Reductase-catalyzed tetrahydrobiopterin regeneration alleviates the anti-competitive inhibition of tyrosine hydroxylation by 7,8-dihydrobiopterin

Abstract

l-Tyrosine hydroxylation by tyrosine hydroxylase is a significant reaction for preparing many nutraceutical and pharmaceutical chemicals.

Combining Precursor-Directed Engineering with Modular Designing: An Effective Strategy for De Novo Biosynthesis of l-DOPA in Bacillus licheniformis

“…It was previously reported that the regeneration of BH4 promoted by DHPR derived from E. coli could help alleviate the anticompetitive inhibition of SrTH through 7,8-dihydrobiopterin (BH2). 14 However, the K m value for q-BH2 and NADH of bacterial DHPR is far higher than that of eukaryotic DHPR (Table S3). Given a YfkO nitroreductase in B. licheniformis similar to NprA nitroreductase, which is also classed as DHPR in Rhodobacter capsulatus, 37,38 it is speculated that YfkO may also play a role in the synthesis of BH4 by catalyzing q-BH2.…”

“…28 However, the two amino acid positions of SrTH should be replaced with Val126 and Phe203, which may reduce the affinity toward cofactor BH4. 14 In this study, the protein structure models of SrTH, SrTH V126L , SrTH F203Y , and SrTH V126L,F203Y were constructed, and the binding effects between the four proteins and the ligand BH4 were analyzed by Autodock 6.9 (Figure 1A−D). In the complex SrTH F203Y -BH4, there was another hydrogen bond (3.1 Å) formed between phenyl oxygen of Tyr203 and O-4 of BH4 (Figure 1B).…”

“…It is presented as a yellow protein with an obvious size of above 25 kDa (Figure 3A,B), which is consistent with the previous reports. 14,37 Pure YfkO was then used to synthesize BH4 with q-BH2 and NADH as the substrate. Notably, YfkO nitroreductase led to the synthesis of 49.68 μmol/L BH4 based on the consumption of 63.8 μmol/L q-BH2 and 1 mmol/L NADH for reaction (Figure 3C−E).…”

“…L-DOPA can be oxidized to L-dopaquinone by the diphenolase (DP) activity and eventually into melanin. 13,14 PHAH can generate up to 0.69 and 25.53 g/L L-DOPA in shake flasks and 5 L bioreactor in E. coli. 10 However, a large amount of melanin would be formed during the fermentation process, and a lot of L-tyrosine remained, resulting in the loss of carbon flux into the downstream high-value products of L-DOPA, such as hydroxytyrosol, plant alkaloids, betalain pigments, and monolignols.…”

“…27 Therefore, we tried to build a modular pathway for producing L-DOPA by tyrosine hydroxylase SrTH in B. licheniformis based on previous research. 14,27 In this study, the work of four functional modules has been completed (Scheme 1): (1) unraveling and deleting a spliced transcript for enhancing L-tyrosine productivity of B. licheniformis; (2) improving the enzyme catalytic ability of SrTH;…”