Substitutions at the cofactor phosphate-binding site of a clostridial alcohol dehydrogenase lead to unexpected changes in substrate specificity.
Protein Eng Des Sel
; 28(8): 251-8, 2015 Aug.
Article
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| MEDLINE
| ID: mdl-26034298
ABSTRACT
Changing the cofactor specificity of an enzyme from nicotinamide adenine dinucleotide 2'-phosphate (NADPH) to the more abundant NADH is a common strategy for increasing overall enzyme efficiency in microbial metabolic engineering. The aim of this study was to switch the cofactor specificity of the primary-secondary alcohol dehydrogenase from Clostridium autoethanogenum, a bacterium with considerable promise for the bio-manufacturing of fuels and other petrochemicals, from strictly NADPH-dependent to NADH-dependent. We used insights from a homology model to build a site-saturation library focussed on residue S199, the position deemed most likely to disrupt binding of the 2'-phosphate of NADPH. Although the CaADH(S199X) library did not yield any NADH-dependent enzymes, it did reveal that substitutions at the cofactor phosphate-binding site can cause unanticipated changes in the substrate specificity of the enzyme. Using consensus-guided site-directed mutagenesis, we were able to create an enzyme that was stringently NADH-dependent, albeit with a concomitant reduction in activity. This study highlights the role that distal residues play in substrate specificity and the complexity of enzyme-cofactor interactions.
Palabras clave
Texto completo:
1
Bases de datos:
MEDLINE
Asunto principal:
Proteínas Bacterianas
/
Alcohol Deshidrogenasa
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Clostridium
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Sustitución de Aminoácidos
Idioma:
En
Revista:
Protein Eng Des Sel
Asunto de la revista:
BIOQUIMICA
/
BIOTECNOLOGIA
Año:
2015
Tipo del documento:
Article
País de afiliación:
Nueva Zelanda