The molybdenum cofactor, a complex of the trace element with an organic molecule termed molybdopterin, ia a common component of the three human molybdoenzymes sulfite oxidase, xanthine dehydrogenase and aldehyde oxidase. The severe neuropathology and fatal consequences of molybdenum cofactor deficiency in humans underscore the essential nature of Mo, molybdopterin and sulfite oxidase for normal human development. The broad goals of this project are to investigate the molecular lesions in patients with genetic deficiency of sulfite oxidase or with molybdenum cofactor deficiency. Using cloned human sulfite oxidase DNA as the probe, the defective DNA from fibroblasts of individual patients will be identified and sequenced to assess the effect of the mutation on the expression of the sulfite oxidase protein. The naturally occurring mutants with single amino acid substitutions and mutants created by site directed mutagenesis will be examined by spectroscopic techniques such as Resonance Raman, Extended X-ray Absorbance Fine Structure Analysis, Electron Paramagnetic Resonance, and Magnetic Circular Dichroism as well as a variety of kinetic techniques to delineate the effects of the mutations on the physicochemical properties of the enzyme. The pathway of molybdopterin biosynthesis in humans will be probed using radioactive precursors in the growth medium of group B molybdopterin- deficient fibroblasts which secrete a precursor, termed precursor Z, into the medium. The human genes codeing for the enzymes in the pathway will be searched in cDNA libraries using cloned E. coli genes as the probes. Any identified gene will be cloned, sequenced and expressed in E. Coli in order to understand the nature of the reactions catalyzed by the encoded proteins. The studies described above should help in understanding the biochemical abnormalities in a group of human genetic disorders relating to biochemical function of molybdenum.
