New Insights into the Biosynthesis of Prenylated Xanthones: Xptb from Aspergillus nidulans Catalyses an O‐Prenylation of Xanthones

Gene‐inactivation experiments have indicated that the putative prenyltransferase XptB from Aspergillus nidulans was likely to be responsible for the prenylation of 1,7‐dihydroxy‐6‐methyl‐8‐hydroxymethylxanthone. Recently, it was suggested that this enzyme might also accept as substrate the benzophenone arugosin H, which is assumed to be a precursor of prenylated xanthones. In this study, five benzophenones and ten xanthones were incubated with purified recombinant XptB in the presence of dimethylallyl diphosphate (DMAPP). XptB accepted four xanthones as substrates, including the proposed natural substrate, and catalysed regiospecific O‐prenylations at C‐7 of the xanthone core. K_m values in the range of 0.081–1.1 mM and turnover numbers (k_cat) between 0.02 and 0.5 s^?1 were determined for the accepted xanthones. The kinetic parameters for DMAPP were found to be 0.024 mM (K_m) and 0.13 s^?1 (k_cat). Arugosin H was not accepted by XptB under the tested conditions. XptB was relatively specific towards its prenyl donor and did not accept geranyl or farnesyl diphosphate as substrate. Mn²⁺ and Co²⁺ strongly enhanced XptB activity (up to eightfold); this has not been reported before for prenyltransferases of the DMATS superfamily.     

Tryptophan C5‐, C6‐ and C7‐Prenylating Enzymes Displaying a Preference for C‐6 of the Indole Ring in the Presence of Unnatural Dimethylallyl Diphosphate Analogues

The behavior of four dimethylallyltryptophan synthases (DMATSs) (5‐DMATS and 5‐DMATS_Sc as tryptophan C5‐prenyltransferases, and 6‐DMATS_Sa and 6‐DMATS_Sv as C6‐prenyltransferases) and one L ‐tyrosine prenyltransferase with a tryptophan C7‐prenyltransferase activity was investigated in the presence of two unnatural alkyl donors (methylallyl and 2‐pentenyl diphosphate) and one benzyl donor (benzyl diphosphate). Detailed biochemical investigations revealed the acceptance of these dimethylallyl diphosphate (DMAPP) analogues by all tested enzymes with different relative activities. Enzyme products with the allyl or benzyl moiety attached to different positions were identified in the reaction mixtures, whereby C‐6 alkylated or benzylated L ‐tryptophan was found as one of the main products. This observation demonstrates a preference of the five prenyltransferases toward C‐6 of the indole ring in the presence of unnatural DMAPP derivatives. Molecular dynamics simulation experiments with a homologous model of 5‐DMATS explained well its reactions with methylallyl and 2‐pentenyl diphosphate. Furthermore this study expands significantly the potential usage of tryptophan prenylating enzymes as biocatalysts for Friedel–Crafts alkylation.

     

Determination of Alkyl‐Donor Promiscuity of Tyrosine‐O‐Prenyltransferase SirD from Leptosphaeria maculans

Natural product prenyltransferases are known to display relaxed acceptor substrate specificity. Although recent studies with a small set of unnatural alkyl donors have revealed that prenyltransferases are flexible with regard to their alkyl donors, the scope of their alkyl donor specificity remains poorly understood. Towards this goal, we report the synthesis of 20 unnatural alkyl pyrophosphate donors and an assessment of the reactions of these synthetic unnatural alkyl pyrophosphate analogues catalyzed by tyrosine O‐prenyltransferase SirD. This study demonstrates that SirD can utilize 16 out of 21 alkyl pyrophosphate analogues (including the natural donor) in catalyzing mostly O‐alkylation of l ‐tyrosine. This study reveals the broad alkyl donor specificity of SirD and opens the door for the interrogation of the alkyl donor specificity of other prenyltransferases for potential utility as biocatalysts for differential alkylation applications.     

Biosynthesis of Sesterterpenes,Head‐to‐Tail Triterpenes,and Sesquarterpenes in Bacillus clausii: Identification of Multifunctional Enzymes and Analysis of Isoprenoid Metabolites

We performed functional analysis of recombinant enzymes and analysis of isoprenoid metabolites in Bacillus clausii to gain insights into the biosynthesis of rare terpenoid groups of sesterterpenes, head‐to‐tail triterpenes, and sesquarterpenes. We have identified an (all‐E)‐isoprenyl diphosphate synthase (E‐IDS) homologue as a trifunctional geranylfarnesyl diphosphate (GFPP)/hexaprenyl diphosphate (HexPP)/heptaprenyl diphosphate (HepPP) synthase. In addition, we have redefined the function of a tetraprenyl‐β‐curcumene synthase homologue as that of a trifunctional sesterterpene/triterpene/sesquarterpene synthase. This study has revealed that GFPP, HexPP, and HepPP, intermediates of two isoprenoid pathways (acyclic terpenes and menaquinones), are biosynthesized by one trifunctional E‐IDS. In addition, GFPP/HexPP and HepPP are the primary substrates for the biosynthesis of acyclic terpenes and menaquinone‐7, respectively.     

Insight into C35 terpene Biosyntheses by Nonpathogenic Mycobacterium Species: Functional Analyses of Three Z‐Prenyltransferases and Identification of Dehydroheptaprenylcyclines

Nonpathogenic Mycobacterium species produce rare cyclic C₃₅ terpenes that are biosynthesized by cyclization of Z‐type C₃₅ polyprenyl diphosphate. To provide deeper insight into the biosynthesis of C₃₅ terpenes, we carried out functional analyses of three Z‐prenyltransferase homologues in M. vanbaalenii identified by genomic analysis. Mvan_3822, a novel bifunctional Z‐prenyltransferase, biosynthesizes C₃₅‐heptaprenyl diphosphate as a main product from (E,E)‐farnesyl diphosphate (E,E‐FPP) and (E,E,E)‐geranylgeranyl diphosphate (E,E,E‐GGPP), but produces a C₅₀‐decaprenyl diphosphate from geranyl diphosphate. Mvan_1705 is a novel Z,E,E‐GGPP synthase. In addition, novel cyclic C₃₅ terpenes, (14E)‐ and (14Z)‐dehydroheptaprenylcycline, were identified as minor metabolites in nonpathogenic Mycobacterium cells. C₃₅ terpenes could be biosynthesized by two routes, in which E and Z geometric isomers of heptaprenyl diphosphate are produced from E,E‐FPP and E,E,E‐GGPP, and the prenylreductase responsible for the biosynthesis of C₃₅ terpenes could reduce both E and Z prenyl residues.     

A Tryptophan 6‐Halogenase and an Amidotransferase Are Involved in Thienodolin Biosynthesis

The biosynthetic gene cluster for the plant growth‐regulating compound thienodolin was identified in and cloned from the producer organism Streptomyces albogriseolus MJ286‐76F7. Sequence analysis of a 27 kb DNA region revealed the presence of 21 ORFs, 14 of which are involved in thienodolin biosynthesis. Three insertional inactivation mutants were generated in the sequenced region to analyze their involvement in thienodolin biosynthesis and to functionally characterize specific genes. The gene inactivation experiments together with enzyme assays with enzymes obtained by heterologous expression and feeding studies showed that the first step in thienodolin biosynthesis is catalyzed by a tryptophan 6‐halogenase and that the last step is the formation of a carboxylic amide group catalyzed by an amidotransferase. The results led to a hypothetical model for thienodolin biosynthesis.     

N‐Acylethanolamines Bind to SIRT6

Sirtuin 6 (SIRT6) is an NAD+‐dependent histone deacetylase enzyme that is involved in multiple molecular pathways related to aging. Initially, it was reported that SIRT6 selectively deacetylated H3K9Ac and H3K56Ac; however, it has more recently been shown to preferentially hydrolyze long‐chain fatty acyl groups over acetyl groups in vitro. Subsequently, fatty acids were demonstrated to increase the catalytic activity of SIRT6. In this study, we investigated whether a series of N‐acylethanolamines (NAEs), quercetin, and luteolin could regulate SIRT6 activity. NAEs increased SIRT6 activity, with oleoylethanolamide having the strongest activity (EC₅₀ value of 3.1 μm ). Quercetin and luteolin were demonstrated to have dual functionality with respect to SIRT6 activity; namely, they inhibited SIRT6 activity with IC₅₀ values of 24 and 2 μm , respectively, and stimulated SIRT6 activity more than sixfold (EC₅₀ values of 990 and 270 μm , respectively).     

Nucleophilic Water Capture or Proton Loss: Single Amino Acid Switch Converts δ‐Cadinene Synthase into Germacradien‐4‐ol Synthase

δ‐Cadinene synthase is a sesquiterpene cyclase that utilises the universal achiral precursor farnesyl diphosphate (FDP) to generate predominantly the bicyclic sesquiterpene δ‐cadinene and about 2 % germacradien‐4‐ol, which is also generated from FDP by the cyclase germacradien‐4‐ol synthase. Herein, the mechanism by which sesquiterpene synthases discriminate between deprotonation and reaction with a nucleophilic water molecule was investigated by site‐directed mutagenesis of δ‐cadinene synthase. If W279 in δ‐cadinene synthase was replaced with various smaller amino acids, the ratio of alcohol versus hydrocarbon product was directly proportional to the van der Waals volume of the amino acid side chain. DCS‐W279A is a catalytically highly efficient germacradien‐4‐ol synthase (k_cat/K_M=1.4×10^?3 μm s^?1) that produces predominantly germacradien‐4‐ol in addition to 11 % δ‐cadinene. Water capture is not achieved through strategic positioning of a water molecule in the active site, but through a coordinated series of loop movements that allow bulk water access to the final carbocation in the active site prior to product release.     

NAD+‐Dependent Dehydrogenase PctP and Pyridoxal 5′‐Phosphate Dependent Aminotransferase PctC Catalyze the First Postglycosylation Modification of the Sugar Intermediate in Pactamycin Biosynthesis

The unique five‐membered aminocyclitol core of the antitumor antibiotic pactamycin originates from d ‐glucose, so unprecedented enzymatic modifications of the sugar intermediate are involved in the biosynthesis. However, the order of the modification reactions remains elusive. Herein, we examined the timing of introduction of an amino group into certain sugar‐derived intermediates by using recombinant enzymes that were encoded in the pactamycin biosynthesis gene cluster. We found that the NAD⁺‐dependent alcohol dehydrogenase PctP and pyridoxal 5′‐phosphate dependent aminotransferase PctC converted N‐acetyl‐d ‐glucosaminyl‐3‐aminoacetophonone into 3′‐amino‐3′‐deoxy‐N‐acetyl‐d ‐glucosaminyl‐3‐aminoacetophenone. Further, N‐acetyl‐d ‐glucosaminyl‐3‐aminophenyl‐β‐oxopropanoic acid ethyl ester was converted into the corresponding 3′‐amino derivative. However, PctP did not oxidize most of the tested d ‐glucose derivatives, including UDP‐GlcNAc. Thus, modification of the GlcNAc moiety in pactamycin biosynthesis appears to occur after the glycosylation of aniline derivatives.     

Identification of the Tirandamycin Biosynthetic Gene Cluster from Streptomyces sp. 307‐9

The structurally intriguing bicyclic ketal moiety of tirandamycin is common to several acyl‐tetramic acid antibiotics, and is a key determinant of biological activity. We have identified the tirandamycin biosynthetic gene cluster from the environmental marine isolate Streptomyces sp. 307–9, thus providing the first genetic insight into the biosynthesis of this natural product scaffold. Sequence analysis revealed a hybrid polyketide synthase–nonribosomal peptide synthetase gene cluster with a colinear domain organization, which is entirely consistent with the core structure of the tirandamycins. We also identified genes within the cluster that encode candidate tailoring enzymes for elaboration and modification of the bicyclic ketal system. Disruption of tamI, which encodes a presumed cytochrome P450, led to a mutant strain deficient in production of late stage tirandamycins that instead accumulated tirandamycin C, an intermediate devoid of any post assembly‐line oxidative modifications.     

Identification of a New Diterpene Biosynthetic Gene Cluster that Produces O‐Methylkolavelool in Herpetosiphon aurantiacus

Diterpenoids are usually found in plants and fungi, but are rare in bacteria. We have previously reported new diterpenes, named tuberculosinol and isotuberculosinol, which are generated from the Mycobacterium tuberculosis gene products Rv3377c and Rv3378c. No homologous gene was found at that time, but we recently found highly homologous proteins in the Herpetosiphon aurantiacus ATCC 23779 genome. Haur_2145 was a class II diterpene cyclase responsible for the conversion of geranylgeranyl diphosphate into kolavenyl diphosphate. Haur_2146, homologous to Rv3378c, synthesized (+)‐kolavelool through the nucleophilic addition of a water molecule to the incipient cation formed after the diphosphate moiety was released. Haur_2147 afforded (+)‐O‐methylkolavelool from (+)‐kolavelool, so this enzyme was an O‐methyltransferase. This new diterpene was indeed detected in H. aurantiacus cells. This is the first report of the identification of a (+)‐O‐methylkolavelool biosynthetic gene cluster.     

4‐Hydroxy‐3‐methyl‐6‐(1‐methyl‐2‐oxoalkyl)pyran‐2‐one Synthesis by a Type III Polyketide Synthase from Rhodospirillum centenum

The purple photosynthetic bacterium Rhodospirillum centenum has a putative type III polyketide synthase gene (rpsA). Although rpsA was known to be transcribed during the formation of dormant cells, the reaction catalyzed by RpsA was unknown. Thus we examined the RpsA reaction in vitro, using various fatty acyl‐CoAs with even numbers of carbons as starter substrates. RpsA produced tetraketide pyranones as major compounds from one C_10–14 fatty acyl‐CoA unit, one malonyl‐CoA unit and two methylmalonyl‐CoA units. We identified these products as 4‐hydroxy‐3‐methyl‐6‐(1‐methyl‐2‐oxoalkyl)pyran‐2‐ones by NMR analysis. RpsA is the first bacterial type III PKS that prefers to incorporate two molecules of methylmalonyl‐CoA as the extender substrate. In addition, in vitro reactions with ¹³C‐labeled malonyl‐CoA revealed that RpsA produced tetraketide 6‐alkyl‐4‐hydroxy‐1,5‐dimethyl‐2‐oxocyclohexa‐3,5‐diene‐1‐carboxylic acids from C_14–20 fatty acyl‐CoAs. This class of compounds is likely synthesized through aldol condensation induced by methine proton abstraction. No type III polyketide synthase that catalyzes this reaction has been reported so far. These two unusual features of RpsA extend the catalytic functions of the type III polyketide synthase family.     

Mechanism‐Based Trapping of the Quinonoid Intermediate by Using the K276R Mutant of PLP‐Dependent 3‐Aminobenzoate Synthase PctV in the Biosynthesis of Pactamycin

Mutational analysis of the pyridoxal 5′‐phosphate (PLP)‐dependent enzyme PctV was carried out to elucidate the multi‐step reaction mechanism for the formation of 3‐aminobenzoate (3‐ABA) from 3‐dehydroshikimate (3‐DSA). Introduction of mutation K276R led to the accumulation of a quinonoid intermediate with an absorption maximum at 580 nm after the reaction of pyridoxamine 5′‐phosphate (PMP) with 3‐DSA. The chemical structure of this intermediate was supported by X‐ray crystallographic analysis of the complex formed between the K276R mutant and the quinonoid intermediate. These results clearly show that a quinonoid intermediate is involved in the formation of 3‐ABA. They also indicate that Lys276 (in the active site of PctV) plays multiple roles, including acid/base catalysis during the dehydration reaction of the quinonoid intermediate.