Event Title

Glyoxylate Reductase: An Unexpected Journey

Mentor 1

Nicholas Silvaggi

Location

Union Wisconsin Room

Start Date

28-4-2017 1:30 PM

End Date

28-4-2017 4:00 PM

Description

We are studying how organisms like Streptomyces hygroscopicus biosynthesize enduracididine from arginine. by three enzymes: Our preliminary data suggest that MppQ catalyzes the last step in the process, the transamination of the ketone form of enduracididine ("keto-End") and an amino group donor, like alanine or glycine. MppQ also reacts with ketoarginine and an amino group donor to recycles the dead-end product back to L-arginine. Alanine is quite a poor substrate, having very low affinity for the enzyme (KM 50 mM). We suspect that glycine will prove to be a more efficient amino donor substrate, but in order to measure the steady state parameters of the reaction we need a coupling enzyme that will work with glyoxylate (the product when glycine donates its amino group to the "keto-End" or ketoarginine). The lactate dehydrogenase that we used to measure the reaction with ketoarginine and alanine would not accept glyoxylate as a substrate. To get around this problem, we cloned glyoxylate reductase (GR) from E. coli, which we reasoned should react well with glyoxylate. Surprisingly, GR has a higher affinity for 2-ketoarginine than its native ligand, glyoxylate. This makes wild-type GR a poor coupling enzyme for monitoring the reaction of MppQ with ketoarginine and glycine. We have undertaken structural studies to guide our efforts to engineer a form of GR that will not react efficiently with ketoarginine. Here we present our structural evidence for the binding mode of ketoarginine to the GR active site, as well as the steady state kinetic characterization of GR reacting with 2-ketoarginine, glyoxylate, α-ketoglutarate, NADH, and NADPH. We have found that there is significant substrate inhibition with glyoxylate, but not with 2-ketoarginine. Given the low concentrations of glyoxylate in our MppQ assays, this substrate inhibition is unlikely to be a problem for our future work with MppQ.

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Apr 28th, 1:30 PM Apr 28th, 4:00 PM

Glyoxylate Reductase: An Unexpected Journey

Union Wisconsin Room

We are studying how organisms like Streptomyces hygroscopicus biosynthesize enduracididine from arginine. by three enzymes: Our preliminary data suggest that MppQ catalyzes the last step in the process, the transamination of the ketone form of enduracididine ("keto-End") and an amino group donor, like alanine or glycine. MppQ also reacts with ketoarginine and an amino group donor to recycles the dead-end product back to L-arginine. Alanine is quite a poor substrate, having very low affinity for the enzyme (KM 50 mM). We suspect that glycine will prove to be a more efficient amino donor substrate, but in order to measure the steady state parameters of the reaction we need a coupling enzyme that will work with glyoxylate (the product when glycine donates its amino group to the "keto-End" or ketoarginine). The lactate dehydrogenase that we used to measure the reaction with ketoarginine and alanine would not accept glyoxylate as a substrate. To get around this problem, we cloned glyoxylate reductase (GR) from E. coli, which we reasoned should react well with glyoxylate. Surprisingly, GR has a higher affinity for 2-ketoarginine than its native ligand, glyoxylate. This makes wild-type GR a poor coupling enzyme for monitoring the reaction of MppQ with ketoarginine and glycine. We have undertaken structural studies to guide our efforts to engineer a form of GR that will not react efficiently with ketoarginine. Here we present our structural evidence for the binding mode of ketoarginine to the GR active site, as well as the steady state kinetic characterization of GR reacting with 2-ketoarginine, glyoxylate, α-ketoglutarate, NADH, and NADPH. We have found that there is significant substrate inhibition with glyoxylate, but not with 2-ketoarginine. Given the low concentrations of glyoxylate in our MppQ assays, this substrate inhibition is unlikely to be a problem for our future work with MppQ.