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  • 2019-09
  • An AmDH is engineered by mutating


    An AmDH is engineered by mutating two conserved amino checkpoint activity residues in the active site of an amino acid dehydrogenase (AADH). Starting from the leucine dehydrogenase (LeuDH) from Bacillus stearothermophilus, substituting a serine and leucine at the K68 and N261 positions, respectively, resulted in the leucine amine dehydrogenase (L-AmDH) [1]. More than a dozen different AmDHs have since been generated by applying homologous mutations to AADHs [9], [10], [11], [12], [13]. In the balanced reaction for L-AmDH in Fig. 1, 2-pentanone (Pent) is reductively aminated to form (R)-2-aminopentane (AmPent). The reaction for LeuDH, also shown in Fig. 1, is the reductive amination of ketoleucine (KLeu) to (S)-leucine (Leu). For both enzymes the NADH cofactor is oxidized to NAD+ through the hydride transfer step [14]. The present work lays the groundwork for reactor engineering, required for proper scaling of synthesis. An enzyme’s kinetic mechanism (the order and manner of substrate binding and product release) determines its kinetic rate law. The rate law defines the turnover rate as a function of substrate concentration and is required for the reactor design equations, which are used to determine proper reactor size and to predict yield. We have used the method of initial rates described by Cook and Cleland [15] to propose rate laws for both the reductive amination and oxidative deamination reactions for L-AmDH and LeuDH. As their primary sequence differs by only a few amino acid residues, the kinetic mechanism of the engineered L-AmDH was assumed to be the same as its parent. However, the results of the present study surprisingly show that the mechanisms and thus the rate laws of the two enzymes are different. Often, viscogens (i.e. small sugars and sugar alcohols) are included in large-scale reaction solutions for enzyme stability and can affect overall enzyme reaction kinetics by way of a kinetic solvent viscosity effect (KSVE) [16]. These KSVEs are often the result of either an enzyme conformational change or the rate-limiting diffusion of substrates or products to or from the enzyme. Therefore, the potential impact of these KSVEs on the industrial scale is examined through the addition of viscogens to small-scale reactions.
    Materials and methods
    Results To develop a kinetic rate law for LeuDH and L-AmDH, initial rates in the reductive amination direction at varied concentrations of NH4Cl, keto/keto acid, and NADH were best fit to Eq. (1) using the method described by Cleland [15]. Terms in the denominator were omitted from the rate law based on their statistical significance (as defined by the P-value). Results of these fits are shown in Table 1. In the global fit for LeuDH, the constant term and Coeffb were found to be insignificant (P = 0.486 and 0.763, respectively) and were excluded from the final model. This result implied a lack of both a quaternary complex and binary enzyme-ketoleucine complex in solution. In the amination direction for L-AmDH, the constant term and Coeffc term were found to be insignificant (P = 0.561 and 0.395, respectively) and were excluded from the final model. Omission of the constant term corresponds to the lack of the quaternary complex and the binary enzyme-NH3 complex in solution. For the deamination of leucine by LeuDH, no terms were excluded from the final model (Table 2), which was consistent with a sequential mechanism. For the deamination of (R)-2-aminopentane by L-AmDH, the constant term was found to be insignificant (P = 0.637) and was excluded from the final model. This corresponds to the lack of a ternary complex in solution. The high values for appKM,NH4 prevented determination of the true value of kcat for either enzyme. The solubility of ammonium chloride in solution was observed to be around 6000 mM, so measurement of enzyme activity at [NH4Cl] > appKM,NH4 value was not possible. Note also the general trend of smaller appKM values in the case of LeuDH when compared to L-AmDH, indicating stronger substrate binding.