Artificial transaminases: Tuning the selectivity and rate of pyridoxamine catalyzed reactions in a protein cavity modified by site directed mutagenesis

Fipid binding proteins are members of a class of small proteins (131 residues) with a simple, architect itro th;it con si si s of two orthogonal planes of beta- sheet secondary structure and a large sequestered cavity. We are using thi<- system to prepare artificial enzymes and recently reported a conjugate that incorpu rates a pyridoxamine moiety that rednctively animates alpha keto acids to the corresponding arnino acids. Here we describe the use of three mutant pro teins that allow the position of pyndoxamine attachment to be moved within the cavity. The rnutanl protein 1FABP PX60 rediictively aminated alpha-keto glutarate to glutamate rapidly: the reaction proceeded to 96% conversion in 21 hours. Further kinetic analysis showed that kcat/Km for this mutant was approximately 200 fold higher than that of free pyridoxamine. As many HS 50 turnovers and an enant ioselect ivity of 96% ee have been observed with this niu tant. Reactions with IFABP-PX72 produced several I) amino acids. Alanitn1 was produced in 169% ee. valine was produced in 63% ee, and leucine was pro duced in 28% ee. Low selectivity was obtained for tyrosine and glutamu acid. These results are strikingly different than those obtained in our original system that produced mainly the F enantiomers. IFABP-PX104 produces alanine and tyrosine from the corresponding koto acid substrates but is unable to produce valine or glutamate from their respective keto acid precursors. A x-ray crystal structure of one of t he conjugates has been solved to 2.6 A resolution.