The deoxyxylulose phosphate pathway of isoprenoid biosynthesis: Studies on the mechanisms of the reactions catalyzed by IspG and IspH protein

<jats:p> Earlier <jats:italic>in vivo</jats:italic> studies have shown that the sequential action of the IspG and IspH proteins is essential for the reductive transformation of 2 <jats:italic>C</jats:italic> -methyl- <jats:sc>d</jats:sc> -erythritol 2,4-cyclodiphosphate into dimethylallyl diphosphate and isopentenyl diphosphate via 1-hydroxy-2-methyl-2-( <jats:italic>E</jats:italic> )-butenyl 4-diphosphate. A recombinant fusion protein comprising maltose binding protein and IspG protein domains was purified from a recombinant <jats:italic>Escherichia coli</jats:italic> strain. The purified protein failed to transform 2 <jats:italic>C</jats:italic> -methyl- <jats:sc>d</jats:sc> -erythritol 2,4-cyclodiphosphate into 1-hydroxy-2-methyl-2-( <jats:italic>E</jats:italic> )-butenyl 4-diphosphate, but catalytic activity could be restored by the addition of crude cell extract from an <jats:italic>ispG</jats:italic> -deficient <jats:italic>E. coli</jats:italic> mutant. This indicates that auxiliary proteins are required, probably as shuttles for redox equivalents. On activation by photoreduced 10-methyl-5-deaza-isoalloxazine, the recombinant protein catalyzed the formation of 1-hydroxy-2-methyl-2-( <jats:italic>E</jats:italic> )-butenyl 4-diphosphate from 2 <jats:italic>C</jats:italic> -methyl- <jats:sc>d</jats:sc> -erythritol 2,4-cyclodiphosphate at a rate of 1 nmol⋅min <jats:sup>−1</jats:sup> ⋅mg <jats:sup>−1</jats:sup> . Similarly, activation by photoreduced 10-methyl-5-deaza-isoalloxazine enabled purified IspH protein to catalyze the conversion of 1-hydroxy-2-methyl-2-( <jats:italic>E</jats:italic> )-butenyl 4-diphosphate into a 6:1 mixture of isopentenyl diphosphate and dimethylallyl diphosphate at a rate of 0.4 μmol⋅min <jats:sup>−1</jats:sup> ⋅mg <jats:sup>−1</jats:sup> . IspH protein could also be activated by a mixture of flavodoxin, flavodoxin reductase, and NADPH at a rate of 3 nmol⋅min <jats:sup>−1</jats:sup> ⋅mg <jats:sup>−1</jats:sup> . The striking similarities of IspG and IspH protein are discussed, and plausible mechanistic schemes are proposed for the two reactions. </jats:p>