Event Title

Expression and Characterization of MppP from Verrucosispora maris

Mentor 1

Dr. Nicholas Silvaggi

Location

Union Wisconsin Room

Start Date

29-4-2016 1:30 PM

End Date

29-4-2016 3:30 PM

Description

MppP is an enzyme involved in the synthesis of the non-proteinogenic amino acid L-enduracididine (L-End), which is a component of a number of naturally produced antibiotics. The marine bacterium Verrucosispora maris produces a glycopeptide antibiotic called mannopeptimycin that contains two L-End residues. Mannopeptimycin is an interesting compound, because it has potent activity against antibiotic-resistant strains like MRSA. It is potentially a useful scaffold for further antibiotic development, but this research has been hampered by the fact the key L-End building block is not commercially available and is not terribly easy to synthesize. In order to develop an enzymatic or chemo-enzymatic route to L-End, we have been working to determine how this amino acid is synthesized in producing organisms. Based on sequence similarity to known ATases, the MppP homolog from V. maris (VmMppP) is predicted to be a PLP-dependent aminotransferase (ATase). However, our structural and kinetics studies show that VmMppP is not an ATase, but instead catalyzes the oxidation of L-Arg to give a mixture of 4-hydroxy-2-ketoarginine (4HKA) and 2-ketoarginine (2KA). Further studies require large quantities of protein. The goal of this research was to develop an efficient protein expression protocol for the production of VmMppP. A number of different media, as well as expression conditions like temperature were tested.

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

Expression and Characterization of MppP from Verrucosispora maris

Union Wisconsin Room

MppP is an enzyme involved in the synthesis of the non-proteinogenic amino acid L-enduracididine (L-End), which is a component of a number of naturally produced antibiotics. The marine bacterium Verrucosispora maris produces a glycopeptide antibiotic called mannopeptimycin that contains two L-End residues. Mannopeptimycin is an interesting compound, because it has potent activity against antibiotic-resistant strains like MRSA. It is potentially a useful scaffold for further antibiotic development, but this research has been hampered by the fact the key L-End building block is not commercially available and is not terribly easy to synthesize. In order to develop an enzymatic or chemo-enzymatic route to L-End, we have been working to determine how this amino acid is synthesized in producing organisms. Based on sequence similarity to known ATases, the MppP homolog from V. maris (VmMppP) is predicted to be a PLP-dependent aminotransferase (ATase). However, our structural and kinetics studies show that VmMppP is not an ATase, but instead catalyzes the oxidation of L-Arg to give a mixture of 4-hydroxy-2-ketoarginine (4HKA) and 2-ketoarginine (2KA). Further studies require large quantities of protein. The goal of this research was to develop an efficient protein expression protocol for the production of VmMppP. A number of different media, as well as expression conditions like temperature were tested.