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Abstract

5-Hydroxy-l-tryptophan (5-HTP) is a naturally occurring aromatic amino acid present in the seeds of the African plant Griffonia simplicifolia. Although 5-HTP has therapeutic effects in various symptoms, efficient method of producing 5-HTP has not been established. In this study, we developed a novel cofactor regeneration process to achieve enhanced synthesis of 5-HTP by using modified l-phenylalanine 4-hydroxylase of Chromobacterium violaceum. For the synthesis of 5-HTP using Escherichia coli whole cell bioconversion, l-tryptophan and 5-HTP degradation by E. coli endogenous catabolic enzymes should be considered. The tryptophanase gene was disrupted using the λ red recombination system, since tryptophanase is postulated as an initial enzyme for the degradation of l-tryptophan and 5-HTP in E. coli. For regeneration of the cofactor pterin, we screened and investigated several key enzymes, including dihydropteridine reductase from E. coli, glucose dehydrogenase from Bacillus subtilis, and pterin-4α-carbinolamine dehydratase from Pseudomonas syringae. Genes encoding these three enzymes were overexpressed in an E. coli tryptophanase-deficient host, resulting in the synthesis of 0.74 mM 5-HTP in the presence of 0.1 mM pterin and the synthesis of 0.07 mM 5-HTP in the absence of regeneration of pterin. These results clearly indicated the successful regeneration of pterin. Following optimization of the reaction conditions, 2.5 mM 5-HTP was synthesized with cofactor regeneration, while 0.8 mM 5-HTP was recovered without cofactor regeneration under the same reaction conditions, suggesting that the principle described here provides a new method for cofactor regeneration.

Animals

Male Sprague–Dawley rats (250–300 g) were purchased from Sankyo Labo Service Co. Inc. (Tokyo, Japan). Three or four animals were housed per cage in a temperature-controlled environment (23 ± 0.5°C) with a 12-h light–dark cycle (light on at 7 am) with food and water ad libitum. One hundred and thirty-three rats (forced swimming test, 25; open-field test, 21; elevated plus-maze test, 28; contextual fear-conditioning test, 23; 5-HTP-induced head-twitches, 18; DOI-induced head-twitches, 18) were used in the present study.

Head-twitch test

The head-twitch test was carried out according a method described by Rojas-Corrales et al. [] with minor modifications. Rats were orally treated with MAK (1 g/kg) or fluvoxamine (10 mg/kg) 60 min before intraperitoneal injection of 5-HTP (200 mg/kg). Immediately after 5-HTP administration, the rat was placed into an observation cage (30 cm × 25 cm × 18 cm) and the cumulative number of head twitches (i.e., rapid movements of the head with little or no involvement of the trunk) counted by an observer blinded to the study protocol for 30 min. Similarly, the effect of MAK on DOI (3 mg/kg)-induced head-twitch responses was measured.

5-HTP- or DOI-induced head twitches

Figure shows the effect of MAK (1 g/kg) on 5-HTP-induced head twitches. One-way ANOVA revealed significant differences in 5-HTP-induced head twitches ([F(2,15) = 33.4, P < 0.01]). Post hoc analyses indicated significant differences between the control group and fluvoxamine-treated group (P < 0.01), but the difference between the control group and MAK-treated group was not significant (P = 0.33). Figure demonstrates the effect of MAK on DOI-induced head twitches. One-way ANOVA revealed significant differences in DOI-induced head twitches ([F(2,15) = 25.2, P < 0.01]). Post hoc analyses indicated significant differences between the control group and MAK-treated group (P < 0.01).