly ordered kinetic mechanism where ATP binds before l-Met and with SAM released prior to random release of phosphate and pyrophosphate. Isothermal titration calorimetry (ITC) showed binding of ATP to Mat2A with a Kd of 80 ± 30 µM, which is close to the Km(ATP) of 50 ± 10 µM. In contrast, l-Met or l-cLeu showed no binding to Mat2A in the absence of ATP; however, binding to l-cLeu was observed in the presence of ATP. The ITC results are fully consistent with the product and dead-inhibition results obtained. We also carried out kinetic studies in the presence of the physiological regulator Mat2B. Under conditions where all Mat2A is found in complex with Mat2B, no significant change in the kinetic parameters was observed despite confirmation of a very high binding affinity of Mat2A to Mat2B (Kd of 6 ± 1 nM). Finally, we found that while Mat2A is unstable at low concentrations (<100 nM), rapidly losing activity at 37 °C, it retained full activity for at least 2 h when Mat2B was present at the known 2:1 Mat2A/Mat2B stoichiometry.

Optimization of Metabolic Oligosaccharide Engineering with Ac4GalNAlk and Ac4GlcNAlk by an Engineered Pyrophosphorylase.

Metabolic oligosaccharide engineering (MOE) has fundamentally contributed to our understanding of protein glycosylation. Efficient MOE reagents are activated into nucleotide-sugars by cellular biosynthetic machineries, introduced into glycoproteins and traceable by bioorthogonal chemistry. Despite their widespread use, the metabolic fate of many MOE reagents is only beginning to be mapped. While metabolic interconnectivity can affect probe specificity, poor uptake by biosynthetic salvage pathways may impact probe sensitivity and trigger side reactions. Here, we use metabolic engineering to turn the weak alkyne-tagged MOE reagents Ac4GalNAlk and Ac4GlcNAlk into efficient chemical tools to probe protein glycosylation. We find that bypassing a metabolic bottleneck with an engineered version of the pyrophosphorylase AGX1 boosts nucleotide-sugar biosynthesis and increases bioorthogonal cell surface labeling by up to two orders of magnitude. A comparison with known azide-tagged MOE reagents reveals major differences in glycoprotein labeling, substantially expanding the toolbox of chemical glycobiology.

Metabolic precision labeling enables selective probing of O-linked N-acetylgalactosamine glycosylation.

Protein glycosylation events that happen early in the secretory pathway are often dysregulated during tumorigenesis. These events can be probed, in principle, by monosaccharides with bioorthogonal tags that would ideally be specific for distinct glycan subtypes. However, metabolic interconversion into other monosaccharides drastically reduces such specificity in the living cell. Here, we use a structure-based design process to develop the monosaccharide probe N-(S)-azidopropionylgalactosamine (GalNAzMe) that is specific for cancer-relevant Ser/Thr(O)-linked N-acetylgalactosamine (GalNAc) glycosylation. By virtue of a branched N-acylamide side chain, GalNAzMe is not interconverted by epimerization to the corresponding N-acetylglucosamine analog by the epimerase N-acetylgalactosamine-4-epimerase (GALE) like conventional GalNAc-based probes. GalNAzMe enters O-GalNAc glycosylation but does not enter other major cell surface glycan types including Asn(N)-linked glycans. We transfect cells with the engineered pyrophosphorylase mut-AGX1 to biosynthesize the nucleotide-sugar donor uridine diphosphate (UDP)-GalNAzMe from a sugar-1-phosphate precursor. Tagged with a bioorthogonal azide group, GalNAzMe serves as an O-glycan-specific reporter in superresolution microscopy, chemical glycoproteomics, a genome-wide CRISPR-knockout (CRISPR-KO) screen, and imaging of intestinal organoids. Additional ectopic expression of an engineered glycosyltransferase, "bump-and-hole" (BH)-GalNAc-T2, boosts labeling in a programmable fashion by increasing incorporation of GalNAzMe into the cell surface glycoproteome. Alleviating the need for GALE-KO cells in metabolic labeling experiments, GalNAzMe is a precision tool that allows a detailed view into the biology of a major type of cancer-relevant protein glycosylation.

D-Cycloserine destruction by alanine racemase and the limit of irreversible inhibition.

The broad-spectrum antibiotic D-cycloserine (DCS) is a key component of regimens used to treat multi- and extensively drug-resistant tuberculosis. DCS, a structural analog of D-alanine, binds to and inactivates two essential enzymes involved in peptidoglycan biosynthesis, alanine racemase (Alr) and D-Ala:D-Ala ligase. Inactivation of Alr is thought to proceed via a mechanism-based irreversible route, forming an adduct with the pyridoxal 5'-phosphate cofactor, leading to bacterial death. Inconsistent with this hypothesis, Mycobacterium tuberculosis Alr activity can be detected after exposure to clinically relevant DCS concentrations. To address this paradox, we investigated the chemical mechanism of Alr inhibition by DCS. Inhibition of M. tuberculosis Alr and other Alrs is reversible, mechanistically revealed by a previously unidentified DCS-adduct hydrolysis. Dissociation and subsequent rearrangement to a stable substituted oxime explains Alr reactivation in the cellular milieu. This knowledge provides a novel route for discovery of improved Alr inhibitors against M. tuberculosis and other bacteria.

An essential bifunctional enzyme in Mycobacterium tuberculosis for itaconate dissimilation and leucine catabolism.

Mycobacterium tuberculosis (Mtb) is the etiological agent of tuberculosis. One-fourth of the global population is estimated to be infected with Mtb, accounting for ∼1.3 million deaths in 2017. As part of the immune response to Mtb infection, macrophages produce metabolites with the purpose of inhibiting or killing the bacterial cell. Itaconate is an abundant host metabolite thought to be both an antimicrobial agent and a modulator of the host inflammatory response. However, the exact mode of action of itaconate remains unclear. Here, we show that Mtb has an itaconate dissimilation pathway and that the last enzyme in this pathway, Rv2498c, also participates in l-leucine catabolism. Our results from phylogenetic analysis, in vitro enzymatic assays, X-ray crystallography, and in vivo Mtb experiments, identified Mtb Rv2498c as a bifunctional ß-hydroxyacyl-CoA lyase and that deletion of the rv2498c gene from the Mtb genome resulted in attenuation in a mouse infection model. Altogether, this report describes an itaconate resistance mechanism in Mtb and an l-leucine catabolic pathway that proceeds via an unprecedented (R)-3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) stereospecific route in nature.

Dietary 18:3omega3 influences immune function and the tissue fatty acid response to antigens and adjuvant.

Alpha-linolenic acid (18:3omega3) has many important physiological functions including being beta-oxidized, serving a precursor to the synthesis of other lipids and it has immunomodulation properties. The objective of the present study was to test the effects of immunization and dietary 18:3omega3 on immune function and the fatty acid profile of immunized pig tissues. Piglets suckled from sows consuming either a control or high 18:3omega3 diet until 14 days old when they were weaned onto a similar diet as the sow and were moved to a segregated nursery for the remainder of the study. At 35 days of age, pigs on both diets (2 x 2 factorial design) received either an injection containing hen eggwhite lysozyme (HEWL), killed Mycobacterium tuberculosis and Freund's complete adjuvant (immunized) or phosphate buffered saline (PBS) (non-immunized) into the neck followed by a booster injection 2 weeks later and induction of delayed-type hypersensitivity (DTH) one week later. Immunization increased (compared to non-immunized) while the high 18:3omega3 diet decreased haptoglobin by 30% compared to pigs consuming the control diet. Immunized pigs had a seven-fold increase in antibodies to HEWL and pigs consuming the high 18:3omega3 diet also had transiently higher levels of serum antibodies. There was a diet by immunization interaction on the DTH reaction such that immunized pigs consuming the high 18:3omega3 had the largest DTH reaction. The neck muscle proximal to the site of injection of immunized pigs had 10-30% lower levels of triglyceride and phospholipid linoleic (18:2omega6) and 18:3omega3 compared to non-immunized pigs. Thus, a high 18:3omega3 intake in pigs modulates immune function and tissue fatty acids in response to immunization.

Whole-body utilization of n-3 PUFA in n-6 PUFA-deficient rats.

We evaluated the utilization of a-linolenic acid (18:3n-3) in growing rats consuming a diet deficient in n-6 PUFA. After 90 d, whole-body 18:3n-3 accumulation was 55% lower, total n-3 PUFA accumulation was 21% lower, and 18:3n-3 disappearance was 14% higher in n-6 PUFA-deficient rats. Part of the reduction of whole-body 18:3n-3 in n-6 PUFA-deficient rats was due to the 25% increase in net conversion of 18:3n-3 to long-chain n-3 PUFA. Despite adequate 18:3n-3 intake, n-6 PUFA deficiency decreased the accumulation of 18:3n-3 and total n-3 PUFA.

Intramuscular injection of antigens and adjuvant preferentially decreases 18:2n-6 and 18:3n-3 in pig neck muscle.

Linoleic (18:2n-6) and alpha-linolenic acids (18:3n-3) have many important physiological functions including immunomodulation. We tested how immunization influences the metabolism of 18:2n-6 and 18:3n-3 in the neck muscle of pigs. At 35 d old, pigs received either an intramuscular neck injection containing hen egg white lysozyme (HEWL), killed Mycobacterium tuberculosis, and Freund's complete adjuvant (immunized) or PBS (control). At 49 d old, immunized pigs received a booster injection of HEWL and Freund's incomplete adjuvant, and the control pigs received PBS into the neck. At 56 d old, all pigs received an intradermal injection of Mycobacterium bovis into the hind leg to induce a delayed-type hypersensitivity (DTH) reaction. At 57 d old, immunized pigs had a twofold increase in serum haptoglobin, a 10-fold increase in antibodies to HEWL, and the skinfold at the DTH reaction site was 10 times thicker than the controls. Both 18:2n-6 and 18:3n-3 (% composition) were approximately 25% lower in muscle TG, 40% lower in FFA, 50% lower in phospholipids, but not different in cholesteryl esters of the neck muscle of immunized pigs. The antigens in this model induce an increased response in the innate (haptoglobin), humoral (antibodies), and cellular (DTH) immune systems as well as a preferential decrease of 18:2n-6 and 18:3n-3 in the inflamed neck muscle. It appears that 18:2n-6 and 18:3n-3 are preferentially metabolized (possibly beta-oxidized) in response to antigens.