METABOLIC ENGINEERING OF ATP SULFURYLASE AND APS REDUCTASE IN THE PLANT SULFUR ASSIMILATION PATHWAY

Samuel McKinney, Geoffrey Ravilious¹, Dr. Hankuil Yi², Corey Westfall¹, Dr. Joseph Jez,^{1  1}Washington University in St. Louis, ²Danforth Plant Science Center

Soy protein is the major protein source for cattle, but millions of dollars are spent every year adding cysteine and methionine to cattle feed because soy is deficient in these sulfur-containing amino acid.  Sulfur is assimilated in plants through the uptake and subsequent activation of sulfate to adenosine 5’ phosphosulfate (APS) by ATP Sulfurylase.  APS can be routed towards becoming a lipid or be reduced by APS Reductase and incorporated into cysteine.  We created a fused construct of ATP Sulfurylase and APS Reductase in order to determine whether fusion of sulfurylase and reductase into a single protein would result in higher enzymatic activity than sulfurylase and reductase alone, and thus a higher production of cysteine in soy.  We also performed Steady-State Kinetics assays to determine the rate of catalysis for sulfurylase and the inhibitory effect of nitrate, a byproduct of cysteine synthesis.  Nitrate significantly inhibited ATP Sulfurylase at a concentration of 3 millimolar such that cysteine production cannot be increase by simple over-expression of sulfurylase.  Future experimentation will involve determining the rate of catalysis for the sulfurylase-reductase hybrid, and removing the nitrate inhibition binding site for the construct.