Abstract
Mandelate Racemase (MR) from Pseudomonas putida catalyzes the Mg2+-dependent 1,1-proton transfer that interconverts the enantiomers of mandelate. MR relies on an “asymmetric” two-base mechanism (Lys 166 and His 297) to catalyze the reaction. Specifically, His 297 facilitates the reaction that changes R-(D)-mandelate into S-(L)-mandelate and Lys 166 facilitates the reaction that changes S-mandelate into R-mandelate. This reaction requires the abstraction of a proton from carbon and is particularly difficult because the pKa of this substrate hydrogen is ~29. Mandelate Racemase was intensively studied. The previous results including structural studies of S-mandelate/atrolactate bound with WT or MR mutants. However, there is no structural information on how R-mandelate/atrolactate is bound in the active site. To address this question,
we designed a double mutant H297G/D270K MR and a single mutant L93A MR. Our H297G/D270K MR mutant successfully traps an R-mandelate or an R-atrolactate in the active site and by overlaying with previous reported S-mandelate/atrolactate bound MR structures, we
reveal how phenyl-ring of the substrates could move during catalysis. Even though the L93A MR mutant fails to trap the desired substrates/ inhibitors, the enzymatic and structural study of the L93A MR mutant reveals the importance of L93 residue to be a part of the hydrophobic
pocket which stabilizing the positions of the phenyl-ring of ligands. Additionally, the only moveable region of MR, the 20s loop, will also be discussed. There are two positions of the 20s loop observed in our structure, positioned “in” or “out”. We propose that 20s loop could a speed
control of MR. Taken together, these results will help to fill some of the blanks for the Mandelate Racemase reaction and further increase our knowledge on how MR achieves its catalytic mission.