Cloning, overexpression, and characterization of a bacterial XXXX -dependent phospholipase D.

@entity71124 @entity71122 @entity462 @entity526 @entity4620 @entity3793 @entity71123 @entity2569 @entity2734 @entity8558 @entity1520 @entity141 @entity71125 @entity493 @entity71121
@entity4620 ( @entity4620 ), an important enzyme involved in signal transduction in mammals, is also secreted by many microorganisms. A highly conserved HKD motif has been identified in most @entity4620 homologs in the @entity4620 superfamily. However, the @entity141 -dependent @entity4620 from Streptomyces chromofuscus exhibits little homology to other PLDs. We have cloned (using DNA isolated from the ATCC type strain), overexpressed in @entity526 (two expression systems, pET-23a(+) and pTYB11), and purified the S. chromofuscus @entity4620 . Based on attempts at sequence alignment with other known @entity141 -independent @entity4620 enzymes from Streptomyces species, we mutated five @entity3793 residues (His72, His171, His187, His200, His226) that could be part of variants of an HKD motif. Only @entity71121 and @entity71122 showed dramatically reduced activity. However, mutation of these @entity3793 residues to @entity493 also significantly altered the secondary structure of @entity4620 . @entity2734 replacements at these positions yielded enzymes with structure and activity similar to the recombinant wild-type @entity4620 . The extent of @entity8558 (PA) activation of PC hydrolysis by the recombinant @entity4620 enzymes differed in magnitude from @entity4620 purified from S. chromofuscus culture medium (a 2-fold activation rather than 4-5-fold). One of the His mutants, @entity71123 , showed a 12-fold enhancement by PA, suggesting this residue is involved in the kinetic activation. Another notable difference of this bacterial @entity4620 from others is that it has a single @entity462 ( @entity2569 123); other Streptomyces @entity141 -independent PLDs have eight @entity2569 involved in intramolecular @entity1520 bonds. Both @entity71124 and @entity71125 , with secondary structure and stability similar to recombinant wild-type @entity4620 , exhibited specific activity reduced by 10(-5) and 10(-4). The @entity2569 mutants still bound @entity141 , so that it is likely that this residue is part of the active site of the @entity141 -dependent @entity4620 . This would suggest that S. chromofuscus @entity4620 is a member of a new class of @entity4620 enzymes.
