Substitution of Zn2+ with alternative metals increases the rate of flavin- reduction in bacterial D-2-hydroxyglutarate dehydrogenase

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Abstract Summary/Description
The enzyme D-2-hydroxyglutarate dehydrogenase is essential for the biosynthesis of L-serine in Pseudomonas aeruginosa PAO1 and offers a significant opportunity for the development of new drugs.(1) Being a metallo-flavoenzyme, the bacterial enzyme employs FAD and Zn2+ to convert D-2-hydroxyglutarate to 2-ketoglutarate.(2,3) The enzyme is also active with the alternative metals Co2+, Ni2+, Mn2+, and Cd2+.(3) In this study, Zn2+ was replaced with Co2+, Ni2+, Cd2+, or Mn2+ to explore the impact of these metals on the rate of flavin reduction and catalysis in the bacterial enzyme. We purified the recombinant His-tagged enzyme with 1 mM chloride salts of the various metals and tested the kinetic properties and metal stoichiometry of all enzyme variants. The metal-to-protein ratio was determined by ICP-MS to evaluate metal binding to the enzyme and all metals were loaded in significant amounts to the enzyme. Aside from the Cd2+-loaded enzyme that showed no D-malate concentration dependence on the rate of flavin reduction, the anaerobic reductive half-reaction showed a ≤ 2-fold increase in rate of flavin reduction (kred) values for the Co2+, Ni2+, and Mn2+-loaded enzymes compared to the Zn2+-loaded enzyme (70 s-1), indicating that alternative metals influence the rate of flavin reduction in the enzyme. Additionally, the Kd values for the D-malate substrate were similar for the Zn2+, Co2+ and Mn2+-loaded enzymes (~10 mM) but was ~2-fold less with the Ni2+-loaded enzyme (4 mM), suggesting that the highest binding affinity is achieved with the least electropositive metal (Ni2+) in the bacterial enzyme. While Ni2+ (24,000 M-1s-1) and Co2+ (43,000 M-1s-1) increased the kcat/Km values of the bacterial enzyme, Mn2+ (3,300 M-1s-1) and Cd2+ (950 M-1s-1) decreased the enzyme’s kcat/Km value compared to Zn2+ (7,000 M-1s-1) with D-malate as substrate. Additionally, all alternative metals yielded a ~4-fold decrease in the rate of enzyme turnover compared to the Zn2+-loaded enzyme, except for Co2+, which yielded similar rates as Zn2+. These results collectively indicate that the alternative metal ions modulate the rate of flavin reduction, substrate specificity and catalysis in the bacterial enzyme.
Abstract ID :
NKDR221
Department Of Chemistry, College Of Arts And Sciences
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