In situ substrate generation to characterize the maltosil-transferase GlgE from Rhodococcus jostii and its promiscuity towards substrates

ALVAREZ HM; IGLESIAS AA; IGLESIAS MJ; ASENCION DIEZ MD

Rosario

Congreso; LIX SAIB ANNUAL MEETING; 2023

Sociedad Argentina de Investigaciones en Bioquimica y Biologia Molecular (SAIB)

In recent years, a new maltosil transferase (EC: 2.4.99.16, GlgE) was discovered in Actinobacteria, leadingto the hypothesis of a novel pathway for glycogen synthesis. GlgE elongates a preformed glycogenmolecules in two glycosidic moieties using maltose-1P as a substrate, contrasting the classic pathwaywhere glycogen is the acceptor of a single glucose subunit from ADP-glucose (ADP-Glc). Maltose-1Pmay be synthesized by the maltosil-1P synthase (EC: 2.4.1.342, GlgM) that uses Glc-1P and ADP-Glc assubstrates. The NDP-sugar is produced from Glc-1P and ATP by ADP-Glc pyrophosphorylase (EC: 2.7.7.27,GlgC). This is the key step in glycogen synthesis as it is regulated by different effectors related to themain carbon pathway(s) in the organism, setting Glc-1P destiny to glycogen storage. So far, thisalternative pathway for glycogen synthesis has only been described in Mycobacteria by thecharacterization of the GlgM and GlgE enzymes. Further studies on this enzymatic route in othermicroorganisms are lacking in the main literature, although glgE was predicted to be in 14% of knownbacterial genomes. Our previous work reports the characterization of GlgC and GlgM from Rhodococcusjostii (RjoGlgC and RjoGlgM). Remarkably, both enzymes were able to use glucosamine-1P (GlcN-1P)alternatively to Glc-1P as a substrate. Here, we present the recombinant obtention, purification and