Catalytic properties of a bacterial acylating acetaldehyde dehydrogenase: evidence for several active oligomeric states and coenzyme A activation upon binding.

Until the last decade, two unrelated aldehyde dehydrogenase (ALDH) superfamilies, i.e. the phosphorylating and non-phosphorylating superfamilies, were known to catalyze the oxidation of aldehydes to activated or non-activated acids. However, a third one was discovered by the crystal structure of a bifunctional enzyme 4-hydroxy-2-ketovalerate aldolase/acylating acetaldehyde dehydrogenase (DmpFG) from Pseudomonas sp. strain CF600 (Manjasetty et al., Proc. Natl. Acad. Sci. USA 100 (2003) 6992-6997). Indeed, DmpF exhibits a non-phosphorylating CoA-dependent ALDH activity, but is structurally related to the phosphorylating superfamily. In this study, we undertook the characterization of the catalytic and structural properties of MhpEF from Escherichia coli, an ortholog of DmpFG in which MhpF converts acetaldehyde, produced by the cleavage of 4-hydroxy-2-ketovalerate by MhpE, into acetyl-CoA. The kinetic data obtained under steady-state and pre-steady-state conditions show that the aldehyde dehydrogenase, MhpF, is active as a monomer, a unique feature relative to the phosphorylating and non-phosphorylating ALDH superfamilies. Our results also reveal that the catalytic properties of MhpF are not dependent on its oligomeric state, supporting the hypothesis of a structurally and catalytically independent entity. Moreover, the transthioesterification is shown to be rate-limiting and, when compared with a chemical model, its catalytic efficiency is increased 10(4)-fold. Therefore, CoA binding to MhpF increases its reactivity and optimizes its positioning relative to the thioacylenzyme intermediate, thus enabling the formation of an efficient deacylation complex.