In a fruitful collaboration with Bill Jacobs and Rainer Kalscheuer at the Albert Einstein College of Medicine in New York, we have identified a new four-step metabolic pathway that is widespread among bacteria (and present in one archaeal species). It is involved in the developmentally regulated management of carbon in Streptomyces coelicolor and is an attractive and novel target for anti-tuberculosis drugs. The pathway converts the abundant disaccharide trehalose (which often serves to protect cells against stress) into an alpha-glucan polysaccharide. Three of the four enzyme activities had been described previously, but we were able to link them together in a new pathway by identifying the novel activity of a fourth enzyme, GlgE.
Karl Syson (JIC) fully characterised the GlgE enzyme. It catalyses the anabolic polymerisation of alpha-maltose 1-phosphate to an alpha-1,4 linked glucan with the release of phosphate in a ping-pong type mechanism. In the new pathway, trehalose (alpha,alpha-1,1-linked diglucose) is converted to maltose (alpha-1,4-linked diglucose) by TreS enzyme and the maltose is phosphorylated by Pep2 maltose kinase. The action of GlgE generates linear glucans that are then acted upon by GlgB enzyme to make a branched glucan product. GlgE works most efficiently with an acceptor molecule with at least four glucose units. The enzyme belongs to the glycoside hydrolase 13_3 family and yet it catalyses a glycosyltransfer reaction. Interestingly, GlgE also exhibits a glycoside hydrolase-type transglucosidation reaction by disproportionating maltose units between chains. Thus it is capable of editing chain lengths as well as extending them with maltose 1-phosphate. With David Lawson, we have solved the structure of a GlgE, which provides a platform for not only understanding how it functions but also for developing inhibitors.
Meanwhile, Rainer Kalscheuer (AECOM) observed that when the GlgE-catalysed step is blocked in Mycobacterium tuberculosis, the toxic build-up of the maltose 1-phosphate intermediate leads to cell killing of the pathogen. Surprisingly, the organism reacts to this poisoning by generating more of the maltose 1-phosphate precursor, trehalose, in a misguided stress response. Blocking the first steps of the pathway leads to lethality when another glucan pathway is simultaneously blocked (a phenomenon known in genetics as synthetic lethality). This implies that the product(s) of the two pathways, which are implicated in cell wall/capsule biogenesis, virulence and persistence, are essential for growth. These two pathways therefore represent novel targets that are ripe for drug targeting with combination therapies that minimises the chance of resistance developing. The development of new therapies against TB is more important than ever given the emergence of extensively-drug-resistant strains of M. tuberculosis that are virtually untreatable with any of the current drugs.
The new pathway was discovered independently by the JIC and AECOM teams using complementary approaches. The identification of the likely role of GlgE in a new pathway in actinomycetes by Steph Bornemann and Karl Syson (JIC) built upon both published and unpublished research carried out in the past at the JIC by Celia Bruton and Keith Chater. The current JIC team used comprehensive in vitro enzymology to support their hypothesis. It was when Steph Bornemann wanted to find a collaborator to carry out the mycobacterial genetics that he discovered through Graham Hatfull that Bill Jacobs and Rainer Kalscheuer (AECOM) were already studying the genetics and had independently identified the role of GlgE using a sophisticated combination of traditional and chemical genetic approaches. The two groups published their complementary studies together. In a neat twist of fate, Bill Jacobs attended an EMBO course on the genetic manipulation of Streptomyces at the JIC in 1985, hosted by David Hopwood, Keith Chater and Celia Bruton, amongst others, before establishing his M. tuberculosis genetics group at AECOM.
