MOLECULAR AND BIOCHEMICAL BASIS OF INTERMEDIATE MAPLE-SYRUP-URINE-DISEASE - OCCURRENCE OF HOMOZYGOUS G245R AND F364C MUTATIONS AT THE E1-ALPHA LOCUS OF HISPANIC-MEXICAN PATIENTS

Maple syrup urine disease (MSUD) is caused by a deficiency of the mito chondrial branched-chain alpha-keto acid dehydrogenase (BCKAD) complex . The multienzyme complex comprises five enzyme components, including the E1 decarboxylase with a heterotetrameric (alpha(2) beta(2)) struct ure. Four unrelated Hispanic-Mexican MSUD patients with the intermedia te clinical phenotype were diagnosed 7 to 22 mo after birth during eva luation for developmental delay. Three of the four patients were found homozygous for G to A transition at base 895 (exon 7) of the E1 alpha locus, which changes Gly-245 to Arg (G245R) in that subunit. The rema ining patient was homozygous for T to G transversion at base 1,253 in the E1 alpha gene, which converts Phe-364 to Cys (F364C) in the gene p roduct. Transfection studies in E1 alpha-deficient lymphoblasts indica te that both G245R and F364C mutant E1 alpha subunits were unable to s ignificantly reconstitute BCKAD activity. Western blotting showed that both mutant E1 alpha subunits in transfected cells failed to efficien tly rescue the normal E1 beta through assembly. The putative assembly defect was confirmed by pulse-chase labeling of E1 subunits in a chape rone-augmented bacterial overexpression system. The kinetics of initia l assembly of the G245R E1 alpha subunit with the normal E1 beta was s hown to be slower than the normal E1 alpha. No detectable assembly of the F364C E1 alpha subunit with normal E1 beta was observed during the 2 h chase. Small amounts of recombinant mutant E1 proteins mere produ ced after 15 h induction with isopropyl thiogalactoside and exhibited very low or no E1 activity. Our study establishes that G245R and F364C mutations in the E1 alpha subunit disrupt both the E1 heterotetrameri c assembly and function of the BCKAD complex. Moreover, the results su ggest that the G245R mutant E1 alpha allele may be important in the Hi spanic-Mexican population.