Structural Determination and Function of Streptomyces griseofuscus
Mentor 1
Nicholas Silvaggi
Location
Union Wisconsin Room
Start Date
5-4-2019 1:30 PM
End Date
5-4-2019 3:30 PM
Description
The enzyme MppP from Streptomyces wadayamensis is the prototype of a family of pyridoxal 5’-phosphate (PLP)-dependent enzymes that use dioxygen to perform a 4-electron oxidation of L-Arg to 4-hydroxy-2-ketoarginine in the biosynthesis of L-enduracididine (L-End). L-End is a non-proteinogenic amino acid found in several peptide antibiotics, such as mannopeptimycin. MppP homologs exist in different genomic contexts and thus likely have different biochemical roles. For example, a group of MppP-like enzymes that are associated with an NADH-dependent reductase produce 4,5-dehydro-D-arginine rather than the hydroxylated product. Another MppP homolog was found in an uncharacterized gene cluster from Streptomyces griseofuscus, along with a hypothetical protein predicted to be a proclavaminate amidinohydrolase (SgrAH), a putative iron- and S-adenosylmethionine (SAM)-dependent methyltransferase (SgrMT), a predicted N-acetyltransferase (SgrNAT), a homolog of hydroxyphenylacetate 3-monooxygenase, and two proteins with no significant homology to proteins of known function. Here we show that SgrMppP has the same catalytic activity as the prototypical enzyme from S. wadayamensis. Initial activity tests show that SgrAH does not possess argininase, agmatinase, guanidinopropionase, or arginine deiminase activities when tested against agmatine, L-Arg, 2-ketoarginine, or the SgrMppP reaction product, 4-hydroxy-2-ketoarginine. The structure of SgrAH was determined and strongly suggests that it is a true amidinohydrolase. Given the lack of SgrAH activity in the preliminary tests, it is likely that SgrMT acts on the MppP product, and this methylated compound is the substrate for SgrAH. SgrMT does bind both iron and SAM. We are currently focused on finding the substrate for SgrAH, determining the structure and activity of SgrMT, and expressing SgrNAT.
Structural Determination and Function of Streptomyces griseofuscus
Union Wisconsin Room
The enzyme MppP from Streptomyces wadayamensis is the prototype of a family of pyridoxal 5’-phosphate (PLP)-dependent enzymes that use dioxygen to perform a 4-electron oxidation of L-Arg to 4-hydroxy-2-ketoarginine in the biosynthesis of L-enduracididine (L-End). L-End is a non-proteinogenic amino acid found in several peptide antibiotics, such as mannopeptimycin. MppP homologs exist in different genomic contexts and thus likely have different biochemical roles. For example, a group of MppP-like enzymes that are associated with an NADH-dependent reductase produce 4,5-dehydro-D-arginine rather than the hydroxylated product. Another MppP homolog was found in an uncharacterized gene cluster from Streptomyces griseofuscus, along with a hypothetical protein predicted to be a proclavaminate amidinohydrolase (SgrAH), a putative iron- and S-adenosylmethionine (SAM)-dependent methyltransferase (SgrMT), a predicted N-acetyltransferase (SgrNAT), a homolog of hydroxyphenylacetate 3-monooxygenase, and two proteins with no significant homology to proteins of known function. Here we show that SgrMppP has the same catalytic activity as the prototypical enzyme from S. wadayamensis. Initial activity tests show that SgrAH does not possess argininase, agmatinase, guanidinopropionase, or arginine deiminase activities when tested against agmatine, L-Arg, 2-ketoarginine, or the SgrMppP reaction product, 4-hydroxy-2-ketoarginine. The structure of SgrAH was determined and strongly suggests that it is a true amidinohydrolase. Given the lack of SgrAH activity in the preliminary tests, it is likely that SgrMT acts on the MppP product, and this methylated compound is the substrate for SgrAH. SgrMT does bind both iron and SAM. We are currently focused on finding the substrate for SgrAH, determining the structure and activity of SgrMT, and expressing SgrNAT.
