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.
Highlights
Human branched-chain aminotransferase is an enzyme that catalyzes the transamination of the branchedchain amino acids leucine, valine and isoleucine and -ketoglutarate to their respective -keto acids and glutamate (Hall et al, 1993; Hutson, 1988)
The Human branched-chain aminotransferase (hBCAT) proteins are PLP-dependent enzymes and their reaction is accompanied by the interconversion of the cofactor pyridoxal 50-phosphate (PLP) and pyridoxamine 50-phosphate (PMP) (Birolo et al, 1995; Yano et al, 1992; Jager et al, 1994; Smith et al, 1989)
Oxidation of hBCATm regulates its ability to form a ‘metabolon’ complex with the E1 subunit of the branched-chain -keto acid dehydrogenase complex (BCKDC) (Islam et al, 2007) and glutamate dehydrogenase (GDH) (Islam et al, 2010), enzymes that are important for the complete oxidation of the branched-chain amino acids
Summary
Human branched-chain aminotransferase (hBCAT) is an enzyme that catalyzes the transamination of the branchedchain amino acids leucine, valine and isoleucine and -ketoglutarate to their respective -keto acids and glutamate (Hall et al, 1993; Hutson, 1988). Structural and kinetic studies have shown that Cys315 is the ‘redox sensor’, while Cys318 allows reversible disulfide-bond formation (Conway et al, 2004) The function of this redox switch has in part been characterized for hBCATm. For instance, oxidation of hBCATm regulates its ability to form a ‘metabolon’ complex with the E1 subunit of the BCKDC (Islam et al, 2007) and glutamate dehydrogenase (GDH) (Islam et al, 2010), enzymes that are important for the complete oxidation of the branched-chain amino acids. With the goal of finding structural differences that contribute to the observed differences in activity, we compared the structure of the C318A/ C315CSD mutant with that of the oxidized form of wild-type hBCATm, which has an abolished activity, and with that of the reduced form of the C318A mutant of hBCATm, which has an almost unchanged activity
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