Flux through Trehalose Synthase Flows from Trehalose to the Alpha Anomer of Maltose in Mycobacteria

Abstract

SummaryTrehalose synthase (TreS) was thought to catalyze flux from maltose to trehalose, a precursor of essential trehalose mycolates in mycobacterial cell walls. However, we now show, using a genetic approach, that TreS is not required for trehalose biosynthesis in Mycobacterium smegmatis, whereas two alternative trehalose-biosynthetic pathways (OtsAB and TreYZ) are crucial. Consistent with this direction of flux, trehalose levels in Mycobacterium tuberculosis decreased when TreS was overexpressed. In addition, TreS was shown to interconvert the α anomer of maltose and trehalose using 1H and 19F-nuclear magnetic resonance spectroscopies using its normal substrates and deoxyfluoromaltose analogs, with the nonenzymatic mutarotation of α/β-maltose being slow. Therefore, flux through TreS in mycobacteria flows from trehalose to α-maltose, which is the appropriate anomer for maltose kinase of the GlgE α-glucan pathway, which in turn contributes to intracellular and/or capsular polysaccharide biosynthesis.

Genetics of Mycobacterial Trehalose Metabolism

“…Although the equilibrium of purified TreS favors the formation of trehalose from maltose in vitro, flux through TreS in vivo is in the opposite direction (24), which is driven by the rapid and irreversible ATP-dependent phosphorylation of the formed maltose to maltose-1-phosphate by the maltose kinase Pep2 (Rv0127) (26,27). The observed finding of the direction of flux through TreS for consumption of trehalose in M. smegmatis contradicts a previous study (9).…”

“…This observation clearly defined that TreS plays no significant role in trehalose biosynthesis in M. smegmatis under the tested in vitro conditions. Furthermore, overexpression of TreS (Rv0126) in M. tuberculosis did not cause an elevation, but rather the opposite, a drastic reduction of the intracellular trehalose level, implying that TreS consumes rather than produces trehalose in mycobacteria (24). In fact, as described later in this article, TreS has recently been recognized as the first enzymatic step of a novel four-step pathway converting trehalose into branched alpha-glucans, which is widespread among prokaryotes (23,25).…”

“…In order to unambiguously readdress the importance of TreS for trehalose biosynthesis in M. smegmatis, defined gene deletion mutants were generated with all possible combinatorial inactivations of the OtsA-OtsB, TreY-TreZ, and TreS pathways by targeting the genes otsA, treY-Z, and treS. These studies revealed that combined inactivation of the OtsA-OtsB and TreYTreZ pathways was sufficient to result in trehalose auxotrophy in M. smegmatis, although in this genetic context the treS gene was still intact (24). This observation clearly defined that TreS plays no significant role in trehalose biosynthesis in M. smegmatis under the tested in vitro conditions.…”

“…5), which is now known as the GlgE pathway and which is widespread among prokaryotes (23,25). TreS interconverts trehalose and maltose with formation of the α-anomer of maltose (24), which is subsequently rapidly and quantitatively phosphorylated in an ATPdependent reaction to maltose-1-phosphate by the maltose kinase Pep2 (Rv0127) (27). Maltose-1-phosphate then acts as the activated donor substrate for the key enzyme of this pathway, the maltosyltransferase GlgE (Rv1327c), which transfers the maltosyl moiety to the nonreducing 4′-hydroxyl group of an α-1,4-glucan acceptor molecule, yielding linear alpha-glucans.…”

α-Glucan biosynthesis and the GlgE pathway in Mycobacterium tuberculosis

“…The final enzyme in the GlgE pathway is an α-1,6 branching enzyme [31,32]. The overall thermodynamics of the four-step GlgE pathway dictate that flux must be in favour of the production of α-glucan from trehalose [33] and this has been confirmed in vivo [34]. Indeed, in the context of the GlgE pathway the name trehalose synthase is misleading because flux through this enzyme is away from trehalose towards α-maltose.…”