Elucidation Of The Biosynthetic Pathway Of Anthraquinone Beticolin

Natural products with diverse chemical structures are important resources for modern drug development,which play an important role in agricultural and pharmaceutical applications.The elucidation of biosynthetic pathways for natural products,particularly the elucidation of key enzymes involved in their synthesis,not only facilitates the discovery of novel enzymes that catalyze complicated reactions and the use of these enzymes as fundamental elements of synthetic biology in microbial cell factories,but also enables the construction of biosynthetic pathways for the efficient heterologous synthesis of natural products and their derivatives.Here,we focus on the biosynthetic gene cluster of beticolin,a natural product first discovered in the plant endophytic fungus Cercospora sp.JNU001.By exploring the catalytic mechanisms of key enzymes,the biosynthetic pathway of beticolin was elucidated,and the synthesis of chiral alcohos as drug intermediates was realized through the structure-guided engineering of some key enzymes.The main results are as follows:1.The molecular mechanism of emodin reduction catalyzed by anthrol reductase BTG9in the biosynthesis of beticolin was analyzedFirstly,the catalytic properties of anthrol reductase BTG9 were studied.The in vitro enzymatic assay indicated that BTG9 was capable of efficiently converting emodin hydroquinone to（R）-3,8,9,10-tetrahydroxy-6-methyl-3,4-dihydroanthracene-1（2H）-one（2）（ee>99%）under an argon atmosphere.The Km and kcat values of BTG9 towards emodin were determined to be 0.2 m M and 102.51 min^-1,respectively.In addition,BTG9 also showed catalytic activity towards estrone（3a）and its derivatives.To reveal the catalytic mechanism of BTG9,the crystal structures of BTG9-NADP⁺and BTG9-NADP⁺-emodin complexes were solved,and the flexible loop region from residue 209 to 216 was identified as the"gate"for substrate recognition and binding.Tyr210 and His162 play a key role in substrate recognition,binding,and stabilization by forming hydrogen bond interactions to ensure that the substrate is in the proper conformation in the substrate-binding pocket of BTG9,which leads to the synthesis of a single chiral product.2.Structure-guided engineering of BTG9 for the preparation of optically pure2,2-disubstituted-3-hydroxycycloketones andβ-halohydrinsTo further explore the catalytic performance of BTG9 and realize the synthesis of optically pure 2,2-disubstituted-3-hydroxycycloketones andβ-halohydrins,structure-guided engineering of BTG9 was carried out using 2-methyl-2-（4-methylbenzyl）cyclopentane-1,3-dione（5a）as the model substrate.The in vitro enzymatic analysis showed that the three mutants（BTG9-H162F,BTG9-Y210A,and BTG9-Y210F）,which disrupted the potential hydrogen bond by mutating Tyr210 and His162,could catalyze the synthesis of optically pure2,2-disubstituted-3-hydroxycycloketones using substrate 5a.Their catalytic activity toward substrate 5a was significantly increased compared with the wild-type,and the best mutant BTG9-H162F had about 45-fold higher catalytic efficiency than that of the wild-type.And,BTG9-H162F was able to convert various 2,2-disubstituted prochiral 1,3-cyclodiketones andα-haloacetophenones into corresponding optically pure（2S,3S）-ketols（dr>99/1,ee>99%）and（R）-β-chiral halohydrins（ee>99%）.In addition,the stereoselectivity mechanism of the reaction catalyzed by BTG9-H162F was explained by the crystal structures of BTG9-H162F-NADP⁺-5a and BTG9-H162F-NADP⁺-2-bromo-1-（4-bromo-2-hydroxyphenyl）ethan-1-one（6e）,which provides a rationale for the study and engineering of anthrol reductases.3.A novel non-heme iron metalloenzyme BTG13 was identified,and its molecular mechanism for catalyzing anthraquinone ring cleavage in the biosynthesis of beticolin was analyzedTo elucidate the mechanism of the anthraquinone ring cleavage of chrysophanol in the biosynthesis of beticolin,bioinformatics analysis and in vitro enzymatic analysis were performed.BTG13,which is derived from Cercospora sp.JNU001,was discovered to be a Questin oxidase that is involved in the anthraquinone ring cleavage of chrysophanol.To better understand the enzymatic mechanism,the crystal structure of BTG13 was solved using single-wavelength anomalous dispersion（SAD）.The electron density map analysis revealed that the active center of BTG13 is composed of His55,His158,His296,His374,potentially modified Lys377,a water molecule,and a metal ion.To determine the types of metal ions in the active center,the purified BTG13 was analyzed with inductively coupled plasma mass spectrometry（ICP-MS）,which revealed that Fe²⁺was the major component and the coordinated metal ion was initially identified as Fe²⁺.Subsequently,the purified BTG13 was treated with EDTA,and through the supplementation of different metal ions combined with biochemical experiments,the coordinated metal ion was determined to be Fe²⁺.Futhermore,through conformational analysis of the electron density map and in vitro biochemical verification,we demonstrated that Lys377 exists in crystal structure in the form of carboxylated lysine（Kcx377）.The above results demonstrate that BTG13 is a novel non-heme iron metalloenzyme that has not yet been reported.Moreover,structure analysis and in vitro reactions suggested that Thr299 and His58 establish hydrogen bonds with Kcx377,which can modulate the catalytic activity of BTG13.To investigate the substrate recognition of BTG13,a complex structure of BTG13 binding with the substrate was obtained through molecular docking.The complex structure,together with in vitro reactions,revealed that Arg48,His53,Phe292,and His181 play key roles in substrate recognition and binding of BTG13,and His53 is the closest residue to the substrate reaction site and may serve as a proton receptor.Finally,the enzymatic mechanism of anthraquinone ring cleavage catalyzed by BTG13 was proposed based on the above results.