Identification and biochemical characterisation of tyrosine aminotransferase from Anthoceros agrestis unveils the conceivable entry point into rosmarinic acid biosynthesis in hornworts

Main conclusion Tyrosine aminotransferase (AaTAT) from the hornwort Anthoceros agrestis Paton (Anthocerotaceae) was amplified and expressed in E. coli. The active enzyme is able to accept a wide range of substrates with distinct preference for l-tyrosine, therefore, possibly catalysing the initial step in rosmarinic acid biosynthesis. Abstract The presence of rosmarinic acid (RA) in the hornwort A. agrestis is well known, and some attempts have been made to clarify the biosynthesis of this caffeic acid ester in lower plants. Parallel to the biosynthesis in vascular plants, the involvement of tyrosine aminotransferase (EC 2.6.1.5; TAT) as the initial step was assumed. The amplification of a nucleotide sequence putatively encoding AaTAT (Genbank MN922307) and expression in E. coli were successful. The enzyme proved to have a high acceptance of l-tyrosine (Km 0.53 mM) whilst slightly preferring 2-oxoglutarate over phenylpyruvate as co-substrate. Applying l-phenylalanine as a potential amino donor or using oxaloacetate or pyruvate as a replacement for 2-oxoglutarate as amino acceptor resulted in significantly lower catalytic efficiencies in each of these cases. To facilitate further substrate search, two methods were introduced, one using ninhydrin after thin-layer chromatography and the other using derivatisation with o-phthalaldehyde followed by HPLC or LC–MS analysis. Both methods proved to be well applicable and helped to confirm the acceptance of further aromatic and aliphatic amino acids. This work presents the first description of a heterologously expressed TAT from a hornwort (A. agrestis) and describes the possible entry into the biosynthesis of RA and other specialised compounds in a so far neglected representative of terrestrial plants and upcoming new model organism. Supplementary Information The online version contains supplementary material available at 10.1007/s00425-021-03623-2.


Method 2:
Determination of temperature optimum To determine temperature-optimum, the range of 5.5 °C to 75 °C was tested. The assays were prepared and analysed based on method 1 using 1 M Tris-HCl buffer pH 8.5.

Determination of PLP dependence
To determine saturation curves for the prosthetic group PLP, concentrations of up to 1.92 mM PLP were tested. Each assay was prepared in a total volume of 250 µl consisting of 1 M Tris-HCl pH 8.5, 80 mM 2-oxoglutarate, 6 mM Ltyrosine and varying PLP-concentrations. After pre-incubation at 40 °C, the reaction was started by adding purified enzyme. The remaining reaction conditions are based on method 1.

Method 4:
Substratesaturation kinetics for L-tyrosine To determine substrate-saturation curves for L-tyrosine, concentrations of up to 6 mM were tested. Each assay was prepared in a total volume of 250 µl consisting of 1 M Tris-HCl pH 8.5, 80 mM 2-oxoglutarate and varying Ltyrosine concentrations. After pre-incubation at 40 °C, a mixture of purified enzyme and PLP was added, resulting in a total PLP concentration of 0.48 mM. Incubation time was adjusted to 2 minutes. The remaining conditions are based on method 1.

Substratesaturation kinetics for 2-oxoglutarate
To determine substrate-saturation curves for 2-oxoglutarate, concentrations of up to 120 mM were tested. Each assay was prepared in a total volume of 250 µl consisting of 1 M Tris-HCl pH 8.5, 6 mM L-tyrosine and varying 2oxoglutarate concentrations. After pre-incubation at 40 °C, a mixture of purified enzyme and PLP was added, resulting in a total PLP concentration of 0.48 mM. The remaining conditions are based on method 1.

Method 6:
Substratesaturation kinetics for oxaloacetate To determine substrate-saturation curves for oxaloacetate, concentrations of up to 120 mM were tested. Each assay was prepared in a total volume of 250 µl consisting of 1 M Tris-HCl pH 8.5, 6 mM L-tyrosine and varying oxaloacetate concentrations. After pre-incubation at 40 °C, a mixture of purified enzyme and PLP was added, resulting in a total PLP concentration of 0.48 mM. Incubation time was adjusted to 2 minutes. The remaining conditions are based on method 1.
Method 7: Substratesaturation kinetics for phenylpyruvate To determine substrate-saturation curves for phenylpyruvate, concentrations of up to 36 mM were tested. Each assay was prepared in a total volume of 250 µl consisting of 1 M Tris-HCl pH 8.5, 6 mM L-tyrosine and varying phenylpyruvate concentrations. After pre-incubation at 40 °C, a mixture of purified enzyme and PLP was added, resulting in a total PLP concentration of 0.48 mM. Incubation time was adjusted to 1 minute. The remaining conditions are based on method 1. In order to optimise pH-value directly before measurement, 1 M Tris-HCl pH 7 was added.

Substratesaturation kinetics for pyruvate
To determine substrate-saturation curves for pyruvate, concentrations of up to 260 mM were tested. Each assay was prepared in a total volume of 250 µl consisting of 1 M Tris-HCl pH 8.5, 6 mM L-tyrosine and varying pyruvate concentrations. After pre-incubation at 40 °C, a mixture of purified enzyme and PLP was added, resulting in a total PLP concentration of 0.48 mM. Incubation time was adjusted to 0.5 minutes. The remaining conditions are based on method 1. In order to optimise pH-value directly before measurement, 1 M Tris-HCl pH 7 was added.
Method 9: Comparison of Ltyrosine and Dtyrosine To compare the specific enzyme activities with L-and D-tyrosine, assays in a total volume of 250 µl consisting of 1 M Tris-HCl pH 8.5, 80 mM 2-oxoglutarate and 6 mM L-or D-tyrosine were prepared. After pre-incubation at 40 °C, a mixture of purified enzyme and PLP was added, resulting in a total PLP concentration of 0.48 mM. The remaining conditions are based on method 1.

Method Assay
Method 10: Substrate-saturation kinetics for Lphenylalanine To determine substrate-saturation curves for L-phenylalanine, concentrations of up to 132 mM were tested. Each assay was prepared in a total volume of 250 µl consisting of 1 M Tris-HCl pH 8.5, 80 mM 2-oxoglutarate and varying L-phenylalanine concentrations. After pre-incubation at 40 °C, a mixture of purified enzyme and PLP was added, resulting in a total PLP concentration of 0.48 mM. The remaining conditions are based on method 1.