Isonitrile Formation by a Non‐Heme Iron(II)‐Dependent Oxidase/Decarboxylase

Harris, Nicholas C.; Born, David A.; Cai, Wenlong; Huang, Yao‐Bing; Martin, Joelle; Khalaf, Ryan; Drennan, Catherine L.; Zhang, Wenjun

doi:10.1002/ange.201804307

Cited by 8 publications

(7 citation statements)

References 18 publications

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“…Identical fragmentation patterns by LC-MS/MS analysis of these metabolites were observed (Figure S7) indicating that these compounds are isomers. Second, click reactions with tetrazine confirmed the presence of unique isonitrile functionality in the compounds (45) (Fig. 5B-C).…”

Section: Resultsmentioning

confidence: 73%

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Adaptation of Mycobacterium tuberculosis to biofilm growth is genetically linked to drug tolerance

Richards

Cai

Zill

et al. 2019

Preprint

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21Mycobacterium tuberculosis (Mtb) spontaneously grows at the air-medium interface 22 forming pellicle biofilms, which harbor more drug tolerant persisters than planktonic 23 cultures. The underlying basis for increased persisters in Mtb biofilms is unknown. 24 Using a Tn-seq approach, we show here that multiple genes that are necessary for 25 fitness of Mtb cells within biofilms, but not in planktonic cultures, are also important for 26 their tolerance to a diverse set of stressors and antibiotics. Thus, development of Mtb 27 biofilms appears to be associated with population enrichment, in which endogenous 28 stresses presumably generated by challenging growth conditions within biofilm 29 architecture select for cells that maintain tolerance to exogenous stresses including 30 antibiotic exposure. We further observed that the intrinsic drug tolerance of constituent 31 cells of biofilms determines the frequency of persisters: morphologically 32 indistinguishable monoculture biofilms of a ΔpstC2A1 mutant hypersensitive to 33 rifampicin harbor ~20-fold fewer persisters than wild-type. These findings together allow 34 us to propose that the selection of elite cells during biofilm development significantly 35 contributes to the persister frequency. Furthermore, probing the possibility that the 36 population enrichment is an outcome of unique environment within biofilms, we 37 demonstrate biofilm-specific induction in the synthesis of isonitrile lipopeptides (INLP). 38 Mutation analysis indicates that INLP is necessary for the architecture development of 39 Mtb biofilms. In summary, the study offers an insight into persistence of Mtb biofilms 40 under antibiotic exposure, while identifying INLP as a biomarker for further investigation 41 of this phenomenon. 42 3 SIGNIFICANCE 43 The tuberculosis (TB) pathogen Mycobacterium tuberculosis (Mtb) is one of the 44 deadliest bacterial pathogens known to mankind, and TB treatment is inefficient. A 45 lengthy chemotherapy for TB is attributed to a small subpopulation of Mtb bacilli 46 exhibiting phenotypic tolerance to antibiotics. Drugs targeting these persisters are 47 55 Treatment of tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), entails a 56 multi-drug regimen administered for at least 6 months. A lengthy treatment is 57presumably necessitated by the persistence of a small subpopulation of bacilli, which 58 exhibit phenotypic tolerance to antibiotics (1, 2). Although the mechanism underlying the 59 development of Mtb persisters during infection is unclear, in vitro studies suggest that 60 these bacilli probably develop through both stochastic and induced mechanisms(3-6). 61The persistence of microbes against antibiotics has been closely linked to their 62 ability to grow as sessile, three-dimensionally organized, matrix encapsulated, 63 multicellular communities called biofilms (7-11). Biofilm-related antibiotic tolerance is 64 rendered particularly relevant by the fact that a majority of chronic microbial infections in 65 humans occur as biofi...

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Section: Resultsmentioning

confidence: 73%

“…Third, weak but noticable incoporation of 13 C-glycine was observed in these four metabolites (Figure S8). Glycine is a direct precursor utilized by INLPS (43, 45). Therefore its incorporation in 1-4 is in agreement with the characterized biosynthetic mechanism for INLP synthesis.…”

Section: Resultsmentioning

confidence: 99%

Adaptation of Mycobacterium tuberculosis to biofilm growth is genetically linked to drug tolerance

Richards

Cai

Zill

et al. 2019

Preprint

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“…A crystal structure (Fig. 1) was resolved at 1.8 Å resolution on a Zn(II)-containing and substrate-devoid system, which structurally resembles the well-characterized active site of taurine/αKG dependent dioxygenase [45,46] and hence was identified as a nonheme iron dioxygenase.…”

Section: Introductionmentioning

confidence: 99%

“…Both substrate and αKG are missing from the pdb file, but acetate is bound and likely is in the position of αKG. The substrate binding pocket is empty, but the Arg 310 side chain has been hypothesized [45] to be involved in substrate binding and positioning in the active site. In addition, a number of aromatic residues lines the substrate binding pocket, namely Phe 110 , Phe 137 and Phe 239 .…”

Section: Introductionmentioning

confidence: 99%

Density Functional Theory Study into the Reaction Mechanism of Isonitrile Biosynthesis by the Nonheme Iron Enzyme ScoE

2021

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The nonheme iron enzyme ScoE catalyzes the biosynthesis of an isonitrile substituent in a peptide chain. To understand details of the reaction mechanism we created a large active site cluster model of 212 atoms that contains substrate, the active oxidant and the first- and second-coordination sphere of the protein and solvent. Several possible reaction mechanisms were tested and it is shown that isonitrile can only be formed through two consecutive catalytic cycles that both use one molecule of dioxygen and α-ketoglutarate. In both cycles the active species is an iron(IV)-oxo species that in the first reaction cycle reacts through two consecutive hydrogen atom abstraction steps: first from the N–H group and thereafter from the C–H group to desaturate the NH-CH2 bond. The alternative ordering of hydrogen atom abstraction steps was also tested but found to be higher in energy. Moreover, the electronic configurations along that pathway implicate an initial hydride transfer followed by proton transfer. We highlight an active site Lys residue that is shown to donate charge in the transition states and influences the relative barrier heights and bifurcation pathways. A second catalytic cycle of the reaction of iron(IV)-oxo with desaturated substrate starts with hydrogen atom abstraction followed by decarboxylation to give isonitrile directly. The catalytic cycle is completed with a proton transfer to iron(II)-hydroxo to generate the iron(II)-water resting state. The work is compared with experimental observation and previous computational studies on this system and put in a larger perspective of nonheme iron chemistry.

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“…[1,4] Deoxyribonuclease and ovalbumin, for example, have identical isoelectricp oints of pI = 5.1, but the formal and measured net charge of both proteins differ by approximately 7u nits at pH 8.4. [4] The systematic absence of experimentally determinedv alues of Z has likely impeded ar igorous understanding of most chemicalp rocesses in which proteinsa re involved including aggregation and self-assembly, [20][21][22][23][24][25][26] ligand binding, [27][28][29][30][31][32][33][34] catalysis, [35][36][37][38][39] electron transfer, [3,6,[40][41][42][43][44][45][46][47] protein crystallization, [14,48] analytical separation, [49,50] and protein engineering. [51][52][53][54][55][56] It is tempting to assume that the formal net chargeo faprotein predicted from generalized residue pK a values (Z seq )issosimilar to the actual net charge that any difference is irrelevant, and the isoelectric point tells us all we need to know about ap rotein's net charge.…”

Section: Introductionmentioning

confidence: 99%

What Are We Missing by Not Measuring the Net Charge of Proteins?

Zahler

Shaw

2019

Chemistry A European J

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The net electrostatic charge (Z) of a folded protein in solution represents a bird's eye view of its surface potentials—including contributions from tightly bound metal, solvent, buffer, and cosolvent ions—and remains one of its most enigmatic properties. Few tools are available to the average biochemist to rapidly and accurately measure Z at pH≠pI. Tools that have been developed more recently seem to go unnoticed. Most scientists are content with this void and estimate the net charge of a protein from its amino acid sequence, using textbook values of pKa. Thus, Z remains unmeasured for nearly all folded proteins at pH≠pI. When marveling at all that has been learned from accurately measuring the other fundamental property of a protein—its mass—one wonders: what are we missing by not measuring the net charge of folded, solvated proteins? A few big questions immediately emerge in bioinorganic chemistry. When a single electron is transferred to a metalloprotein, does the net charge of the protein change by approximately one elementary unit of charge or does charge regulation dominate, that is, do the pKa values of most ionizable residues (or just a few residues) adjust in response to (or in concert with) electron transfer? Would the free energy of charge regulation (ΔΔGz) account for most of the outer sphere reorganization energy associated with electron transfer? Or would ΔΔGz contribute more to the redox potential? And what about metal binding itself? When an apo‐metalloprotein, bearing minimal net negative charge (e.g., Z=−2.0) binds one or more metal cations, is the net charge abolished or inverted to positive? Or do metalloproteins regulate net charge when coordinating metal ions? The author's group has recently dusted off a relatively obscure tool—the “protein charge ladder”—and used it to begin to answer these basic questions.

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Isonitrile Formation by a Non‐Heme Iron(II)‐Dependent Oxidase/Decarboxylase

Cited by 8 publications

References 18 publications

Adaptation of Mycobacterium tuberculosis to biofilm growth is genetically linked to drug tolerance

Adaptation of Mycobacterium tuberculosis to biofilm growth is genetically linked to drug tolerance

Density Functional Theory Study into the Reaction Mechanism of Isonitrile Biosynthesis by the Nonheme Iron Enzyme ScoE

What Are We Missing by Not Measuring the Net Charge of Proteins?

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