Classic Severe Maple Syrup Urine Disease Albert Yu

Introduction Maple Syrup Urine Disease, also known as Branched-Chain Ketoaciduria, is a metabolic disease inherited in an autosomal recessive pattern 1. Victims of Maple Syrup Urine Disease are often diagnosed as infants or young children at the very latest 2.

Symptoms Poor Feeding Lethargy Vomiting Poor Weight Gain Maple Syrup Scented Urine Muscle Spasms/Seizures Brain Damage and Coma Death 2

Normal Branched Chain Amino Acid Metabolism Branched Chain Amino Acids (Valine, Leucine, and Isoleucine) are essential in our diets since they cannot be synthesized by the body. Excess Branched Chain Amino Acids (BCAA’s) are metabolized and used for ATP generation 3.

Normal Branched Chain Amino Acid Metabolism (cont.) Catabolism of BCAA’s has two major steps: – Transfer of an amino group from the amino acid to α-ketoglutarate to form branched chain α-keto acids. – Oxidation of α-keto acids by branched chain amino acid dehydrogenase complex to form CoA derivatives 4.

Branched Chain Amino Acid Dehydrogenase Complex (BCKD) A complex of enzymes that forms Acyl-CoA derivatives from α-keto acids. – E1- α-ketoacid Dehydrogenase – E2- Dihydrolipoyl Transacylase – E3- Dihydrolipoamid Dehydrogenase Ranges in molecular mass from 4 to 10 million daltons, depending on source and type 5.

Maple Syrup Urine Disease- On the Molecular Level Failure to properly catabolize excess BCAA’s causes toxic build-ups of BCAA’s and their α- ketoacid derivatives 2. The NCBI has recognizes four major genes that, if mutated, produce Maple Syrup Urine Disease: – BCKDHA – BCKDHB – DBT – DLD 6

Structural Causes of the Disease Any mutations resulting in structural anomalies that result in inhibition of catalytic functions or proper assembly of the BCKD complex result in Maple Syrup Urine Disease 6.

BCKDHA Gene encodes the E1 alpha subunit of the BCKD complex. The E1 alpha subunit forms a heterotetramer with the beta subunit to form the E1 catalytic subunit of the BCKD complex. The E1 subunit catalyzes the decarboxylation of branched chain α-ketoacids 7.

E1 α Subunit (1DTW)

BCKDHB Gene encodes the E1 beta subunit of the BCKD complex 8.

E1 β Subunit (1DTW)

E1 Subunit (1DTW) α Subunit β Subunit

BCKDHA Mutation Mutations in the BCKDHA gene produce mutant E1 α subunits. Mutant E1 α subunits typically show drastically reduced assembly kinetics, inability to form proper tetramers, improper folding, and no enzymatic activity 7.

BCKDHA Mutation- Y393N Mutation Caused by an A-T Transversion in the BCKDHA gene. Y393N mutation is most prevalent in the Old Order Mennonite population. Mutation affects the assembly kinetics of the E1 subunit; it takes over two hours to assemble the same amount of Y393N mutant E1 as wild-type E1 forms in 10 minutes. Y393 mutant E1 also forms dimers instead of the normal tetramer, thus completely abolishing enzyme activity 7. Since the dimers are α-subunit dimers, Y393 is proposed to be integral to proper association of the α-subunit to the β- subunit 13.

α-Subunits Y393

α-Subunits β-Subunits Y393

F324 W330

BCKDHB Mutation Mutant β subunits demonstrate improper folding, impaired assembly with α subunits, severely inhibited cofactor association, and mitochondrial targeting mutations 8.

BCKDHB Mutation- H156Y C to T transition in the BCKDHB gene results in Histidine being replaced by Tyrosine at the 156 th residue. Produced polypeptides are expressed at 18% of the wild-type. Produced polypeptides are show assembly defects, forming dimers, trimers, or tetramers. Produced β subunits also show drastically reduced affinity for thiamine, an essential cofactor 8. Mutation introduces structural changes by interfering with hydrogen bonding of Histidine 156 and Threonine

α-subunit β-subunit Thiamine Diphosphate

β-subunit Thiamine Diphosphate H156 T284

β-subunit Thiamine Diphosphate H156 T284

Dihydrolipoamide Branched-Chain Transacylase (DBT) Gene codes for the E2 subunit (Dihydrolipoamide Branched-Chain Transacylase) of BCKD. Catalyzes the transfer of an acyl-group from S- acyldihydrolipoamide to CoA to form an Acyl- CoA. Forms a homo-24-meric complex 9.

E2 Monomer (2IHW)

E2 Trimer (2IHW)

E2 Complex (2IHW)

DBT Mutation Majority of DBT Mutations affect the sequence of the produced E2 subunit. These mutations include frameshift mutations, deletions, and insertions 9.

DBT Mutation- Single Guanine Base Deletion A single Guanine base is deleted from an intron-exon junction resulting in the inability of splicing machinery to recognize the exon. Ultimately, a 78 base pair exon is excluded from the mature mRNA transcript, resulting in a nonfunctional BCKD Complex 12.

E2 Trimer Deleted Region

E2 Trimer Deleted Region

E2 Trimer No Interactions with Amino Terminal Tail

Dihydrolipoamide Dehydrogenase (DLD) Gene codes for an enzyme that degrades lipoamide and produces dihydrolipoamide. In the BCKD Complex, DLD processes one of the by-products of Acyl-CoA formation from branched chain amino acids 10. Two E3 monomers dimerize to form the dimeric catalytic subunit E3 11.

E3 Monomer

E3 Catalytic Subunit

DLD Mutation Mutations in the E3 subunit that cause MSUD affect the enzyme’s ability to catalyze lipoamide degradation by altering the active site, cofactor binding, or dimer association 10.

DLD Mutation- K37E and P453L Mutations The two mutations resulted in the altered active site of the E3 subunit and inhibited the enzyme’s ability to bind FAD, an essential cofactor for the enzyme’s function 10.

K37 P453

K37 P453 FAD

K37 P453 FAD

Social Impact Maple Syrup Disease is most common in populations with limited gene pools. If not properly treated, victims show low IQ’s and mental disabilities. Treatment involves dietary restrictions, which, under the proper administration, allow MSUD patients to live full and productive lives 2.

Works Cited 1.Podebrad F, Heil M, Reichert S, Mosandl A, Sewell AC, Böhles H (April 1999). "4,5-dimethyl-3-hydroxy-25H- furanone (sotolone)--the odour of maple syrup urine disease". Journal of inherited metabolic disease 22(2): 107– Kugler, Mary. "Maple Syrup Urine Disease Metabolic Disorder." About.com Health's Disease and Condition : n. pag. Web. 25 Mar King, Micheal. "Introduction to Amino Acid Metabolism." The Medical Biochemistry Page. Indiana University School of Medicine, 24 Mar Web. 29 Mar "Diseases of Amino Acid Metabolism." Inherited and Neurodegenerative Diseases. American Society for Neurochemistry, Web. 29 Mar Ævarsson, Arnthor. "Crystal structure of human branched-chain α-ketoacid dehydrogenase and the molecular basis of multienzyme complex deficiency in maple syrup urine disease." Structure 8.3 (2000): Web. 30 Mar "MAPLE SYRUP URINE DISEASE." Online Mendelian Inheritance in Man. Online Mendelian Inheritance in Man, 02 Nov Web. 30 Mar "BRANCHED-CHAIN KETO ACID DEHYDROGENASE E1, ALPHA POLYPEPTIDE; BCKDHA." Online Mendelian Inheritance in Man. Online Mendelian Inheritance in Man, 06 Nov Web. 30 Mar

Works Cited (cont.) 8."BRANCHED-CHAIN KETO ACID DEHYDROGENASE E1, BETA POLYPEPTIDE; BCKDHB." Online Mendelian Inheritance in Man. Online Mendelian Inheritance in Man, 28 Jun Web. 30 Mar "DIHYDROLIPOAMIDE BRANCHED-CHAIN TRANSACYLASE; DBT." Online Mendelian Inheritance in Man. Online Mendelian Inheritance in Man, 17 Sep Web. 30 Mar "DIHYDROLIPOAMIDE DEHYDROGENASE; DLD." Online Mendelian Inheritance in Man. Online Mendelian Inheritance in Man, 13 Jun Web. 30 Mar Brautigam, Chad. "Crystal Structure of Human Dihydrolipoamide Dehydrogenase: NAD+/NADH Binding and the Structural Basis of Disease-causing Mutations." Journal of Molecular Biology (2005): Web. 30 Mar Mitsubuchi, H. "Maple syrup urine disease caused by a partial deletion in the inner E2 core domain of the branched chain alpha-keto acid dehydrogenase complex due to aberrant splicing. A single base deletion at a 5'- splice donor site of an intron of the E2 gene disrupts the consensus sequence in this region.." Journal of Clinical Investigation 87.4 (1991): Web. 2 Apr Fisher, Carolyn. "Maple Syrup Urine Disease in Mennonites Evidence that the Y393N Mutation in Ela Impedes Assembly of the El Component of Branched-chain a-Keto Acid Dehydrogenase Complex." Journal of Clinical Investigation 88.3 (1991): Web. 6 Apr Chuang, Jacinta. "Structural and Biochemical Basis for Novel Mutations in Homozygous Israeli Maple Syrup Urine Disease Patients A PROPOSED MECHANISM FOR THE THIAMIN-RESPONSIVE PHENOTYPE." THE JOURNAL OF BIOLOGICAL CHEMISTRY (2004): Web. 6 Apr