Despite the astonishing breadth of enzymes in nature, no enzymes are known for many of the valuable catalytic transformations discovered by chemists. Recent work in enzyme design and evolution, ...however, gives us good reason to think that this will change. We describe a chemomimetic biocatalysis approach that draws from small-molecule catalysis and synthetic chemistry, enzymology, and molecular evolution to discover or create enzymes with non-natural reactivities. We illustrate how cofactor-dependent enzymes can be exploited to promote reactions first established with related chemical catalysts. The cofactors can be biological, or they can be non-biological to further expand catalytic possibilities. The ability of enzymes to amplify and precisely control the reactivity of their cofactors together with the ability to optimize non-natural reactivity by directed evolution promises to yield exceptional catalysts for challenging transformations that have no biological counterparts.
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IJS, KILJ, NUK, PNG, UL, UM
In methanogenic archaea, the carbon dioxide (CO₂) fixation and methane-forming steps are linked through the heterodisulfide reductase (HdrABC)–NiFe-hydrogenase (MvhAGD) complex that uses flavin-based ...electron bifurcation to reduce ferredoxin and the heterodisulfide of coenzymes M and B. Here, we present the structure of the native heterododecameric HdrABC-MvhAGD complex at 2.15-angstrom resolution. HdrB contains two noncubane 4Fe-4S clusters composed of fused 3Fe-4S-2Fe-2S units sharing 1 iron (Fe) and 1 sulfur (S), which were coordinated at the CCG motifs. Soaking experiments showed that the heterodisulfide is clamped between the two noncubane 4Fe-4S clusters and homolytically cleaved, forming coenzyme M and B bound to each iron. Coenzymes are consecutively released upon one-by-one electron transfer. The HdrABC-MvhAGD atomic model serves as a structural template for numerous HdrABC homologs involved in diverse microbial metabolic pathways.
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BFBNIB, NMLJ, NUK, ODKLJ, PNG, SAZU, UL, UM, UPUK
Absolute quantification of intracellular coenzyme A (CoA), coenzyme A disulfide, and short-chain acyl-coenzyme A thioesters was addressed by developing a tailored metabolite profiling method based on ...liquid chromatography in combination with tandem mass spectrometric detection (LC-MS/MS). A reversed phase chromatographic separation was established which is capable of separating a broad spectrum of CoA, its corresponding derivatives, and their isomers despite the fact that no ion-pairing reagent was used (which was considered as a key advantage of the method). Excellent analytical figures of merit such as high sensitivity (LODs in the nM to sub-nM range) and high repeatability (routinely 4 %; N = 15) were obtained. Method validation comprised a study on standard purity, stability, and recoveries during sample preparation. Uniformly labeled U¹³C yeast cell extracts offered ideal internal standards for validation purposes and for a quantification exercise in the rumen bacterium Megasphaera elsdenii.
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DOBA, EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, IZUM, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UILJ, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Saccharomyces cerevisiae is an important industrial cell factory and an attractive experimental model for evaluating novel metabolic engineering strategies. Many current and potential products of ...this yeast require acetyl coenzyme A (acetyl-CoA) as a precursor and pathways towards these products are generally expressed in its cytosol. The native S. cerevisiae pathway for production of cytosolic acetyl-CoA consumes 2 ATP equivalents in the acetyl-CoA synthetase reaction. Catabolism of additional sugar substrate, which may be required to generate this ATP, negatively affects product yields. Here, we review alternative pathways that can be engineered into yeast to optimize supply of cytosolic acetyl-CoA as a precursor for product formation. Particular attention is paid to reaction stoichiometry, free-energy conservation and redox-cofactor balancing of alternative pathways for acetyl-CoA synthesis from glucose. A theoretical analysis of maximally attainable yields on glucose of four compounds (n-butanol, citric acid, palmitic acid and farnesene) showed a strong product dependency of the optimal pathway configuration for acetyl-CoA synthesis. Moreover, this analysis showed that combination of different acetyl-CoA production pathways may be required to achieve optimal product yields. This review underlines that an integral analysis of energy coupling and redox-cofactor balancing in precursor-supply and product-formation pathways is crucial for the design of efficient cell factories.
•Precursor supply strongly influences the yield of acetyl-CoA-derived products on sugars.•The native yeast pathway for cytosolic acetyl-CoA formation is ATP inefficient.•Pathways from glucose to acetyl-CoA have different ATP, NAD(P)H and carbon stoichiometries.•Optimal pathway configuration for acetyl-CoA generation is strongly product dependent.•Combinations of acetyl-CoA forming reactions should be considered in pathway design.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Coenzyme Q10 (CoQ10) is essential for mitochondrial ATP production and functions as an important antioxidant in every biomembrane and lipoprotein. Due to its hydrophobicity, a binding and transfer ...protein for CoQ10 is plausible, and we previously described saposin B as a CoQ10-binding and transfer protein. Here, we report that prosaposin, the precursor of saposin B, also binds CoQ10. As prosaposin is both a secretory protein and integral membrane protein, it is ubiquitous in the body. Prosaposin was isolated from human seminal plasma, and CoQ10 was extracted from hexane solution into the water phase. It was additionally found that immunoprecipitates of mouse brain cytosol generated using two different anti-prosaposin antibodies contained coenzyme Q9. Furthermore, mouse liver cytosol and mouse kidney cytosol also contained prosaposin–coenzyme Q9 complex. These results suggest that prosaposin binds CoQ10 in human cells and body fluids. The significance and role of the Psap-CoQ10 complex in vivo is also discussed.
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GEOZS, IJS, IMTLJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBMB, UL, UM, UPUK
7.
Molybdenum cofactor biology, evolution and deficiency Mayr, Simon J.; Mendel, Ralf-R.; Schwarz, Guenter
Biochimica et biophysica acta. Molecular cell research,
January 2021, 2021-01-00, 20210101, Volume:
1868, Issue:
1
Journal Article
Peer reviewed
Open access
The molybdenum cofactor (Moco) represents an ancient metal‑sulfur cofactor, which participates as catalyst in carbon, nitrogen and sulfur cycles, both on individual and global scale. Given the ...diversity of biological processes dependent on Moco and their evolutionary age, Moco is traced back to the last universal common ancestor (LUCA), while Moco biosynthetic genes underwent significant changes through evolution and acquired additional functions. In this review, focused on eukaryotic Moco biology, we elucidate the benefits of gene fusions on Moco biosynthesis and beyond. While originally the gene fusions were driven by biosynthetic advantages such as coordinated expression of functionally related proteins and product/substrate channeling, they also served as origin for the development of novel functions. Today, Moco biosynthetic genes are involved in a multitude of cellular processes and loss of the according gene products result in severe disorders, both related to Moco biosynthesis and secondary enzyme functions.
•Molybdenum cofactor (Moco) biosynthesis is an ancient and highly conserved pathway.•Various gene fusions gave rise to novel protein functions.•Alternative splicing regulates MOCS1 and gephyrin properties.•The radical SAM enzyme MOCS1A receives 4Fe-4S in the cytosol and mitochondria.•Moco deficiency causes neurodegeneration via primary and secondary metabolic defects.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
In vitro biotransformation (ivBT) facilitated by in vitro synthetic enzymatic biosystems (ivSEBs) has emerged as a highly promising biosynthetic platform. Several ivSEBs have been constructed to ...produce poly-3-hydroxybutyrate (PHB) via acetyl-coenzyme A (acetyl-CoA). However, some systems are hindered by their reliance on costly ATP, limiting their practicality. This study presents the design of an ATP-free ivSEB for one-pot PHB biosynthesis via acetyl-CoA utilizing starch-derived maltodextrin as the sole substrate. Stoichiometric analysis indicates this ivSEB can self-maintain NADP
/NADPH balance and achieve a theoretical molar yield of 133.3%. Leveraging simple one-pot reactions, our ivSEBs achieved a near-theoretical molar yield of 125.5%, the highest PHB titer (208.3 mM, approximately 17.9 g/L) and the fastest PHB production rate (9.4 mM/h, approximately 0.8 g/L/h) among all the reported ivSEBs to date, and demonstrated easy scalability. This study unveils the promising potential of ivBT for the industrial-scale production of PHB and other acetyl-CoA-derived chemicals from starch.
In most organisms, transition metal ions are necessary cofactors of ribonucleotide reductase (RNR), the enzyme responsible for biosynthesis of the 2′-deoxynucleotide building blocks of DNA. The metal ...ion generates an oxidant for an active site cysteine (Cys), yielding a thiyl radical that is necessary for initiation of catalysis in all RNRs. Class I enzymes, widespread in eukaryotes and aerobic microbes, share a common requirement for dioxygen in assembly of the active Cys oxidant and a unique quaternary structure, in which the metallo- or radical-cofactor is found in a separate subunit, β, from the catalytic α subunit. The first class I RNRs, the class Ia enzymes, discovered and characterized more than 30 years ago, were found to use a diiron(III)-tyrosyl-radical Cys oxidant. Although class Ia RNRs have historically served as the model for understanding enzyme mechanism and function, more recently, remarkably diverse bioinorganic and radical cofactors have been discovered in class I RNRs from pathogenic microbes. These enzymes use alternative transition metal ions, such as manganese, or posttranslationally installed tyrosyl radicals for initiation of ribonucleotide reduction. Here we summarize the recent progress in discovery and characterization of novel class I RNR radical-initiating cofactors, their mechanisms of assembly, and how they might function in the context of the active class I holoenzyme complex.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Summary
Unlike bibenzyls derived from the vascular plants, lunularic acid (LA), a key precursor for macrocyclic bisbibenzyl synthesis in nonvascular liverworts, exhibits the absence of one hydroxy ...group within the A ring. It was hypothesized that both polyketide reductase (PKR) and stilbenecarboxylate synthase 1 (STCS1) were involved in the LA biosynthesis, but the underlined mechanisms have not been clarified.
This study used bioinformatics analysis with molecular, biochemical and physiological approaches to characterize STCS1s and PKRs involved in the biosynthesis of LA.
The results indicated that MpSTCS1s from Marchantia polymorpha catalyzed both C2→C7 aldol‐type and C6→C1 Claisen‐type cyclization using dihydro‐p‐coumaroyl‐coenzyme A (CoA) and malonyl‐CoA as substrates to yield a C6‐C2‐C6 skeleton of dihydro‐resveratrol following decarboxylation and the C6‐C3‐C6 type of phloretin in vitro. The protein–protein interaction of PKRs with STCS1 (PPI‐PS) was revealed and proved essential for LA accumulation when transiently co‐expressed in Nicotiana benthamiana. Moreover, replacement of the active domain of STCS1 with an 18‐amino‐acid fragment from the chalcone synthase led to the PPI‐PS greatly decreasing and diminishing the formation of LA. The replacement also increased the chalcone formation in STCS1s.
Our results highlight a previously unrecognized PPI in planta that is indispensable for the formation of LA.
See also the Commentary on this article by Davies & Andre, 237: 371–373.
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