Identification of functional paralog shift mutations: conversion of Escherichia coli malate dehydrogenase to a lactate dehydrogenase.
Five positions in the Escherichia coli malate dehydrogenase (eMDH) sequence, which distinguish MDH from lactate dehydrogenase (LDH) activity, were identified through a combination of Venn diagrams constructed from whole genomic data and from unbiased representative sequences from terminal clades. Incorporation of the five changes in eMDH sufficed to convert the enzyme from one with (k(cat)/K(m)(pyruvate))/(k(cat)/K(m)(oxaloacetate)) = 6.1 x 10(-9) to one with that ratio = 28. The substrate specificity was thus changed by a factor of 4.6 x 10(9). The k(cat)/K(m)(pyruvate) value for the pentamutant (eMDH I12V/R81Q/M85E/G210A/V214I) is 3,500 M(-1).s(-1), which is approximately equal 1/1,000 of the values found for typical wild-type LDHs. The procedure isolates an intersection of "strong forcing sets" that should prove to be of general use in switching paralog function.
Yin
Yifeng
Y
QB3 Institute, 572 Stanley Hall, University of California, Berkeley, CA 94720-3220, USA.
Kirsch
Jack F
JF
D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000235:genetics* / Q000378:metabolism*; D008291:Malate Dehydrogenase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015394:Molecular Structure; D009154:Mutation / Q000235:genetics*; D009243:NAD / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D012680:Sensitivity and Specificity
D009243:NAD; D007770:L-Lactate Dehydrogenase; D008291:Malate Dehydrogenase