Functional analysis of the methylerythritol phosphate pathway terminal enzymes IspG and IspH from .

Unlabelled: Isoprenoids are a diverse family of compounds that are synthesized from two isomeric compounds, isopentenyl diphosphate and dimethylallyl diphosphate. In most bacteria, isoprenoids are produced from the essential methylerythritol phosphate (MEP) pathway. The terminal enzymes of the MEP pathway IspG and IspH are [4Fe-4S] cluster proteins, and in the substrates of IspG and IspH accumulate in cells in response to O, suggesting possible lability of their [4Fe-4S] clusters. Here, we show using complementation assays in that even under anaerobic conditions, IspG and IspH are not as functional as their counterparts, requiring higher levels of expression to rescue viability. A deficit of the sulfur utilization factor (SUF) Fe-S cluster biogenesis pathway did not explain the reduced function of IspG and IspH since no improvement in viability was observed in expressing the SUF pathway or having increased expression of the SUF pathway. Complementation of single and double mutants with various combinations of and IspG and IspH indicated that optimal growth required the pairing of IspG and IspH from the same species. Furthermore, IspH conferred an O-sensitive growth defect to that could be partially rescued by co-expression of IspG. analysis showed O sensitivity of the [4Fe-4S] cluster of both IspG and IspH. Altogether, our data indicate an important role of the cognate protein IspG in IspH function under both aerobic and anaerobic conditions.

Importance: Isoprenoids are one of the largest classes of natural products, exhibiting diversity in structure and function. They also include compounds that are essential for cellular life across the biological world. In bacteria, isoprenoids are derived from two precursors, isopentenyl diphosphate and dimethylallyl diphosphate, synthesized primarily by the methylerythritol phosphate pathway. The aerotolerant has the potential for methylerythritol phosphate pathway engineering by diverting some of the glucose that is typically efficiently converted into ethanol to produce isoprenoid precursors to make bioproducts and biofuels. Our data revealed the surprising finding that IspG and IspH need to be co-optimized to improve flux via the methyl erythritol phosphate pathway in part to evade the oxygen sensitivity of IspH.