A structural mapping of mutations causing succinyl-CoA:3-ketoacid CoA transferase (SCOT) deficiency

Succinyl-CoA:3-ketoacid CoA transferase (SCOT) deficiency is a rare inherited metabolic disorder of ketone metabolism, characterized by ketoacidotic episodes and often permanent ketosis. To date there are ∼20 disease-associated alleles on the OXCT1 gene that encodes the mitochondrial enzyme SCOT. SCOT catalyzes the first, rate-limiting step of ketone body utilization in peripheral tissues, by transferring a CoA moiety from succinyl-CoA to form acetoacetyl-CoA, for entry into the tricarboxylic acid cycle for energy production. We have determined the crystal structure of human SCOT, providing a molecular understanding of the reported mutations based on their potential structural effects. An interactive version of this manuscript (which may contain additional mutations appended after acceptance of this manuscript) may be found on the web address: http://www.thesgc.org/jimd/SCOT. Electronic supplementary material The online version of this article (doi:10.1007/s10545-013-9589-z) contains supplementary material, which is available to authorized users.


Introduction
Ketone bodies (acetoacetate, 3-hydroxybutyrate, acetone), predominantly produced in the liver, provide extrahepatic organs such as heart and brain with energy when glucose supply is limited (Sass 2012). Any enzyme malfunction in ketone body utilization (ketolysis) could lead to a buildup of unused ketone bodies and result in ketoacidosis. Succinyl-CoA:3-ketoacid CoA transferase (SCOT; gene name OXCT1; EC 2.8.3.5) catalyzes the first and rate-determining Communicated by: Johannes Zschocke Database reference OMIM, 245050; GenBank, 4557817; URL, http://www.thesgc.org/SCOT; PDB code, 3DLX Electronic supplementary material The online version of this article (doi:10.1007/s10545-013-9589-z) contains supplementary material, which is available to authorized users. step of ketolysis (Fukao et al 2000;Kassovska-Bratinova et al 1996). SCOT is a member of the CoA transferase family I that activates acetoacetate by transferring a CoA moiety from succinyl-CoA to form acetoacetyl-CoA. Acetoacetyl-CoA is further metabolized by acetoacetyl-CoA thiolase into two acetyl-CoA molecules which enter the citric acid cycle for energy production (Williamson et al 1971). SCOT is a mitochondrial enzyme expressed in all extrahepatic tissues, but abundant in the heart, brain and kidney (Fukao et al 1997). There exists also a testis-specific isoform SCOT-t (gene name OXCT2), sharing 74 % amino acid identity with SCOT (Tanaka et al 2002).
Mutations in the human OXCT1 gene on chromosome location 5p13 result in the rare autosomal recessive deficiency of SCOT (OMIM 245050) . SCOT-deficient patients, often with neonatal onset, present with recurrent ketoacidosis episodes that could be life-threatening, but with no symptoms between episodes (Niezen-Koning et al 1997). The number of reported cases is few, and symptoms, which may vary among individuals, include vomiting, lethargy and tachypnea, as well as unconsciousness caused by severe ketoacidosis (Berry et al 2001;Sakazaki et al 1995;Snyderman et al 1998). Permanent ketosis or ketonuria is a characteristic symptom but may be absent in patients retaining residual SCOT activity (Fukao et al 2010;Fukao et al 2011;Fukao et al 2004). Approximately 30 affected probands were reported to date and 24 mutations identified (Table 1). Here we report the crystal structure of human SCOT, and present an interactive mapping of missense mutations to understand the molecular basis of SCOT deficiency.
Purified protein was concentrated to 21 mg/ml and stored in 10 mM HEPES pH7.5, 500 mM NaCl, 5 % (w/v) glycerol and 0.5 mM TCEP at −80°C. Crystals were grown by vapour diffusion at 20°C, in sitting drops mixing 100 nl protein pre-incubated with 2 mM acetyl-CoA and 300 nl reservoir solution containing 0.20 M sodium chloride, 0.1 M Tris pH9.0 and 25 % (w/v) polyethylene glycol 3,350. Crystals were cryo-protected in mother liquor containing 20 % (w/v) glycerol and flash-frozen in liquid nitrogen.  (Bateman et al 2002). Initial automated model building was performed with ARP/wARP (Perrakis et al 2001), followed by cycles of iterative manual model building with COOT (Emsley and Cowtan 2004) and REFMAC5 refinement (Murshudov et al 1997). The refined model consists of protein residues 40-285 and 297-519. No electron density was observed for part of the inter-domain linker (residues 286-296). No ligand density for acetyl-CoA was found in the active site though it was added during crystallization. Structure factors and coordinates were deposited in the Protein Data Bank under the accession code 3DLX.

Results and discussion
We have determined the crystal structure of human SCOT which exhibits a homodimer architecture containing two active sites (Fig. 1). Each monomer consists of the amino-terminal (N-; aa 40-272) and carboxy-terminal (C-; aa 298-510) domains, connected by a linker region (aa 273-297). The Nand C-domains share a common α/β structural fold for CoA transferase family I members (Heider 2001), as previously seen in the pig SCOT structure (89 % sequence identity) (Bateman et al 2002). The active site of each monomer is situated at the interface of the two domains, where a strictly conserved residue Glu344 (Fig. 1, orange sticks) attacks the incoming succinyl-CoA substrate and forms an enzyme-CoA thioester intermediate, as an integral part of the catalytic mechanism (Solomon and Jencks 1969). Residues in the active site of human SCOT are also conserved in the testis-specific isoform SCOT-t, suggesting it may have enzymatic activity. To date, three nonsense, two insertion and 19 missense mutations leading to SCOT deficiency are known from literature or newly reported here (Table 1 and Supplementary text), although six missense mutations have not been confirmed as pathogenic mutations by expression analysis. There is a polymorphism c.173C>T (T58M) which retains full enzyme activity (Song et al 1998). The three nonsense mutations (R217X, E273X, S283X) are expected to cause premature translation termination, resulting in truncated SCOT proteins that lack completely the C-domain and hence abolish part of the active site. Another mutation c.1561T>C at the termination codon results in X521R and adds 20 amino acids in the C terminus of SCOT peptide (Alkén 2008). The missense mutations are broadly distributed between the N-and C-domains of the protein, although two clusters of 'mutational hotspots' can be observed (Fig. 2). One cluster is close to the interface between two SCOT subunits in a dimer. A duplication mutation (N220-I222dup) is also present in this region (Fukao et al 2010). The other cluster is located in secondary structure elements that make up the active site and CoA-binding site of the enzyme.
The mapping of missense mutations onto the human SCOT structure allows us to classify their potential structural consequences broadly into three types. In the first type, amino acids tightly packed in the enzyme are substituted to bulkier and/or charged residues ( Fig. 2 and Table 1, #2-7,11,14,16,17). This likely results in severe steric and electrostatic clashes in the local environment which in turn can compromise severely the folding, architecture and stability of the enzyme. These mutants often result in the more severe phenotype (permanent acidosis), consistent with their much diminished enzyme activity (Fukao et al 2000;Song et al 1998). The second type disrupts the integrity of a secondary structure element, either by introducing a conformationallyrestrained residue (e.g. Pro, Gly) into an α-helix/β-strand ( Fig. 2 and Table 1, #12,15), or by removing such residues from their critical involvement in a loop/turn segment ( Fig. 2 and Table 1, #8). The third type involves the substitution of arginine residues where their guanidinium side-chains are involved in salt bridge formation. These charged interactions contributed to stabilizing two neighbouring regions in 3D space that are distant apart in the polypeptide sequence. Substitution of arginine to a weakly positive-charged ( Fig. 2 and Table 1, #10) or uncharged amino acid ( Fig. 2 and Table 1, #9,19) will abolish these salt bridges. Though retaining partial enzyme activities, these mutant proteins are thermally less stable compared to wild-type Fukao et al 2011).