Distinctive properties of ADP-glucose pyrophosphorylases from Gram-positive bacteria

ASENCIÓN DIEZ, MATÍAS D.; DEMONTE, ANA MARÍA; PEIRÚ, SALVADOR; GRAMAJO, HUGO; GUERRERO, SERGIO A.; IGLESIAS, ALBERTO A.

San Miguel de Tucumán

Congreso; VII Congreso de Microbiología General “SAMIGE del Bicentenario”; 2011

Sociedad Argentina de Microbiología General -SAMIGE-

Glycogen or similar &alpha;-1,4-polyglucans were reported in more than 50 different bacterial species. This is not restricted to any class of bacteria, as many Gram-negative and Gram-positive bacteria as well as archaebacteria have been reported to accumulate the polysaccharide. The process occurs through ADP‑glucose (ADPGlc) as glucosyl donor for elongation of the polyglucan. ADPGlc is produced via ADPGlc pyrophosphorylase (ADPGlcPPase, EC 2.7.7.27) in a key regulatory step of the biosynthetic pathway. ADPGlcPPase has been purified from a number of microorganisms, and as a rule is was found homotetrameric in structure. The one exception is the enzyme from <i>Bacillus</i> sp., as in <i>B. stearothermophilus</i> it is also a tetramer, but composed of two subunits: GlgC (active) and GlgD. Also, ADPGlcPPases from bacteria are allosterically regulated enzymes. In general, ADPGlcPPase effectors are main metabolites of the major pathway for carbon assimilation in the respective organism. Studies of enzymes involved in glycogen metabolism in Gram‑positive bacteria are scarce, which represents an important deficit for the understanding of carbon metabolism in these organisms. We characterized the kinetic, regulatory and structural properties of ADPGlcPPases from <i>Mycobacterium tuberculosis</i> (<i>Mtu</i>), <i>Streptomyces coelicolor</i> (<i>Sco</i>) and <i>Streptococcus mutans</i> (<i>Smu</i>). <i>Mtu</i> and <i>Sco</i> ADPGlcPPases share 60% identity at the protein level and both were activated by PEP and Glc6P, the latter being a novel activator for bacterial ADPGlcPPases. In contrast, we found differences concerning affinity for substrates and enzyme activity, as well as sensitivity to allosteric effectors. Particularly, the <i>Sco</i> enzyme is 20-fold less active than the <i>Mtu</i> counterpart, but the former has 5-fold more affinity for substrates and exhibits more promiscuity to effectors, being also inhibited by NADPH and Pi. Concerning the <i>Smu</i> ADPGlcPPase, it is composed by two subunits: GlgC and GlgD [this being a characteristic of the enzyme from firmicutes], and we determined that GlgC, but not GlgD, is the catalytic one. Also, the heteromeric conformation GlgCD has activity one order of magnitude higher than homomeric GlgC and both conformations have distinctive allosteric effectors. Fru-1,6-bisP activated 2-fold GlgC activity, but it was ineffective on GlgCD. As well, PEP inhibited GlgCD but not GlgC activity. Our results represent key differences regarding the enzyme from <i>B. stearothermophilus</i>, which is an unregulated ADPGlcPPase exhibiting no difference in <i>V</i>_max between GlgC and GlgCD conformations. The whole view supports that regulation of the key regulatory enzyme for glycogen synthesis in Gram-positive bacteria shows peculiarities. These seem closely related to particular metabolic routes in the respective organism operating in carbon and energy accumulation as well as in Glc1P partitioning.