2140-48-9Relevant academic research and scientific papers
Reduction of flavodoxin by electron bifurcation and sodium Ion-dependent reoxidation by NAD+ catalyzed by ferredoxin-NAD+ reductase (Rnf)?
Chowdhury, Nilanjan Pal,Klomann, Katharina,Seubert, Andreas,Buckel, Wolfgang
, p. 11993 - 12002 (2016)
Electron-transferring flavoprotein (Etf) and butyryl-CoA dehydrogenase (Bcd) from Acidaminococcus fermentans catalyze the endergonic reduction of ferredoxin by NADH, which is also driven by the concomitant reduction of crotonyl-CoA by NADH, a process called electron bifurcation. Here we show that recombinant flavodoxin from A. fermentans produced in Escherichia coli can replace ferredoxin with almost equal efficiency. After complete reduction of the yellow quinone to the blue semiquinone, a second 1.4 times faster electron transfer affords the colorless hydroquinone. Mediated by a hydrogenase, protons reoxidize the fully reduced flavodoxin or ferredoxin to the semi-reduced species. In this hydrogen-generating system, both electron carriers act catalytically with apparent Km = 0.26 μM ferredoxin or 0.42 μM flavodoxin. Membrane preparations of A. fermentans contain a highly active ferredoxin/flavodoxin-NAD+ reductase (Rnf) that catalyzes the irreversible reduction of flavodoxin by NADH to the blue semiquinone. Using flavodoxin hydroquinone or reduced ferredoxin obtained by electron bifurcation, Rnf can be measured in the forward direction, whereby one NADH is recycled, resulting in the simple equation: crotonyl-CoA + NADH+ H + = butyryl-CoA + NAD+ with Km= 1.4 μM ferredoxin or 2.0 μM flavodoxin. This reaction requires Na + (Km= 0.12 mM)orLi + (Km = 0.25 mM) for activity, indicating that Rnf acts as a Na + pump. The redox potential of the quinone/semiquinone couple of flavodoxin (Fld) is much higher than that of the semiquinone/hydroquinone couple. With free riboflavin, the opposite is the case. Based on this behavior, we refine our previous mechanism of electron bifurcation.
Substrate specificity, substrate channeling, and allostery in BphJ: An acylating aldehyde dehydrogenase associated with the pyruvate aldolase BphI
Baker, Perrin,Carere, Jason,Seah, Stephen Y. K.
, p. 4558 - 4567 (2012)
BphJ, a nonphosphorylating acylating aldehyde dehydrogenase, catalyzes the conversion of aldehydes to form acyl-coenzyme A in the presence of NAD + and coenzyme A (CoA). The enzyme is structurally related to the nonacylating aldehyde dehydrogenases, aspartate-β-semialdehyde dehydrogenase and phosphorylating glyceraldehyde-3-phosphate dehydrogenase. Cys-131 was identified as the catalytic thiol in BphJ, and pH profiles together with site-specific mutagenesis data demonstrated that the catalytic thiol is not activated by an aspartate residue, as previously proposed. In contrast to the wild-type enzyme that had similar specificities for two- or three-carbon aldehydes, an I195A variant was observed to have a 20-fold higher catalytic efficiency for butyraldehyde and pentaldehyde compared to the catalytic efficiency of the wild type toward its natural substrate, acetaldehyde. BphJ forms a heterotetrameric complex with the class II aldolase BphI that channels aldehydes produced in the aldol cleavage reaction to the dehydrogenase via a molecular tunnel. Replacement of Ile-171 and Ile-195 with bulkier amino acid residues resulted in no more than a 35% reduction in acetaldehyde channeling efficiency, showing that these residues are not critical in gating the exit of the channel. Likewise, the replacement of Asn-170 in BphJ with alanine and aspartate did not substantially alter aldehyde channeling efficiencies. Levels of activation of BphI by BphJ N170A, N170D, and I171A were reduced by ≥3-fold in the presence of NADH and ≥4.5-fold when BphJ was undergoing turnover, indicating that allosteric activation of the aldolase has been compromised in these variants. The results demonstrate that the dehydrogenase coordinates the catalytic activity of BphI through allostery rather than through aldehyde channeling. (Figure Presented).
IcmF is a fusion between the radical B12 enzyme isobutyryl-CoA mutase and its G-protein chaperone
Cracan, Valentin,Padovani, Dominique,Banerjee, Ruma
, p. 655 - 666 (2010)
Coenzyme B12 is used by two highly similar radical enzymes, which catalyze carbon skeleton rearrangements, methylmalonyl-CoA mutase and isobutyryl-CoA mutase (ICM). ICM catalyzes the reversible interconversion of isobutyryl-CoA and n-butyryl-CoA and exists as a heterotetramer. In this study, we have identified >70 bacterial proteins, which represent fusions between the subunits of ICM and a P-loop GTPase and are currently misannotated as methylmalonyl-CoA mutases.Wedesignate this fusion protein as IcmF (isobutyryl-CoA mutase fused). All IcmFs are composed of the following three domains: the N-terminal 5′-deoxyadenosylcobalamin binding region that is homologous to the small subunit of ICM (IcmB), a middle P-loop GTPase domain, and a C-terminal part that is homologous to the large subunit of ICM (IcmA). The P-loop GTPase domain has very high sequence similarity to the Methylobacterium extorquens MeaB, which is a chaperone for methylmalonyl-CoA mutase. We have demonstrated that IcmF is an active ICM by cloning, expressing, and purifying the IcmFs from Geobacillus kaustophilus, Nocardia farcinica, and Burkholderia xenovorans. This finding expands the known distribution of ICM activity well beyond the genus Streptomyces, where it is involved in polyketides biosynthesis, and suggests a role for this enzyme in novel bacterial pathways for amino acid degradation, myxalamid biosynthesis, and acetyl-CoA assimilation.
Identification of an α-Oxoamine Synthase and a One-Pot Two-Step Enzymatic Synthesis of α-Amino Ketones
Zhou, Ting,Gao, Du,Li, Jia-Xin,Xu, Min-Juan,Xu, Jun
supporting information, p. 37 - 41 (2020/12/21)
Alb29, an α-oxoamine synthase involved in albogrisin biosynthesis in Streptomyces albogriseolus MGR072, was characterized and responsible for the incorporation of l-glutamate to acyl-coenzyme A substrates. Combined with Alb29 and Mgr36 (an acyl-coenzyme A ligase), a one-pot enzymatic system was established to synthesize seven α-amino ketones. When these α-amino ketones were fed into the alb29 knockout strain Δalb29, respectively, the albogrisin analogs with different side chains were observed.
Repurposing the 3-Isocyanobutanoic Acid Adenylation Enzyme SfaB for Versatile Amidation and Thioesterification
Zhu, Mengyi,Wang, Lijuan,He, Jing
supporting information, p. 2030 - 2035 (2020/11/30)
Genome mining of microbial natural products enables chemists not only to discover the bioactive molecules with novel skeletons, but also to identify the enzymes that catalyze diverse chemical reactions. Exploring the substrate promiscuity and catalytic mechanism of those biosynthetic enzymes facilitates the development of potential biocatalysts. SfaB is an acyl adenylate-forming enzyme that adenylates a unique building block, 3-isocyanobutanoic acid, in the biosynthetic pathway of the diisonitrile natural product SF2768 produced by Streptomyces thioluteus, and this AMP-ligase was demonstrated to accept a broad range of short-chain fatty acids (SCFAs). Herein, we repurpose SfaB to catalyze amidation or thioesterification between those SCFAs and various amine or thiol nucleophiles, thereby providing an alternative enzymatic approach to prepare the corresponding amides and thioesters in vitro.
A Dual Role Reductase from Phytosterols Catabolism Enables the Efficient Production of Valuable Steroid Precursors
Peng, Haidong,Wang, Yaya,Jiang, Kai,Chen, Xinru,Zhang, Wenlu,Zhang, Yanan,Deng, Zixin,Qu, Xudong
supporting information, p. 5414 - 5420 (2021/02/05)
4-Androstenedione (4-AD) and progesterone (PG) are two of the most important precursors for synthesis of steroid drugs, however their current manufacturing processes suffer from low efficiency and severe environmental issues. In this study, we decipher a dual-role reductase (mnOpccR) in the phytosterols catabolism, which engages in two different metabolic branches to produce the key intermediate 20-hydroxymethyl pregn-4-ene-3-one (4-HBC) through a 4-e reduction of 3-oxo-4-pregnene-20-carboxyl-CoA (3-OPC-CoA) and 2-e reduction of 3-oxo-4-pregnene-20-carboxyl aldehyde (3-OPA), respectively. Inactivation or overexpression of mnOpccR in the Mycobacterium neoaurum can achieve exclusive production of either 4-AD or 4-HBC from phytosterols. By utilizing a two-step synthesis, 4-HBC can be efficiently converted into PG in a scalable manner (100 gram scale). This study deciphers a pivotal biosynthetic mechanism of phytosterol catabolism and provides very efficient production routes of 4-AD and PG.
ATP Regeneration System in Chemoenzymatic Amide Bond Formation with Thermophilic CoA Ligase
Lelièvre, Chloé M.,Balandras, Mélanie,Petit, Jean-Louis,Vergne-Vaxelaire, Carine,Zaparucha, Anne
, p. 1184 - 1189 (2020/01/22)
CoA ligases are enzymes catalyzing the ATP-dependent addition of coenzyme A to carboxylic acids in two steps through an adenylate intermediate. This intermediate can be diverted by a nucleophilic non enzymatic addition of amine to get the corresponding amide for synthetic purposes. To this end, we selected thermophilic CoA ligases to study the conversion of various carboxylic acids into their amide counterparts. To limit the use of ATP, we implemented an ATP regeneration system combining polyphosphate kinase 2 (PPK2 Class III) and inorganic pyrophosphatase. Suitability of this system was illustrated by the lab-scale chemoenzymatic synthesis of N-methylbutyrylamide in 77 % yield using low enzyme loading and 5 % molar ATP.
Rational Control of Polyketide Extender Units by Structure-Based Engineering of a Crotonyl-CoA Carboxylase/Reductase in Antimycin Biosynthesis
Zhang, Lihan,Mori, Takahiro,Zheng, Qingfei,Awakawa, Takayoshi,Yan, Yan,Liu, Wen,Abe, Ikuro
supporting information, p. 13462 - 13465 (2015/11/09)
Bioengineering of natural product biosynthesis is a powerful approach to expand the structural diversity of bioactive molecules. However, in polyketide biosynthesis, the modification of polyketide extender units, which form the carbon skeletons, has remained challenging. Herein, we report the rational control of polyketide extender units by the structure-based engineering of a crotonyl-CoA carboxylase/reductase (CCR), in the biosynthesis of antimycin. Site-directed mutagenesis of the CCR enzyme AntE, guided by the crystal structure solved at 1.5 ? resolution, expanded its substrate scope to afford indolylmethylmalonyl-CoA by the V350G mutation. The mutant A182L selectively catalyzed carboxylation over the regular reduction. Furthermore, the combinatorial biosynthesis of heterocycle- and substituted arene-bearing antimycins was achieved by an engineered Streptomyces strain bearing AntEV350G. These findings deepen our understanding of the molecular mechanisms of the CCRs, which will serve as versatile biocatalysts for the manipulation of building blocks, and set the stage for the rational design of polyketide biosynthesis.
Screening and Engineering the Synthetic Potential of Carboxylating Reductases from Central Metabolism and Polyketide Biosynthesis
Peter, Dominik M.,Schada Von Borzyskowski, Lennart,Kiefer, Patrick,Christen, Philipp,Vorholt, Julia A.,Erb, Tobias J.
supporting information, p. 13457 - 13461 (2015/11/09)
Carboxylating enoyl-thioester reductases (ECRs) are a recently discovered class of enzymes. They catalyze the highly efficient addition of CO2 to the double bond of α,β-unsaturated CoA-thioesters and serve two biological functions. In primary metabolism of many bacteria they produce ethylmalonyl-CoA during assimilation of the central metabolite acetyl-CoA. In secondary metabolism they provide distinct α-carboxyl-acyl-thioesters to vary the backbone of numerous polyketide natural products. Different ECRs were systematically assessed with a diverse library of potential substrates. We identified three active site residues that distinguish ECRs restricted to C4 and C5-enoyl-CoAs from highly promiscuous ECRs and successfully engineered a selected ECR as proof-of-principle. This study defines the molecular basis of ECR reactivity, allowing for predicting and manipulating a key reaction in natural product diversification.
Uncovering the formation and selection of benzylmalonyl-CoA from the biosynthesis of splenocin and enterocin reveals a versatile way to introduce amino acids into polyketide carbon scaffolds
Chang, Chenchen,Huang, Rong,Yan, Yan,Ma, Hongmin,Dai, Zheng,Zhang, Benying,Deng, Zixin,Liu, Wen,Qu, Xudong
supporting information, p. 4183 - 4190 (2015/04/14)
Selective modification of carbon scaffolds via biosynthetic engineering is important for polyketide structural diversification. Yet, this scope is currently restricted to simple aliphatic groups due to (1) limited variety of CoA-linked extender units, which lack aromatic structures and chemical reactivity, and (2) narrow acyltransferase (AT) specificity, which is limited to aliphatic CoA-linked extender units. In this report, we uncovered and characterized the first aromatic CoA-linked extender unit benzylmalonyl-CoA from the biosynthetic pathways of splenocin and enterocin in Streptomyces sp. CNQ431. Its synthesis employs a deamination/reductive carboxylation strategy to convert phenylalanine into benzylmalonyl-CoA, providing a link between amino acid and CoA-linked extender unit synthesis. By characterization of its selection, we further validated that AT domains of splenocin, and antimycin polyketide synthases are able to select this extender unit to introduce the phenyl group into their dilactone scaffolds. The biosynthetic machinery involved in the formation of this extender unit is highly versatile and can be potentially tailored for tyrosine, histidine and aspartic acid. The disclosed aromatic extender unit, amino acid-oriented synthetic pathway, and aromatic-selective AT domains provides a systematic breakthrough toward current knowledge of polyketide extender unit formation and selection, and also opens a route for further engineering of polyketide carbon scaffolds using amino acids.
