Thiamine-dependent actinobacterial 2-hydroxyacyl-CoA lyase (AcHACL) catalyzes the reversible cleavage of 2-hydroxyacyl-CoAs to formyl-CoA and carbonyl compounds. To exploit the enzyme's biotechnological potential, a deeper understanding of the catalysis is required. Previously, AcHACL E493 was identified as an important acid/base catalyst. Here, wild-type and E493 mutant crystal structures representing Michaelis complexes with 2-hydroxyisobutyryl-CoA and (S)-2-methylglyceryl-CoA are provided. Although E493 guarantees high rates of essential proton transfers in AcAHCL-catalyzed on-pathway cleavage of 2-hydroxyacyl-CoAs and off-pathway carboligations with short-chain aldehydes and ketones, wild-type substrate accommodation is suboptimal. Not E493D, but E493A and E493S mutations improved KM. However, kcat is substantially reduced in the mutants. These tradeoffs are discussed by comparing active sites of AcHACL and related enzymes either lacking or possessing an E493 homolog.
Zahn et al. (Wed,) studied this question.