Impaired Assembly of E1 Decarboxylase of the Branched-chain α-Ketoacid Dehydrogenase Complex in Type IA Maple Syrup Urine Disease

Abstract

The E1 decarboxylase component of the human branched-chain ketoacid dehydrogenase complex comprises two E1␣ (45.5 kDa) and two E1␤ (37.5 kDa) subunits forming an ␣ 2 ␤ 2 tetramer. In patients with type IA maple syrup urine disease, the E1␣ subunit is affected, resulting in the loss of E1 and branched-chain ketoacid dehydrogenase catalytic activities. To study the effect of human E1␣ missense mutations on E1 subunit assembly, we have developed a pulse-chase labeling protocol based on efficient expression and assembly of human (His) 6 -E1␣ and untagged E1␤ subunits in Escherichia coli in the presence of overexpressed chaperonins GroEL and GroES. Assembly of the two 35 S-labeled E1 subunits was indicated by their co-extraction with Ni 2؉ -nitrilotriacetic acid resin. The nine E1␣ maple syrup urine disease mutants studied showed aberrant kinetics of assembly with normal E1␤ in the 2-h chase compared with the wild type and can be classified into four categories of normal (N222S-␣ and R220W-␣), moderately slow (G245R-␣), slow (G204S-␣, A240P-␣, F364C-␣, Y368C-␣, and Y393N-␣), and no (T265R-␣) assembly. Prolonged induction in E. coli grown in the YTGK medium or lowering of induction temperature from 37 to 28°C (in the case of T265R-␣), however, resulted in the production of mutant E1 proteins. Separation of purified E1 proteins by sucrose density gradient centrifugation showed that the wild-type E1 existed entirely as ␣ 2 ␤ 2 tetramers. In contrast, a subset of E1␣ missense mutations caused the occurrence of exclusive ␣␤ dimers (Y393N-␣ and F364C-␣) or of both ␣ 2 ␤ 2 tetramers and lower molecular weight species (Y368C-␣ and T265R-␣). Thermal denaturation at 50°C indicated that mutant E1 proteins aggregated more rapidly than wild type (rate constant, 0.19 min ؊1 ), with the T265R-␣ mutant E1 most severely affected (rate constant, 4.45 min ؊1 ). The results establish that the human E1␣ mutations in the putative thiamine pyrophosphate-binding pocket that are studied, with the exception of G204S-␣, have no effect on E1 subunit assembly. The T265R-␣ mutation adversely impacts both E1␣ folding and subunit interactions. The mutations involving the C-terminal aromatic residues impede both the kinetics of subunit assembly and the formation of the native ␣ 2 ␤ 2 structure.The mammalian mitochondrial branched-chain ␣-ketoacid dehydrogenase (BCKD) 1 complex catalyzes the oxidative decarboxylation of the branched-chain ␣-ketoacids derived from the branched-chain amino acids, leucine, isoleucine, and valine (1). This multienzyme complex is organized around a dihydrolipoyl transacylase (E2) core, to which a branched-chained ␣-ketoacid decarboxylase (E1), a dihydrolipoamide dehydrogenase (E3), a specific kinase, and a specific phosphatase are attached through ionic interactions (2, 3). E1 is a thiamine pyrophosphate (TPP)-dependent enzyme comprising two E1␣ and two E1␤ subunits that assemble into an ␣ 2 ␤ 2 tetramer. E2 is a 24-meric protein consisting of identical lipoic acid-bearing subunits arranged on octahedral 4,3,2-point group...

Structure of the photolyase-like domain of cryptochrome 1 from Arabidopsis thaliana

“…5 1-509). Escherichia coli cells containing this plasmid were grown in YTGK medium (10 g/liter Bacto-tryptone/16 g/liter yeast extract/10 ml/liter glycerol/5 g/liter NcCl/0.75 g/liter KCl) (20) at 30°C to an OD 600 of 0.6. The temperature of the culture was then lowered to 25°C, and the expression of CRY1-PHR was induced by the addition of 0.1 mM isopropyl ␤-D-thiogalactoside to the medium.…”

GroEL/GroES-dependent Reconstitution of α2β2 Tetramers of Human Mitochondrial Branched Chain α-Ketoacid Decarboxylase

“…The pTrcHisB expression vector was obtained from Invitrogen (Carlsbad, CA). The pHisT-hE1 plasmid for co-expression of His 6 -␣ and untagged ␤ subunits of human E1 was described previously (28). The His 6 -␣ subunit contained a His 6 tag and a tobacco etch virus protease cleavable linker fused to the N terminus of the ␣ subunit.…”

“…The expression of GroEL and GroES was induced by IPTG overnight at 37°C. Cell lysates were prepared by sonication in a lysis buffer containing 1 mM phenylmethylsulfonyl fluoride and 1 mM benzamidine as described previously (28). GroEL was purified from the lysates according to the method described by Clark et al (29) that included a critical Reactive Red column step with the following modifications.…”

“…Expression of His 6 -␣ and untagged ␤ subunits and GroEL/GroES was induced by IPTG. Assembled human His 6 -E1 was isolated from cell lysates and purified by Ni-NTA (Qiagen, Chatsworth, CA) column chromatography as described previously (28). To remove the His 6 tag, the purified fusion human E1 was digested with the tobacco etch virus protease at 4°C overnight.…”

The Two Active Sites in Human Branched-chain α-Keto Acid Dehydrogenase Operate Independently without an Obligatory Alternating-site Mechanism

“…Wild-type E1b shows two distinct melting temperatures (T m ) at 52.6 and 65.4°C, which likely represent transitions from heterotetramers to heterodimers and from heterodimers to the random-coil structure, respectively (32). The E46Q␤ E1b mutant shows two T m values at 53.3 and 64.0°C, similar to the wild-type E1b.…”