Targeted Mutation of a Non-catalytic Gating Residue Increases the Rate of Pseudomonas aeruginosa d-Arginine Dehydrogenase Catalytic Turnover

Abstract

Commercial food and l-amino acid industries rely on bioengineered d-amino acid oxidizing enzymes to detect and remove d-amino acid contaminants. However, the bioengineering of enzymes to generate faster biological catalysts has proven difficult as a result of the failure to target specific kinetic steps that limit enzyme turnover, k cat, and the poor understanding of loop dynamics critical for catalysis. Pseudomonas aeruginosa d-arginine dehydrogenase (PaDADH) oxidizes most d-amino acids and is a good candidate for application in the l-amino acid and food industries. The side chain of the loop L2 E246 residue located at the entrance of the PaDADH active site pocket potentially favors the closed active site conformation and secures the substrate upon binding. This study used site-directed mutagenesis, steady-state, and rapid reaction kinetics to generate the glutamine, glycine, and leucine variants and investigate whether increasing the rate of product release could translate to an increased enzyme turnover rate. Upon E246 mutation to glycine, there was an increased rate of d-arginine turnover k cat from 122 to 500 s–1. Likewise, the k cat values increased 2-fold for the glutamine or leucine variants. Thus, we have engineered a faster biocatalyst for industrial applications by selectively increasing the rate of the PaDADH product release.

An active site mutation induces oxygen reactivity in D-arginine dehydrogenase: A case of superoxide diverting protons

“…By comparison, this feature is not observed with the Pa DADH wildtype enzyme under both aerobic and anaerobic conditions ( 42 , 44 , 66 , 67 , 69 , 76 , 77 ). In the same way, the flavin semiquinone species was not observed with the E 246 L variant enzyme under anaerobic conditions, although similar kinetic parameters as reported for the aerobic flavin reduction in this study were observed: k red = ∼50 s −1 and K d = 12 mM ( Table 1 ) ( 70 ). The data are consistent with the Pa DADH E 246 L variant reacting with O 2 to yield the flavin semiquinone after flavin reduction and product release, as evidenced by the observed transient increase in the λ 466 nm absorbance of the reduced flavin at 8 s ( Fig.…”

“…The steady-state kinetic studies of the Pa DADH E246L variant enzyme reported in a previous study yielded a k cat value of 265 ± 5 s −1 , a k cat / K m value of 871,000 ± 35,000 M −1 s −1 , and a K m value of 0.30 ± 0.02 mM with D-arginine ( 70 ). Hence, to determine the effect of the enzyme-generated O 2 • ˉ on Pa DADH E 246 L turnover with D-arginine, assays of the variant enzyme with O 2 as electron acceptor and 0.5 mM D-arginine as substrate were carried out under steady-state conditions.…”

“…Assays were performed in 20 mM NaPP i , pH 10.0, at 25 o C. The kinetic parameters’ values were obtained after fitting the kinetic data with Equation 2 . b Previously reported data for the Pa DADH E 246 L variant enzyme under anaerobic conditions ( 70 ). …”

“… b Previously reported data for the Pa DADH E 246 L variant enzyme under anaerobic conditions ( 70 ). …”

“…2 ) ( 67 , 69 ). Residue E 246 of loop L2 is located 7.6 Å from the flavin N5 atom, 8.2 Å from the flavin C(4a) atom, and does not participate in flavin reduction ( 70 ).

Figure 1 General reaction scheme of Pa DADH.

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