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Enhancement of coenzyme binding by a single point mutation at the coenzyme binding domain of E. coli lactaldehyde dehydrogenase

Departamento de Bioquímica, Instituto Nacional de Cardiología "Ignacio Chávez," Tlalpan D.F. 14080, Mexico

(RECEIVED October 3, 2007; FINAL REVISION November 20, 2007; ACCEPTED November 20, 2007)

Phenylacetaldehyde dehydrogenase (PAD) and lactaldehyde dehydrogenase (ALD) share some structural and kinetic properties. One difference is that PAD can use NAD⁺ and NADP⁺, whereas ALD only uses NAD⁺. An acidic residue has been involved in the exclusion of NADP⁺ from the active site in pyridine nucleotide-dependent dehydrogenases. However, other factors may participate in NADP⁺ exclusion. In the present work, analysis of the sequence of the region involved in coenzyme binding showed that residue F180 of ALD might participate in coenzyme specificity. Interestingly, F180T mutation rendered an enzyme (ALD-F180T) with the ability to use NADP⁺. This enzyme showed an activity of 0.87 µmol/(min * mg) and K_m for NADP⁺ of 78 µM. Furthermore, ALD-F180T exhibited a 16-fold increase in the V_m /K_m ratio with NAD⁺ as the coenzyme, from 12.8 to 211. This increase in catalytic efficiency was due to a diminution in K_m for NAD⁺ from 47 to 7 µM and a higher V_m from 0.51 to 1.48 µmol/(min * mg). In addition, an increased K_d for NADH from 175 (wild-type) to 460 µM (mutant) indicates a faster product release and possibly a change in the rate-limiting step. For wild-type ALD it is described that the rate-limiting step is shared between deacylation and coenzyme dissociation. In contrast, in the present report the rate-limiting step in ALD-F180T was determined to be exclusively deacylation. In conclusion, residue F180 participates in the exclusion of NADP⁺ from the coenzyme binding site and disturbs the binding of NAD⁺.

Keywords: ALDHs; aldehyde dehydrogenases; ALD; lactaldehyde dehydrogenase; catalytic efficiency; burst; coenzyme binding domain

Reprint requests to: José Salud Rodríguez-Zavala, Departamento de Bioquímica, Instituto Nacional de Cardiología, Tlalpan, México D.F. 14080, Mexico; e-mail: rodjos{at}cardiologia.org.mx; fax: 52 55 55 73 09 26.

Abbreviations: ALDHs, aldehyde dehydrogenases; ALD, lactaldehyde dehydrogenase; PAD, phenylacetaldehyde dehydrogenase; Vh-ALDH, Vibrio harveyi aldehyde dehydrogenase; ALDH1, class 1 aldehyde dehydrogenase; ALDH3, class 3 aldehyde dehydrogenase.

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.073277108.

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