Characterization of SpnQ from the Spinosyn Biosynthetic Pathway of <i>Saccharopolyspora spinosa</i>:  Mechanistic and Evolutionary Implications for C-3 Deoxygenation in Deoxysugar Biosynthesis

2006-11-08T00:00:00Z (GMT) by Lin Hong Zongbao Zhao Hung-wen Liu
The C-3 deoxygenation step in the biosynthesis of d-forosamine (4-<i>N</i>,<i>N</i>-dimethylamino-2,3,4,6-tetradeoxy-d-<i>threo</i>-hexopyranose), a constituent of spinosyn produced by <i>Saccharopolyspora spinosa</i>, was investigated. The <i>spnQ</i> gene, proposed to encode a TDP-4-keto-2,6-dideoxy-d-glucose 3-dehydratase was cloned and overexpressed in <i>E. coli</i>. Characterization of the purified enzyme established that it is a PMP and iron-sulfur containing enzyme which catalyzes the C-3 deoxygenation in a reductase-dependent manner similar to that of the previously well characterized hexose 3-dehydrase E<sub>1</sub> from <i>Yersinia pseudotuberculosis</i>. However, unlike E<sub>1</sub>, which has evolved to work with a specific reductase partner present in its gene cluster, SpnQ lacks a specific reductase, and works efficiently with general cellular reductases ferredoxin/ferredoxin reductase or flavodoxin/flavodoxin reductase. SpnQ also catalyzes C-4 transamination in the absence of an electron transfer intermediary and in the presence of PLP and l-glutamate. Under the same conditions, both E<sub>1</sub> and the related hexose 3-dehydrase, ColD, catalyze C-3 deoxygenation. Thus, SpnQ possesses important features which distinguish it from other well studied homologues, suggesting unique evolutionary pathways for each of the three hexose 3-dehydrases studied thus far.