Active site mutagenesis of the putative Diels–Alderase macrophomate synthase

Serafimov, Jörg M.; Lehmann, Hans Christian; Oikawa, Hideaki; Hilvert, Donald

doi:10.1039/b703177g

Cited by 8 publications

(9 citation statements)

References 25 publications

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“…The conserved “glycerate utilization operon” kijABCDE is involved in the activation of glycerate, its condensation with the kijanolide linear polyketide precursor via C−C bond formation to give a branched chain intermediate, and subsequent lactonization, dehydration, and cyclization to form the mature spirotetronate group. By analogy to putative Diels-Alderases lovastatin nonaketide synthase (LNKS), solanopyrone synthase, , and macrophomate synthase, − the putative Diels−Alder-type cyclases in kijanimicin biosynthesis form or activate a dienophile prior to cyclization. The steps involved in the formation of the highly modified deoxysugar d -kijanose have also been proposed, and a likely candidate for 3-amine oxidation of a d -kijanose pathway intermediate, the flavoprotein KijD3, has been identified.…”

Section: Discussionmentioning

confidence: 99%

Elucidation of the Kijanimicin Gene Cluster: Insights into the Biosynthesis of Spirotetronate Antibiotics and Nitrosugars

et al. 2007

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The antibiotic kijanimicin produced by the actinomycete Actinomadura kijaniata has a broad spectrum of bioactivities as well as a number of interesting biosynthetic features. To understand the molecular basis for its formation and to develop a combinatorial biosynthetic system for this class of compounds, a 107.6 kb segment of the Actinomadura kijaniata chromosome containing the kijanimicin biosynthetic locus was identified, cloned, and sequenced. The complete pathway for the formation of TDP-L-digitoxose, one of the two sugar donors used in construction of kijanimicin, was elucidated through biochemical analysis of four enzymes encoded in the gene cluster. Sequence analysis indicates that the aglycone kijanolide is formed by the combined action of a modular Type-I polyketide synthase (PKS) and a conserved operon involved attachment and intramolecular cyclization of a glycerate-derived three-carbon unit, which forms the core of the spirotetronate moiety. The genes involved in the biosynthesis of the unusual deoxysugar D-kijanose [2,3,4,6-tetradeoxy-4-(methylcarbamyl)-3-C-methyl-3-nitro-D-xylo-hexopyranose], including one encoding a flavoenzyme predicted to catalyze the formation of the nitro group, have also been identified. This work has implications for the biosynthesis of other spirotetronate antibiotics and nitro sugar-bearing natural products, as well as for future mechanistic and biosynthetic engineering efforts.Kijanimicin (1) is a spirotetronate antibiotic isolated from Actinomadura kijaniata, a soil actinomycete. It has a broad spectrum of antimicrobial activity against Gram-positive bacteria, anaerobes, and the malaria parasite Plasmodium falciparum, 1 and also shows antitumor activity. 2 The structure of kijanimicin (1) consists of a pentacyclic core, which is equipped with four L-digitoxose (2) units and a rare nitro sugar, 2,3,4,6-tetradeoxy-4-(methylcarbamyl)-3-C-methyl-3-nitro-D-xylo-hexopyranose, commonly known as D-kijanose (3). More than sixty kijanimicin-related spirotetronate-type compounds have been reported. Most are made by strains of high-GC Gram positive bacteria (Actinomycetes), including Streptomyces, 3-8 Micromonospora, 9-12 Actinomadura, 1,13,14 Saccharothrix, 15 and Verrucosispora. 16 A species of Bacillus has also been identified as a producer of a member of this class of compounds. 17 Nearly all members of this class exhibit both antibacterial and antitumor activities, and many possess other biological activities. Well-known examples include chlorothricins (4), the anticholesterolemic agents; 18,19 tetronothiodin, a cholecystokinin B (CCK-B) inhibitor; 4 MM46115, an antiviral drug effective against parainfluenzae virus 1 and 2; 13 and tetrocarcins (5) and arisostatins, both of which have been shown to have therapeutic potential as inducers of apoptosis. 20-23 In a recent study, a collection of tetrocarcin analogues was prepared synthetically and some of them showed improved apoptosis-inducing activity. 24 Hence, compounds of this class have broad therapeutic potential wort...

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Section: Discussionmentioning

confidence: 99%

Elucidation of the Kijanimicin Gene Cluster: Insights into the Biosynthesis of Spirotetronate Antibiotics and Nitrosugars

et al. 2007

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“…However, later quantum mechanics/molecular mechanics (QM/MM) calculations on the MPS-catalyzed transformation indicated that a two-step Michael-aldol sequence is energetically preferred over a concerted Diels-Alder reaction [15]. Subsequent biochemical studies also supported this conclusion [16,17]. Thus, although MPS is not a bona fide Diels-Alderase, the series of studies on MPS have provided significant insights into the concertedness of two-bond formations in Diels-Alderase-catalyzed reactions.…”

Section: Introductionmentioning

confidence: 99%

Mechanistic insights into Diels-Alder reactions in natural product biosynthesis

Hashimoto

Kuzuyama

2016

Current Opinion in Chemical Biology

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Natural enzymes that catalyze Diels-Alder reactions have long been sought after, yet few enzymes have been experimentally confirmed to perform this reaction. In the past five years, several stand-alone enzymes that can catalyze the Diels-Alder reaction had been identified and characterized. Among which, the crystal structures of SpnF, PyrI4 and AbyU have been determined. The structures of PyrI4 and AbyU, which are involved in spirotetronate/spirotetramate biosynthesis, are particularly informative since they shed light on how a natural catalyst captures the flexible substrate and facilitates the intramolecular Diels-Alder reaction through stabilization of the transition state in catalysis. These pioneering studies will inspire the design of artificial catalysts for Diels-Alder reactions.

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“…The modest S-selectivity we observe for the promiscuous aldol reactions is consistent with a relatively open binding pocket and further underscores the evolutionary link between MPS and type II pyruvate aldolases, which also preferentially cleave S-configured carbinols. 10 The higher selectivity observed with glyceraldehyde derivative 7a, and the tolerance of the MPS active site to mutation, 15 suggests that relatively minor modification of the substrate or the binding pocket might suffice to reengineer the specificity of this enzyme.…”

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confidence: 99%

The Putative Diels−Alderase Macrophomate Synthase is an Efficient Aldolase

et al. 2008

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We find that the putative Diels-Alderase macrophomate synthase (MPS) catalyzes the addition of pyruvate enolate, generated by decarboxylation of oxaloacetate, to a variety of aldehydes. Alkyl, aryl, and heteroaryl aldehydes are accepted as substrates, providing gamma-hydroxy-alpha-ketoacids in 35-95% yield with modest levels of stereochemical control. These aldol products, which are difficult to synthesize by other methods, are formed with efficiency comparable to that of macrophomate. Our results thus provide evidence that a two-step Michael-aldol pathway is a plausible alternative to the postulated [4 + 2] cycloaddition in the MPS-catalyzed addition of pyruvate enolate to 2-pyrones. They are also relevant to understanding the divergent evolution of type II pyruvate aldolases.

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Active site mutagenesis of the putative Diels–Alderase macrophomate synthase

Cited by 8 publications

References 25 publications

Elucidation of the Kijanimicin Gene Cluster: Insights into the Biosynthesis of Spirotetronate Antibiotics and Nitrosugars

Elucidation of the Kijanimicin Gene Cluster: Insights into the Biosynthesis of Spirotetronate Antibiotics and Nitrosugars

Mechanistic insights into Diels-Alder reactions in natural product biosynthesis

The Putative Diels−Alderase Macrophomate Synthase is an Efficient Aldolase

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