Phage non-canonical DNA

Glycyl Radical Enzymes (GREs)

Pyrimidine degradation pathways

Purine degradation pathways

Sulfonate metabolism

Synthetic Biology

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Phage non-canonical DNA

Selected Papers

1. A widespread pathway for substitution of adenine by diaminopurine in phage genomes. Science 2021, 372(6541):512-516.

DNA modifications vary in form and function but generally do not alter Watson-Crick base pairing. In 1977 Kirnos et al. reported complete substitution of adenine (A) by diaminopurine (Z) in the genome of cyanophage S-2L. The biosynthesis, prevalence, and importance of Z genomes remain unexplored.

Here, we report a multienzyme system that supports Zgenome synthesis. We identified dozens of globally widespread phages harboring such enzymes, and we further verified the Z genome in one of these phages, Acinetobacter phage SH-Ab 15497, by using liquid chromatography with ultraviolet and mass spectrometry. The Z genome endows phages with evolutionary advantages for evading the attack of host restriction enzymes, and the characterization of its biosynthetic pathway enables dZ-DNA production on a large scale for a diverse range of applications.

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2.Nat Microbiol 2023, Jun 12.

Many bacteriophages evade bacterial immune recognition by substituting adenine with 2,6-diaminopurine (Z) in their genomes. The Z-genome biosynthetic pathway involves PurZ that belongs to the PurA (adenylosuccinate synthetase) family and bears particular similarity to archaeal PurA. However, how the transition of PurA to PurZ occurred during evolution is not clear; recapturing this process may shed light on the origin of Z-containing phages. Here we describe the computer-guided identifcation and biochemical characterization of a naturally existing PurZ variant, PurZ0, which uses guanosine triphosphate as the phosphate donor rather than the ATP used by PurZ. The atomic resolution structure of PurZ0 reveals a guanine nucleotide binding pocket highly analogous to that of archaeal PurA. Phylogenetic analyses suggest PurZ0 as an intermediate during the evolution of archaeal PurA to phage PurZ. Maintaining the balance of diferent purines necessitates further evolvement of guanosine triphosphate-using PurZ0 to ATP-using PurZ in adaptation to Z-genome life.

3.Biotechnol. Bioeng. 2023 DOI: 10.1002/bit.28642

2,6‐diaminopurine (Z), a naturally occurring noncanonical nucleotide base found in bacteriophages, enhances DNA hybridization by forming three hydrogen bonds with thymine (T). These distinct biochemical characteristics make it particularly valuable in applications that rely on the thermodynamics of DNA hybridization. However, the practical use of Z‐containing oligos is limited by their high production cost and the challenges associated with their synthesis. Here, we developed an efficient and costeffective approach to synthesize Z‐containing oligos of high quality based on an isothermal strand displacement reaction. These newly synthesized Z‐oligos are then employed as toehold‐blockers in an isothermal genotyping assay designed to detect rare single nucleotide variations (SNV). When compared with their counterparts containing the standard adenine (A) base, the Z‐containing blockers significantly enhance the accuracy of identifying SNV.

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Glycyl Radical Enzymes (GREs)

Selected Papers

1. Annual Rev Biochem 2021, 90:817-846.

Sulfonates include diverse natural products and anthropogenic chemicals and are widespread in the environment. Many bacteria can degrade sulfonates and obtain sulfur, carbon, and energy for growth, playing important roles in the biogeochemical sulfur cycle. Cleavage of the inert sulfonate C–S bond involves a variety of enzymes, cofactors, and oxygen-dependent and oxygen-independent catalytic mechanisms. Sulfonate degradation by strictly anaerobic bacteria was recently found to involve C–S bond cleavage through O2-sensitive free radical chemistry, catalyzed by glycyl radical enzymes (GREs). The associated discoveries of new enzymes and metabolic pathways for sulfonate metabolism in diverse anaerobic bacteria have enriched our understanding of sulfonate chemistry in the anaerobic biosphere. An anaerobic environment of particular interest is the human gut microbiome where sulfonate degradation by sulfate- and sulfite-reducing bacteria (SSRB) produces H₂S, a process linked to certain chronic diseases and conditions.

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2. Nat. Comm. 2018 Oct 11;9(1):4224.

Here we report the use of comparative genomics for the discovery of indoleacetate decarboxylase, an O₂-sensitive glycyl radical enzyme catalysing the decarboxylation of indoleacetate to form skatole as the terminal step of tryptophan fermentation in certain anaerobic bacteria. We describe its biochemical characterization and compare it to other glycyl radical decarboxylases. Indoleacetate decarboxylase may serve as a genetic marker for the identification of skatole-producing environmental and human-associated bacteria, with impacts on human health and the livestock industry.

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4. Proc Natl Acad Sci U S A. 2020 Jul 7;117(27):15599-15608.

Here, we report the discovery and charac- terization of two O₂-sensitive glycyl radical enzymes that use distinct mechanisms for DHPS degradation. DHPS-sulfolyase (HpsG) in sulfate- and sulfite-reducing bacteria catalyzes C–S cleavage to release sulfite for use as a terminal electron acceptor in respiration, producing H₂S. DHPS-dehydratase (HpfG), in fermenting bacteria, catalyzes C–O cleavage to generate 3-sulfopropionaldehyde, subsequently reduced by the NADH-dependent sulfopropionaldehyde reductase (HpfD).

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5. ACS Catalysis 2021, 11(9) 5789–5794.

Here, we report the discovery and structural and biochemical investigation of a fourth GRE arylacetate decarboxylase (AAD) from Olsenella scatoligenes that catalyzes HPA decarboxylation. AAD also catalyzes the decarboxylation of p-aminophenylacetate, which is not a substrate of HPAD, and lacks the Fe−S cluster containing small subunit.

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6. J. Am. Chem. Soc. 2022, 144, 22, 9715–9722

Here, we report a pathway for anaerobic hydroxyproline degradation that involves a new GRE, trans-4-hydroxy-D-proline (t4D-HP) C−N-lyase (HplG). In this pathway, cis-4-hydroxy-L-proline (c4L HP)isfirstisomerized to t4D-HP, followed by radical-mediated ring opening by HplG to give 2-amino-4-ketopentanoate (AKP), the first example of a ring opening reaction catalyzed by a GRE 1,2-eliminase. Subsequent cleavage by AKP thiolase (OrtAB) yields acetyl-CoA and D-alanine.

7.ACS Catal. 2024 DOI: 10.1021/acscatal.4c00216

Here, we report the discovery of two more GREs, N-methyl c4L-HP dehydratase (HpyG) and N-methyl c4D-HP dehydratase (HpzG), which catalyze radical-mediated dehydration of the two N-methyl-c4HP enantiomers, while also displaying significant activities toward their unmethylated substrates. Both GREs are associated with homologues of pyrroline-5-carboxylate reductase, which catalyze reduction of their products N-methylpyrroline-5-carboxylate to form N-methyl-proline.

8.J Am Chem Soc. 2024 DOI: 10.1021/jacs.4c07718

Here, we report that YbiW and PflD catalyze ring-opening C−O cleavage of 1,5-anhydroglucitol-6-phosphate (AG6P) and 1,5-anhydromannitol-6-phosphate (AM6P), respectively. The product of both enzymes, 1-deoxy-fructose-6-phosphate (DF6P), is then cleaved by the aldolases FsaA or FsaB to form glyceraldehyde-3-phosphate (G3P) and hydroxyacetone (HA), which are then reduced by the NADH-dependent dehydrogenase GldA to form 1,2-propanediol (1,2-PDO). Crystal structures of YbiW and PflD in complex with their substrates provided insights into the mechanism of radical-mediated C−O cleavage. This “anhydroglycolysis” pathway enables anaerobic growth of E. coli on 1,5-anhydroglucitol (AG) and 1,5-anhydromannitol (AM), and we probe the feasibility of harnessing this pathway for the production of 1,2-PDO, a highly demanded chiral chemical feedstock, from inexpensive starch.

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Pyrimidine degradation pathways

Selected Papers

1. J Biol Chem. 2019 Oct 25; 294(43): 15662-15671.

Here, we report the biochemical characterization of a β-alanine:2-oxoglutarate aminotransferase (PydD) and a NAD(P)H-dependent malonic semialdehyde reductase (PydE) from a pyrimidine degradation gene cluster in the bacterium Lysinibacillus massiliensis.

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2. Appl & Environ Microbiol. 2020 Mar 18;86(7):e02837-19.

Here we report the characterization of a β-alanine:pyruvate aminotransferase (PydD2), and a NAD+-dependent malonic semialdehyde dehydrogenase (MSDH), from a reductive pyrimidine catabolism gene cluster in Bacillus megaterium.

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3. Biosci Rep. 2020 Jul 31;40(7):BSR20201642.

Here, we report the biochemical characterization of a Clostridial PydA homolog (PydAc) from a Pyd gene cluster in the strict anaerobic bacterium Clostridium chromiireducens.

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4. ACS Catalysis 2021, 11(14), 8895–8901.

The URC (uracil catabolism) pathway, present in fungi and bacteria, enables pyrimidines to be used as nitrogen sources for growth. Although its mechanistic details are unclear, uracil is thought to be first converted into a uridine nucleotide, followed by pyrimidine ring cleavage catalyzed by Urc1p, a distant homologue of GTP cyclohydrolase II (RibA). Here we report a biochemical investigation of Urc1p from Pichia pastoris (Komagataella phaffii) (PpUrc1p) and its bacterial homologue from Rhodococcus wratislaviensis (RwUrcA). Like RibA, the substrate of recombinant PpUrc1p was found to be a nucleoside triphosphate (UTP), and turnover was accompanied by the release of pyrophosphate. The products phosphoribosylurea and malonic semialdehyde were confirmed by mass spectrometry. We also determined a 1.60 Å crystal structure of RwUrcA, which included a C-terminal segment that is not resolved in RibA structures reported to date, containing a conserved Lys residue positioned to interact with the UTP α-phosphate. Further mutagenesis studies revealed roles of active site residues in substrate binding and catalysis, providing insights into the mechanism by which Urc1p catalyzes two sequential hydrolyses at C6 and C4 for the uracil ring.

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Purine degradation pathways

Selected Works

1. Cell Chemical Biology. 2023 July 20; 30: 1–11.

Here we report the identification and characterization of these enzymes, which include four hydrolases belonging to different enzyme families, and a prenyl-flavin mononucleotide-dependent decarboxylase. Introduction of the first two hydrolases to Escherichia coli Nissle 1917 enabled its anaerobic growth on xanthine as the sole nitrogen source. Oral supplementation of these engineered probiotics ameliorated hyperuricemia in a Drosophila melanogaster model, including the formation of renal uric acid stones and a shortened lifespan, providing a route toward the development of purinolytic probiotics.

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Sulfonate metabolism

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Selected Papers

1. Biochem J. 2019 Feb 28;476(4):733-746.

Sulfoacetaldehyde reductase (IsfD) is a member of the short-chain dehydrogenase/reduc- tase (SDR) family, involved in nitrogen assimilation from aminoethylsulfonate (taurine) in certain environmental and human commensal bacteria.

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2. Biochem J. 2019 Jun 11;476(11):1605-1619.

Here we report the biochemical and structural characterization of a new taurine:2-oxoglutarate aminotransferase from the human gut bacterium Bifidobacterium kashiwanohense (BkToa).

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3. Biosci Rep. 2019 Jun 20;39(6).

Here we report the structural and biochemical characterization of a sulfoacetaldehyde reductase from the human gut fermenting bacterium Bifidobacterium kashiwanohense (BkTauF). 

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4. Biochem J. 2019 Aug 15;476(15):2271-2279.

Here we report the biochemical characterization of an anaerobic pathway for taurine sulfur assimilation in a strain of Clostridium butyricum from the human gut. In this pathway, taurine is first converted to hydroxyethylsulfonate (isethionate), followed by C–S cleavage by the O₂-sensitive isethionate sulfolyase IseG, recently identified in sulfate- and sulfite-reducing bacteria.

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6. ACS Catalysis 2021, 11: 14740-14750.

Here, we report the discovery of three additional sulfoglycolysis pathways, the first involving a transketolase (sulfo-TK) related to that in the pentose phosphate pathway, the second involving oxygenolytic C−S cleavage of SQ by a flavin-dependent alkanesulfonate monooxygenase (sulfo-ASMO), and the third being a variant of the sulfo-EMP pathway in Gram-positive bacteria (sulfo-EMP2). Our findings underscore the diversity of mechanisms through which bacteria degrade and utilize this ubiquitous organosulfur compound as a nutrient source.

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7. Biochemistry 2022 DOI: 10.1021/acs.biochem.2c00102.

Here we report the identification and biochemical characterization of the capnine biosynthetic enzymes cysteate synthase (CapA) and cysteate-C-fatty acyltransferase (CapB) from the pathogenic gliding bacterium Capnocytophaga ochracea and NAD(P)H-dependent dehydrocapnine reductase CapC from the avian pathogen Ornithobacterium rhinotracheale.

8. Appl Environ Microbiol. 2023 DOI: 10.1128/aem.00617-23

Here, we describe a gene cluster in an Acholeplasma sp., from a metagenome derived from deeply circulating subsurface aquifer fluids (GenBank accession no. QZKD01000037), encoding a variant of the recently discovered sulfoglycolytic transketolase (sulfo-TK) pathway that produces sulfoacetate instead of isethionate as a by-product. We report the biochemical characterization of a coenzyme A (CoA)-acylating sulfoacetaldehyde dehydrogenase (SqwD) and an ADP-forming sulfoacetate-CoA ligase (SqwKL), which collectively catalyze the oxidation of the transketolase product sulfoacetaldehyde into sulfoacetate, coupled with ATP formation.

9.J Biol Chem. 2023 DOI: 10.1016/j.jbc.2023.105010

Here, we report bioinformatics investigations and in vitro biochemical assays that uncover the molecular basis for the utilization of sulfoacetate as a source of TEA (STEA) for B. wadsworthia, involving conversion to sulfoacetyl-CoA by an ADP-forming sulfoacetate-CoA ligase (SauCD), and stepwise reduction to isethionate by NAD(P)H-dependent enzymes sulfoacetaldehyde dehydrogenase (SauS) and sulfoacetaldehyde reductase (TauF). Isethionate is then cleaved by the O2-sensitive isethionate sulfolyase (IseG), releasing sulfite for dissimilatory reduction to H2S.

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10. iScience.2023 DOI: 10.1016/j.isci.2023.107803

Here we report bioinformatics and biochemical studies revealing an alternative mechanism for oxygenolytic cleavage of the SQ C-S bond, catalyzed by an iron and a-ketoglutarate-dependent alkanesulfonate dioxygenase (SqoD, sulfo-ASDO pathway). In both the ASMO and ASDO pathways, the product 6-dehydroglucose is reduced to glucose by NAD(P)H-dependent SquF.

11.J Biol Chem. 2024 DOI: 10.1016/j.jbc.2024.107371

Here we report a pathway for L-cysteate dissimilation in B. wadsworthia RZATAU, involving isomerization of L-cysteate to D-cysteate by a cysteate racemase (BwCuyB), followed by cleavage into pyruvate, ammonia and sulfite by a D-cysteate sulfo-lyase (BwCuyA). The strong selectivity of BwCuyA for D-cysteate over L-cysteate was rationalized by protein structural modeling. A homolog of BwCuyA in the marine bacterium Silicibacter pomeroyi (SpCuyA) was previously reported to be a L-cysteate sulfo-lyase, but our experiments confirm that SpCuyA too displays a strong selectivity for D-cysteate.

12. iScience. 2024 DOI: 10.1016/j.isci.2024.111010

Here, we report a variant sulfo-TAL pathway in Enterococcus gilvus, involving additional enzymes, a NAD+-dependent 3-SPA dehydrogenase HpfX, and a 3-sulfopropionyl-CoA synthetase HpfYZ, which oxidize 3-SPA to 3-sulfopropionate (3-SP) coupled with ATP formation. E. gilvus grown on SQ or DHPS produced a mixture of 3-HPS and 3-SP, indicating the bifurcated pathway. Similar genes are found in various Firmicutes, including gut bacteria. Importantly, 3-SP, but not 3-HPS, can serve as a respiratory terminal electron acceptor for Bilophila wadsworthia, a common intestinal pathobiont, resulting in the production of toxic H2S.

Synthetic Biology

Selected Works

1. ACS Synth. Biol. 2019 Aug 16;8(8):1698-1704.

Rose has been entwined with human culture and history. “Blue rose” in English signifies unattainable hope or an impossible mission as it does not exist naturally and is not breedable regardless of centuries of effort by gardeners. With the knowledge of genes and enzymes involved in flower pigmentation and modern genetic technologies, synthetic biologists have undertaken the challenge of producing blue rose by engineering the complicated vacuolar flavonoid pigmentation pathway and resulted in a mauve-colored rose. A completely different strategy presented in this study employs a dual expression plasmid containing bacterial idgS and sfp genes. The holoIdgS, activated by Sfp from its apo-form, is a functional nonribosomal
peptide synthetase that converts L-glutamine into the blue pigment indigoidine. Expression of these genes upon petal injection with agroinfiltration solution generates blue-hued rose flowers. We envision that implementing this proof-of-concept with obligatory modifications may have tremendous impact in floriculture to achieve a historic milestone in rose breeding.

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2. ChemBioChem 2022 DOI：10.1002/cbic.202200295

Here we report the enzymatic conversion of 2’-deoxyxanthosine 5’-monophosphate (dXMP) intoACS Catalysis 2021, 11(9) 5789–5794.deoxyisoguanosine monophosphate (dBMP), a precursor of the unnatural isoguanine-isocytosine base pair. The reaction is catalyzed by the bacteriophage enzyme PurZ and bacterial PurB, and is a key addition to the toolbox for de novo biosynthesis of unnatural DNA.