T4 DNA ligase structure reveals a prototypical ATP-dependent ligase with a unique mode of sliding clamp interaction

Shi, Ke; Bohl, Thomas E.; Park, Jeonghyun; Zasada, Andrew; Malik, Shray; Banerjee, Surajit; Tran, Vincent; Li, Na; Yin, Zhiqi; Kurniawan, Fredy; Orellana, Kayo; Aihara, Hideki

doi:10.1093/nar/gky776

Cited by 61 publications

(68 citation statements)

References 75 publications

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“…In this structure, the terminal hydroxyl group of Fo is ideally positioned for nucleophilic attack of the ␤-phosphate of EPPG, strongly suggesting that DH-F 420 -0 biosynthesis occurs through direct transfer of PEP to Fo, via a pentavalent phosphate intermediate that is stabilized by the catalytic metal ion. The positioning of the Fo terminal hydroxyl group in our structure in relation to EPPG is strikingly similar to that of the attacking ribose in the final step in DNA ligation by T4 ligase, recently resolved by X-ray crystallography (33). This is consistent with both reactions resulting in the formation of a phosphodiester bond through direct nucleophilic attack of a diphosphonucleoside intermediate.…”

Section: Discussionsupporting

confidence: 84%

Cellular and Structural Basis of Synthesis of the Unique Intermediate Dehydro-F ₄₂₀ -0 in Mycobacteria

et al. 2020

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F420 is a low-potential redox cofactor used by diverse bacteria and archaea. In mycobacteria, this cofactor has multiple roles, including adaptation to redox stress, cell wall biosynthesis, and activation of the clinical antitubercular prodrugs pretomanid and delamanid. A recent biochemical study proposed a revised biosynthesis pathway for F420 in mycobacteria; it was suggested that phosphoenolpyruvate served as a metabolic precursor for this pathway, rather than 2-phospholactate as long proposed, but these findings were subsequently challenged. In this work, we combined metabolomic, genetic, and structural analyses to resolve these discrepancies and determine the basis of F420 biosynthesis in mycobacterial cells. We show that, in whole cells of Mycobacterium smegmatis, phosphoenolpyruvate rather than 2-phospholactate stimulates F420 biosynthesis. Analysis of F420 biosynthesis intermediates present in M. smegmatis cells harboring genetic deletions at each step of the biosynthetic pathway confirmed that phosphoenolpyruvate is then used to produce the novel precursor compound dehydro-F420-0. To determine the structural basis of dehydro-F420-0 production, we solved high-resolution crystal structures of the enzyme responsible (FbiA) in apo-, substrate-, and product-bound forms. These data show the essential role of a single divalent cation in coordinating the catalytic precomplex of this enzyme and demonstrate that dehydro-F420-0 synthesis occurs through a direct substrate transfer mechanism. Together, these findings resolve the biosynthetic pathway of F420 in mycobacteria and have significant implications for understanding the emergence of antitubercular prodrug resistance. IMPORTANCE Mycobacteria are major environmental microorganisms and cause many significant diseases, including tuberculosis. Mycobacteria make an unusual vitamin-like compound, F420, and use it to both persist during stress and resist antibiotic treatment. Understanding how mycobacteria make F420 is important, as this process can be targeted to create new drugs to combat infections like tuberculosis. In this study, we show that mycobacteria make F420 in a way that is different from other bacteria. We studied the molecular machinery that mycobacteria use to make F420, determining the chemical mechanism for this process and identifying a novel chemical intermediate. These findings also have clinical relevance, given that two new prodrugs for tuberculosis treatment are activated by F420.

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Section: Discussionsupporting

confidence: 84%

Cellular and Structural Basis of Synthesis of the Unique Intermediate Dehydro-F ₄₂₀ -0 in Mycobacteria

et al. 2020

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“…In this structure, the terminal hydroxyl group of Fo is ideally positioned for nucleophilic attack of the β-phosphate of EPPG, strongly suggesting that DH-F 420 -0 biosynthesis occurs through direct transfer of PEP to Fo, via a pentavalent phosphate intermediate that is stabilized by the catalytic metal ion. The positioning of the Fo terminal hydroxyl group in our structure in relation to EPPG is strikingly similar to that of the attacking ribose in the final step in DNA ligation by T4 ligase, recently resolved by X-ray crystallography (34). This is consistent with both reactions resulting in the formation of a phosphodiester bond through direct nucleophilic attack of a diphosphonucleoside intermediate.…”

Section: Discussionsupporting

confidence: 72%

Cellular and structural basis of synthesis of the unique intermediate dehydro-F₄₂₀-0 in mycobacteria

Grinter

Ney

Brammananth

et al. 2020

Preprint

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28F420 is a low-potential redox cofactor used by diverse bacteria and archaea. In mycobacteria, 29 this cofactor has multiple roles, including adaptation to redox stress, cell wall biosynthesis, and 30 activation of the clinical antitubercular prodrugs pretomanid and delamanid. A recent 31 biochemical study proposed a revised biosynthesis pathway for F420 in mycobacteria; it was 32 suggested that phosphoenolpyruvate served as a metabolic precursor for this pathway, rather 33 than 2-phospholactate as long proposed, but these findings were subsequently challenged. In 34 this work, we combined metabolomic, genetic, and structural analyses to resolve these 35 discrepancies and determine the basis of F420 biosynthesis in mycobacterial cells. We show that, 36 in whole cells of Mycobacterium smegmatis, phosphoenolpyruvate rather than 2-37 phospholactate stimulates F420 biosynthesis. Analysis of F420 biosynthesis intermediates 38 present in M. smegmatis cells harboring genetic deletions at each step of the biosynthetic 39 pathway confirmed that phosphoenolpyruvate is then used to produce the novel precursor 40 compound dehydro-F420-0. To determine the structural basis of dehydro-F420-0 production, we 41 solved high-resolution crystal structures of the enzyme responsible (FbiA) in apo, substrate, 42 and product bound forms. These data show the essential role of a single divalent cation in 43 coordinating the catalytic pre-complex of this enzyme and demonstrate that dehydro-F420-0 44 synthesis occurs through a direct substrate transfer mechanism. Together, these findings 45 resolve the biosynthetic pathway of F420 in mycobacteria and have significant implications for 46 understanding the emergence of antitubercular prodrug resistance. 47 48 49 50 51 52 53 54 55 56Factor 420 (F420) is a deazaflavin cofactor that mediates diverse redox reactions in bacteria and 57 archaea (1). Chemically, F420 consists of a redox-active deazaflavin headgroup (derived from 58 the chromophore Fo) that is conjugated to a variable-length polyglutamate tail via a 59 phosphoester linkage (2). While the Fo headgroup of F420 superficially resembles flavins (e.g. 60FAD, FMN), three chemical substitutions in the isoalloxazine ring give it distinct chemical 61 properties more reminiscent of nicotinamides (e.g. NADH, NADPH) (1). These include a low 62 standard potential (-350 mV) and obligate two-electron (hydride) transfer chemistry (3, 4). The 63 electrochemical properties of F420 make it ideal to reduce a wide range of otherwise 64 recalcitrant organic compounds (5-7). Diverse prokaryotes are known to synthesize F420, but 65 the compound is best characterised for its roles in methanogenesis in archaea, antibiotic 66 biosynthesis in streptomycetes, and metabolic adaptation of mycobacteria (1,(8)(9)(10)(11). In 67 mycobacteria, F420 is involved in a plethora of processes: central carbon metabolism, cell wall 68 synthesis, recovery from dormancy, resistance to oxidative stress, and inactivation of certain 69 bactericidal agents (7,(12)(13)(1...

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“…Although this study was intended to address the concern of the long linker length used during LOOPER, this study has also indirectly highlighted the delicate balance of LOOPER. Although new insights into the observed limitations of LOOPER are expected to come to light with the recently solved crystal structure of T4 DNA ligase, practitioners of the method are encouraged, to conduct duplex DNA sequencing when adjusting chemical modifications, linker length, or codon set sequence beyond reported specifications before pursuing in vitro selections.…”

Section: Resultsmentioning

confidence: 99%

Influence of Linker Length on Ligase‐Catalyzed Oligonucleotide Polymerization

2019

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Ligase‐catalyzed oligonucleotide polymerization (LOOPER) that enables the sequence‐defined generation of DNA with up to 16 different modifications has recently been developed. This approach was used to develop new classes of diversely modified DNA aptamers for molecular recognition through in vitro evolution. The modifications in LOOPER are appended by use of a long hexane‐1,6‐diamine linker, which could negatively impact binding thermodynamics. Here we explore the incorporation of modifications with the aid of shorter linkers and the use of commercially available phosphoramidites and assess their efficiency and fidelity of incorporation. We observed that shorter linkers are less tolerated during LOOPER, with very short linkers providing high levels of error and sequence bias. An ethane‐1,2‐diamine linker was found to be optimal in terms of yield, efficiency, and bias; however, codon adjustment was necessary. This shorter‐linker anticodon set for LOOPER should prove valuable in exploring the impact of diverse chemical modifications on the molecular function of DNA.

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T4 DNA ligase structure reveals a prototypical ATP-dependent ligase with a unique mode of sliding clamp interaction

Cited by 61 publications

References 75 publications

Cellular and Structural Basis of Synthesis of the Unique Intermediate Dehydro-F ₄₂₀ -0 in Mycobacteria

Cellular and Structural Basis of Synthesis of the Unique Intermediate Dehydro-F ₄₂₀ -0 in Mycobacteria

Cellular and structural basis of synthesis of the unique intermediate dehydro-F₄₂₀-0 in mycobacteria

Influence of Linker Length on Ligase‐Catalyzed Oligonucleotide Polymerization

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T4 DNA ligase structure reveals a prototypical ATP-dependent ligase with a unique mode of sliding clamp interaction

Cited by 61 publications

References 75 publications

Cellular and Structural Basis of Synthesis of the Unique Intermediate Dehydro-F 420 -0 in Mycobacteria

Cellular and Structural Basis of Synthesis of the Unique Intermediate Dehydro-F 420 -0 in Mycobacteria

Cellular and structural basis of synthesis of the unique intermediate dehydro-F420-0 in mycobacteria

Influence of Linker Length on Ligase‐Catalyzed Oligonucleotide Polymerization

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Cellular and Structural Basis of Synthesis of the Unique Intermediate Dehydro-F ₄₂₀ -0 in Mycobacteria

Cellular and Structural Basis of Synthesis of the Unique Intermediate Dehydro-F ₄₂₀ -0 in Mycobacteria

Cellular and structural basis of synthesis of the unique intermediate dehydro-F₄₂₀-0 in mycobacteria