Crystal structure of an oxidized mutant of human mitochondrial branched-chain aminotransferase

Abstract

This study presents the crystal structure of a thiol variant of the human mitochondrial branched-chain aminotransferase protein. Human branched-chain aminotransferase (hBCAT) catalyzes the transamination of the branched-chain amino acids leucine, valine and isoleucine and α-ketoglutarate to their respective α-keto acids and glutamate. hBCAT activity is regulated by a CXXC center located approximately 10 Å from the active site. This redox-active center facilitates recycling between the reduced and oxidized states, representing hBCAT in its active and inactive forms, respectively. Site-directed mutagenesis of the redox sensor (Cys315) results in a significant loss of activity, with no loss of activity reported on the mutation of the resolving cysteine (Cys318), which allows the reversible formation of a disulfide bond between Cys315 and Cys318. The crystal structure of the oxidized form of the C318A variant was used to better understand the contributions of the individual cysteines and their oxidation states. The structure reveals the modified CXXC center in a conformation similar to that in the oxidized wild type, supporting the notion that its regulatory mechanism depends on switching the Cys315 side chain between active and inactive conformations. Moreover, the structure reveals conformational differences in the N-terminal and inter-domain region that may correlate with the inactivated state of the CXXC center.

Keywords: CXXC center; N-terminal loop; human mitochondrial branched-chain aminotransferase; interdomain loop; redox regulation; transaminases.

Figure 1

The overall structure of the C318A/C315CSD mutant of hBCATm (cartoon; chain A, blue; chain B, light blue) resembles that of wild-type hBCATm. The structure is a homodimer with the PLP linked to Lys202 by a Schiff base. The active-site residues and the CXXC center are shown in stick representation. The phosphate of PLP is shown in orange. (Atom color scheme: carbon, gray; nitrogen, blue; oxygen, red; phosphate, orange; sulfur, yellow.)

Figure 2

(a, b) Close-up of an inter-domain loop overlay of the crystal structures of (a) the C318A (gray; PDB entry 2hgw; Yennawar et al., 2006 ▸) and C318A/C315CSD (blue) mutants and (b) the oxidized wild type (orange/yellow; PDB entry 2hhf; Yennawar et al., 2006 ▸) and the C318A/C315CSD mutant (blue). The interdomain loop (residues 171–181, highlighted by the circle), which was missing in the C318A mutant owing to a lack of electron density, was interpreted in one of the monomers of the C318A/C315CSD mutant (a) and mimics the interdomain loop of oxidized wild-type hBCATm (b). (c, d) Close-up of an N-terminal loop overlay of the crystal structures of (c) the C318A (gray; PDB entry 2hgw) and C318A/C315CSD (blue) mutants and (d) the oxidized wild type (orange/yellow; PDB entry 2hhf) and the C318A/C315CSD mutant (blue). The N-terminal loop (residues 15–32, highlighted by the square) of the C318A mutant is interpreted in both monomers, whereas it is much more flexible and remains uninterpreted in both monomers of the C318A/C315CSD mutant (c) and one monomer of the oxidized wild type (d).

Figure 3

Close-up of the overlay of the CXXC center of the crystal structures of oxidized wild-type hBCATm (yellow ribbon with Cys315 and Cys318 in light gray) and the C318A/C315CSD mutant (blue ribbon with C315CSD and C318A in dark gray). The orientation of C315CSD resembles that of Cys315 in the disulfide-bonded form. (Atom color scheme: carbon, gray and light gray; oxygen, red; sulfur, yellow.)