The genomes of broad host range insect pathogenic fungi, including
A
These data reveal host‐dependent effects of a protease implicated in cuticle degradation on
Chondroitin sulfates are the glycosaminoglycan chains of proteoglycans critical in the normal development and pathophysiology of all animals. Chondroitinase ACII, a polysaccharide lyase originally isolated from
- NSF-PAR ID:
- 10040229
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Biotechnology Journal
- Volume:
- 12
- Issue:
- 10
- ISSN:
- 1860-6768
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract BACKGROUND Cordyceps fumosorosea , encode for a suite of secreted proteases implicated in targeting, penetration, and degradation of the host exoskeleton or cuticle. These cuticle‐degrading proteases act as critical virulence factors, but their functions within the biological context, particularly in relation to host specificity, remain poorly characterized.RESULT C. fumosorosea protease gene,Cfcdp1 , was identified and a targeted gene‐knockout strain constructed. Minor growth defects were observed for theΔCfcdp1 strain when compared to the wild‐type parent and complemented (ΔCfcdp1::Cfcdp1 ) strains, with delayed and decreased sporulation noted for the mutant. Decreased subtilisin‐like protease activity was seen for theΔCfcdp1 strain, although total secreted protease activity was similar between the mutant and wild‐type strains. Insect bioassays using whitefly,Bemisia tabaci , and cabbageworm,Pieris rapae , showed decreased infectivity, i.e. 2.4–3.4‐fold increase in lethal dose (LC50) and an increased time to death (LT50), for theΔCfcdp1 strain. In contrast, insect bioassays using the diamondback moth,Plutella xylostella , or the brown planthopper,Nilaparvata lugens , showed increased infectivity, i.e. a 3–5‐fold decrease in LC50, and a decreased LT50. Differential effects were also seen on the fecundity ofB. tabaci infected by the different fungal strains.CONCLUSION C. fumosorosea virulence. The implications of these findings in suggesting context‐dependent requirements of cuticle‐degrading enzymes and their potentially differential roles in mediating virulence towards different hosts are discussed. © 2019 Society of Chemical Industry -
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Chondroitin sulfates (CSs) are linear glycosaminoglycans that have important applications in the medical and food industries. Engineering bacteria for the microbial production of CS will facilitate a one‐step, scalable production with good control over sulfation levels and positions in contrast to extraction from animal sources. To achieve this goal,
Escherichia coli (E. coli ) is engineered in this study using traditional metabolic engineering approaches to accumulate 3′‐phosphoadenosine‐5′‐phosphosulfate (PAPS), the universal sulfate donor. PAPS is one of the least‐explored components required for the biosynthesis of CS. The resulting engineeredE. coli strain shows an ≈1000‐fold increase in intracellular PAPS concentrations. This study also reports, for the first time, in vitro biotransformation of CS using PAPS, chondroitin, and chondroitin‐4‐sulfotransferase (C4ST), all synthesized from different engineeredE. coli strains. A 10.4‐fold increase is observed in the amount of CS produced by biotransformation by employing PAPS from the engineered PAPS‐accumulating strain. The data from the biotransformation experiments also help evaluate the reaction components that need improved production to achieve a one‐step microbial synthesis of CS. This will provide a new platform to produce CS. -
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Rationale Purification of recombinant proteins is a necessary step for functional or structural studies and other applications. Immobilized metal affinity chromatography is a common recombinant protein purification method. Mass spectrometry (MS) allows for confirmation of identity of expressed proteins and unambiguous detection of enzymatic substrates and reaction products. We demonstrate the detection of enzymes purified on immobilized metal affinity surfaces by direct or ambient ionization MS, and follow their enzymatic reactions by direct electrospray ionization (ESI) or desorption electrospray ionization (DESI).
Methods A protein standard, His‐Ubq, and two recombinant proteins, His‐SHAN and His‐CS, expressed in
Escherichia coli Results Small proteins (His‐Ubq) and medium proteins (His‐SAHN) could readily be detected from 96‐well plates by direct infusion ESI, or from microscope slides by DESI‐MS after purification on surface from clarified
E. coli cell lysate. Protein oxidation was observed for immobilized proteins on both Cu‐NTA and Ni‐NTA; however, this did not hamper the enzymatic reactions of these proteins. Both the nucleosidase reaction products for His‐SAHN and the methylation product of His‐CS (theobromine to caffeine) were detected.Conclusions The immobilization, purification, release and detection of His‐tagged recombinant proteins using immobilized metal affinity surfaces for direct infusion ESI‐MS or ambient DESI‐MS analyses were successfully demonstrated. Recombinant proteins were purified to allow identification directly out of clarified cell lysate. Biological activities of the recombinant proteins were preserved allowing the investigation of enzymatic activity via MS.
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Abstract 7‐Carboxy‐7‐deazaguanine synthase, QueE, catalyzes the radical mediated ring contraction of 6‐carboxy‐5,6,7,8‐tetrahydropterin, forming the characteristic pyrrolopyrimidine core of all 7‐deazaguanine natural products. QueE is a member of the
S ‐adenosyl‐L‐methionine (AdoMet) radical enzyme superfamily, which harnesses the reactivity of radical intermediates to perform challenging chemical reactions. Members of the AdoMet radical enzyme superfamily utilize a canonical binding motif, a CX3CXϕC motif, to bind a [4Fe‐4S] cluster, and a partial (β/α)6TIM barrel fold for the arrangement of AdoMet and substrates for catalysis. Although variations to both the cluster‐binding motif and the core fold have been observed, visualization of drastic variations in the structure of QueE fromBurkholderia multivorans called into question whether a re‐haul of the defining characteristics of this superfamily was in order. Surprisingly, the structure of QueE fromBacillus subtilis revealed an architecture more reminiscent of the classical AdoMet radical enzyme. With these two QueE structures revealing varying degrees of alterations to the classical AdoMet fold, a new question arises: what is the purpose of these alterations? Here, we present the structure of a third QueE enzyme fromEscherichia coli, which establishes the middle range of the spectrum of variation observed in these homologs. With these three homologs, we compare and contrast the structural architecture and make hypotheses about the role of these structural variations in binding and recognizing the biological reductant, flavodoxin.Broader impact statement: We know more about how enzymes are tailored for catalytic activity than about how enzymes are tailored to react with a physiological reductant. Here, we consider structural differences between three 7‐carboxy‐7‐deazaguanine synthases and how these differences may be related to the interaction between these enzymes and their biological reductant, flavodoxin.
