Modification of substrate specificity in single point mutants of Agrobacterium tumefaciens type II NADH dehydrogenase.
Type II NADH dehydrogenases (NDH-2) are monomeric flavoenzymes catalyzing electron transfer from NADH to quinones. While most NDH-2 preferentially oxidize NADH, some of these enzymes have been reported to efficiently oxidize NADPH. With the aim to modify the NADPH vs NADH specificity of the relatively NADH specific Agrobacterium tumefaciens NDH-2, two conserved residues (E and A) of the substrate binding domain were, respectively, mutated to Q and S. We show that when E was replaced by Q at position 203 the enzyme was able to oxidize NADPH as efficiently as NADH. Growth on a minimal medium of an Escherichia coli double mutant lacking both NDH-1 and NDH-2 was restored more efficiently when mutated proteins able to oxidize NADPH were expressed. The biotechnological interest of expressing such modified enzymes in photosynthetic organisms is discussed.
CEA, Direction des Sciences du Vivant, Institut de Biologie Environnementale et de Biotechnologie, Laboratoire de Bioénergétique et Biotechnologie des Bactéries et Microalgues, CEA Cadarache, Saint-Paul-lez-Durance F-13108, France.
C
C
Desplats
A
A
Beyly
S
S
Cuiné
L
L
Bernard
L
L
Cournac
G
G
Peltier
D016960:Agrobacterium tumefaciens / Q000201:enzymology* / Q000235:genetics; D019943:Amino Acid Substitution; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D015870:Gene Expression; D020125:Mutation, Missense; D009243:NAD / Q000235:genetics / Q000378:metabolism; D009245:NADH Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009249:NADP / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D010788:Photosynthesis / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000235:genetics; D013379:Substrate Specificity / Q000235:genetics
D009243:NAD; D009249:NADP; D009245:NADH Dehydrogenase