Event Title

Structure and Function of PbrMppP from Pseudomonas brassicacearum

Mentor 1

Nicholas Silvaggi

Location

Union Wisconsin Room

Start Date

29-4-2016 1:30 PM

End Date

29-4-2016 3:30 PM

Description

Antibiotic resistance is a substantial problem facing our world that requires new antibiotic development. A number of interesting antibiotic compounds are produced by bacteria and fungi that contain the non-proteinogenic amino acid L-enduracididine (L-End). These include the recently discovered antibiotic Teixobactin and two older antibiotics, enduracidin and mannopeptimycin, that are currently in human trials for specific types of infections. It is difficult to develop new compounds based on these scaffolds, since L-End is not commercially available and is prohibitively expensive to make in large quantities. We are interested in learning how L-End is made in nature in order to have a relatively inexpensive source of this unusual building block for antibiotic development research. The biosynthetic cluster for mannopeptimycin contains three enzymes, designated MppP, Q, and R, that synthesize L-End. We are currently studying MppP from Pseudomonas brassicacearum (PbrMppP), which uses the cofactor pyridoxal-5’-phosphate (PLP) to catalyze the oxidation of L-arginine to give a mixture of 2-ketoarginine and 4-hydroxy-2-ketoarginine. This is the first enzyme known to use PLP in a hydroxylation reaction. We have determined the structure of PbrMppP using X-ray crystallography. The tertiary structure of PbrMppP is similar to typical PLP-dependent enzymes like aspartate aminotransferase. There is no metal ion in the active site, in agreement with preliminary enzyme kinetics experiments. That continue there is no metal ion (unlike other hydroxylases which often use a metal ion). The structure of PbrMppP with the final product bound shows how the enzyme likely interacts with its substrate. The structure of PbrMppP was at a resolution of 1.8 Å and the structure of PbrMppP with 4-hydroxy-2-ketoarginine had a resolution of 2.0 Å. Preliminary kinetics studies also indicate that PbrMppP is most active at pH 8.0 and that there is a buffer effect, where the enzyme is most active with TRIS.

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Apr 29th, 1:30 PM Apr 29th, 3:30 PM

Structure and Function of PbrMppP from Pseudomonas brassicacearum

Union Wisconsin Room

Antibiotic resistance is a substantial problem facing our world that requires new antibiotic development. A number of interesting antibiotic compounds are produced by bacteria and fungi that contain the non-proteinogenic amino acid L-enduracididine (L-End). These include the recently discovered antibiotic Teixobactin and two older antibiotics, enduracidin and mannopeptimycin, that are currently in human trials for specific types of infections. It is difficult to develop new compounds based on these scaffolds, since L-End is not commercially available and is prohibitively expensive to make in large quantities. We are interested in learning how L-End is made in nature in order to have a relatively inexpensive source of this unusual building block for antibiotic development research. The biosynthetic cluster for mannopeptimycin contains three enzymes, designated MppP, Q, and R, that synthesize L-End. We are currently studying MppP from Pseudomonas brassicacearum (PbrMppP), which uses the cofactor pyridoxal-5’-phosphate (PLP) to catalyze the oxidation of L-arginine to give a mixture of 2-ketoarginine and 4-hydroxy-2-ketoarginine. This is the first enzyme known to use PLP in a hydroxylation reaction. We have determined the structure of PbrMppP using X-ray crystallography. The tertiary structure of PbrMppP is similar to typical PLP-dependent enzymes like aspartate aminotransferase. There is no metal ion in the active site, in agreement with preliminary enzyme kinetics experiments. That continue there is no metal ion (unlike other hydroxylases which often use a metal ion). The structure of PbrMppP with the final product bound shows how the enzyme likely interacts with its substrate. The structure of PbrMppP was at a resolution of 1.8 Å and the structure of PbrMppP with 4-hydroxy-2-ketoarginine had a resolution of 2.0 Å. Preliminary kinetics studies also indicate that PbrMppP is most active at pH 8.0 and that there is a buffer effect, where the enzyme is most active with TRIS.