Active site pocket of Streptomyces d-stereospecific amidohydrolase has functional roles in aminolysis activity

Elyas, Yasmeen Yousif Ahmed; Miyatani, Kazusa; Bito, Tomohiro; Uraji, Misugi; Hatanaka, Tadashi; Shimizu, Katsuhiko; Arima, Jiro

doi:10.1016/j.jbiosc.2018.03.004

Cited by 3 publications

(1 citation statement)

References 33 publications

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“…S. albus::cos4∆O6 x pUWLR1R2R3 still produces rishirilide B ( Figure S2), indicating that the flavin-dependent oxidoreductase RslO6 is not essential for rishirilide B production. Additionally, 6deletion of rslH, putatively encoding an amidohydrolase [9], also did not influence rishirilide B production ( Figure S2), indicating that the gene does not belong to the cluster or that RslH activity is also not essential for rishirilide production.…”

Section: Rishirilide Production Is Not Strongly Affected By Deletionsmentioning

confidence: 99%

Early Steps in the Biosynthetic Pathway of Rishirilide B

et al. 2020

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The biological active compound rishirilide B is produced by Streptomyces bottropensis. The cosmid cos4 contains the complete rishirilide B biosynthesis gene cluster. Its heterologous expression in the host Streptomyces albus J1074 led to the production of rishirilide B as a major compound and to small amounts of rishirilide A, rishirilide D and lupinacidin A. In order to gain more insights into the biosynthesis, gene inactivation experiments and gene expression experiments were carried out. This study lays the focus on the functional elucidation of the genes involved in the early biosynthetic pathway. A total of eight genes were deleted and six gene cassettes were generated. Rishirilide production was not strongly affected by mutations in rslO2, rslO6 and rslH. The deletion of rslK4 and rslO3 led to the formation of polyketides with novel structures. These results indicated that RslK4 and RslO3 are involved in the generation or selection of the starter unit for rishirilide biosynthesis. In the rslO10 mutant strain, two novel compounds were detected, which were also produced by a strain containing solely the genes rslK1, rslK2, rslK3, rslK4, and rslA. rslO1 and rslO4 mutants predominately produce galvaquinones. Therefore, the ketoreductase RslO10 is involved in an early step of rishirilide biosynthesis and the oxygenases RslO1 and RslO4 are most probably acting on an anthracene moiety. This study led to the functional elucidation of several genes of the rishirilide pathway, including rslK4, which is involved in selecting the unusual starter unit for polyketide synthesis.Molecules 2020, 25, 1955 2 of 15 the initiation module, can be found, consisting of a ketosynthase, a malonyl CoA:ACP transferase and an acyl carrier protein (ACP). The initiation module selects the starter unit and catalyzes the first elongation of the growing polyketide, before the molecule is loaded onto the minPKS for further elongation reactions. Additional enzymes, like ketoreductases, cyclases and aromatases complement the minPKS and determine the folding of the respective polyketide [1]. The aromatic polyketide rishirilide B (Figure 1) was first discovered by Iwaki et al. in 1984. Rishirilide B was isolated from Streptomyces rishiriensis OFR-1056 in the course of screening for new α2-macroglobulin inhibitors [2]. This compound was also described as a product of S. olivaceus SCSIO T05, which also produced rishirilide C, lupinacidin A and galvaquinone A and B [3] (Figure 1). The biosynthetic gene cluster of this strain responsible for the formation of all four compounds was cloned and biosynthetic studies were performed [3]. S. bottropensis (formerly S. sp. Gö C4/4) also produces the compounds rishirilide A, B, D and lupinacidin A (Figure 1). The corresponding rishirilide biosynthetic gene cluster was cloned on a cosmid (cos4), which was subsequently expressed in the heterologous host S. albus J1074. Rishirilide B was produced as a major compound. Moreover, rishirilide A, D and lupinacidin A were also produced in small amounts [3] (Figure 1)....

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Section: Rishirilide Production Is Not Strongly Affected By Deletionsmentioning

confidence: 99%

Early Steps in the Biosynthetic Pathway of Rishirilide B

et al. 2020

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Effect of Active Site Pocket Structure Modification of d-Stereospecific Amidohydrolase on the Recognition of Stereospecific and Hydrophobic Substrates

et al. 2018

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D-Stereospecific amidohydrolase (DAH) from Streptomyces sp. 82F2 has potential utility for the synthesis of D/L configuration dipeptides by an aminolysis reaction. Structural comparison of DAH with substrate-bound D-amino acid amidase revealed that three residues located in the active site pocket of DAH (Thr145, Ala267, and Gly271) might be involved in interactions with D-phenylalanine substrate. We substituted Ala267 and Gly271, which are located at the bottom of the hydrophobic pocket of DAH, with Phe and observed changes in the stereoselectivity and specific activity toward the free and acetylated forms of D/L-Phe-methyl esters. In contrast, the mutation of Thr145, which likely supplies negative charge for recognition of the amino group of the substrate, hardly affected the stereoselectivity of the enzyme. A similar effect was observed in an investigation of hydrolysis and aminolysis reactions using the acetylated forms of D/L-Phe-methyl esters and 1,8-diaminooctane as an acyl-donor and acyl-acceptor, respectively. Substrate binding by DAH was disrupted by the mutation of Ala267 to Val or Trp and kinetic analysis showed that the hydrophobicity of the bottom of the active site pocket (Ala267 and Gly271) is important for both stereoselectivity and recognition of hydrophobic substrates.

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State-of-the-art strategies and research advances for the biosynthesis of D-amino acids

Wang

et al. 2023

Critical Reviews in Biotechnology

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Active site pocket of Streptomyces d-stereospecific amidohydrolase has functional roles in aminolysis activity

Cited by 3 publications

References 33 publications

Early Steps in the Biosynthetic Pathway of Rishirilide B

Early Steps in the Biosynthetic Pathway of Rishirilide B

Effect of Active Site Pocket Structure Modification of d-Stereospecific Amidohydrolase on the Recognition of Stereospecific and Hydrophobic Substrates

State-of-the-art strategies and research advances for the biosynthesis of D-amino acids

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