Expression, Purification, and Crystallization of the Human Oxidoreductase, Pyrox-D1: A New Described Cause of Early-Onset Myopathy in Humans

Abstract

Mutations in the oxidoreductase, Pyrox-D1, have been linked to a newly described, early-onset recessive myopathy from five families with four different recessive variants. This new myopathy presents with a histopathology that is distinctive in that it combines multiple pathological hallmarks characteristic of different myopathies; central and minicore disease, centronuclear, myofibrillar and nemaline myopathy. Patients present in infancy with slowly progressive proximal and distal weakness, facial weakness, nasal speech, swallowing difficulties, and mild to moderately elevated serum Creatine Kinase (CK) levels. The link between the mutations in the Pyrox-D1 gene and the disease phenotype are not yet understood. This project involves expression, purification, and crystallization of Pyrox-D1 for eventual characterization by X-ray diffraction. Following crystallization, preliminary characterization will involve spectroscopic and enzymatic analysis to determine the co-factors used by this enzyme, and eventual structure determination by X-ray crystallography. Understanding the co-factors and biophysical characteristic of this protein could help in developing new treatments in the future for this form of myopathy. To date, we have purified and crystallized Pyrox-d1. Going forward, the goal is to determine the molecular structure of Pyrox-D1, which will help elucidate the protein's function. The structure and function can be utilized to develop pharmaceutical treatments for patients presenting with this idiopathic dystrophy. Currently we know that a cofactor is present in Pyrox-D1, and we will use an FAD assay to further understand the structure. A potential homologous structure has been created for Pyrox-D1. Two mutations have been identified in idiopathic muscular dystrophy patients, which we believe will be found in Pyrox-D1 once we have determined the molecular structure.