Mechanistic Investigations of Unsaturated Glucuronyl Hydrolase from Clostridium perfringens

Jongkees, Seino A. K.; Yoo, Hayoung; Withers, Stephen G.

doi:10.1074/jbc.m113.545293

Cited by 8 publications

(15 citation statements)

References 56 publications

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“…The discovery of GH105 enzymes, which are unsaturated uronyl hydrolases that cleave the 4,5-unsaturated residues from the nonreducing ends of PL-generated oligouronates (21,22), has hinted at the potential for an alternate model of pectin metabolism. The mechanism of GH105 catalysis is thought to parallel that of GH88 members, which results in release of the linearized monosaccharide directly (23,24). The direct release of DKI by GH105 enzymes would obviate the need for a PL22 (or potentially an equivalent) and KdgF to convert the unsaturated nonreducing ends of PL-generated oligouronates into DKI.…”

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confidence: 99%

Biochemical Reconstruction of a Metabolic Pathway from a Marine Bacterium Reveals Its Mechanism of Pectin Depolymerization

Hobbs

Hettle

Vickers

et al. 2019

Appl Environ Microbiol

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Pectin is a complex uronic acid-containing polysaccharide typically found in plant cell walls, though forms of pectin are also found in marine diatoms and seagrasses. Genetic loci that target pectin have recently been identified in two phyla of marine bacteria. These loci appear to encode a pectin saccharification pathway that is distinct from the canonical pathway typically associated with phytopathogenic terrestrial bacteria. However, very few components of the marine pectin metabolism pathway have been experimentally validated. Here, we biochemically reconstructed the pectin saccharification pathway from a marine Pseudoalteromonas sp. in vitro and show that it results in the production of galacturonate and the key metabolic intermediate 5-keto-4-deoxyuronate (DKI). We demonstrate the sequential de-esterification and depolymerization of pectin into oligosaccharides and the synergistic action of glycoside hydrolases (GHs) to fully degrade these oligosaccharides into monosaccharides. Furthermore, we show that this pathway relies on enzymes belonging to GH family 105 to carry out the equivalent chemistry afforded by an exolytic polysaccharide lyase (PL) and KdgF in the canonical pectin pathway. Finally, we synthesize our findings into a model of marine pectin degradation and compare it with the canonical pathway. Our results underline the shifting view of pectin as a solely terrestrial polysaccharide and highlight the importance of marine pectin as a carbon source for suitably adapted marine heterotrophs. This alternate pathway has the potential to be exploited in the growing field of biofuel production from plant waste. IMPORTANCE Marine polysaccharides, found in the cell walls of seaweeds and other marine macrophytes, represent a vast sink of photosynthetically fixed carbon. As such, their breakdown by marine microbes contributes significantly to global carbon cycling. Pectin is an abundant polysaccharide found in the cell walls of terrestrial plants, but it has recently been reported that some marine bacteria possess the genetic capacity to degrade it. In this study, we biochemically characterized seven key enzymes from a marine bacterium that, together, fully degrade the backbone of pectin into its constituent monosaccharides. Our findings highlight the importance of pectin as a marine carbon source available to bacteria that possess this pathway. The characterized enzymes also have the potential to be utilized in the production of biofuels from plant waste.

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confidence: 99%

Biochemical Reconstruction of a Metabolic Pathway from a Marine Bacterium Reveals Its Mechanism of Pectin Depolymerization

Hobbs

Hettle

Vickers

et al. 2019

Appl Environ Microbiol

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“…[14] Interestingly,this makes UGLs relatively promiscuous enzymes that are capable of accepting ar ange of substrates with different aglycones,s ince the aglycone is not directly involved in the rate determining step of this mechanism. [15,16] UGLs represent an excellent and challenging test case for design of screens for unconventional glycosidases.Firstly,they are relatively rare enzymes,w ith only 1449 entries in the GH88 family and 2562 entries in the GH105 family (compared to for example more than 12 000 entries in GH2, the family that contains most of the known b-glucuronidases). Secondly,a ss hown in the present study,t he conventional MU-glycoside screening substrates of UGLs are cleaved, albeit slowly,b yb-glucuronidases.S ince b-glucuronidase activity occurs much more frequently than UGL activity in metagenomic genes,s creening for UGL activity with MUglycosides of unsaturated glucuronides can lead to false positive hits that contain genes for b-glucuronidases and not UGLs.F urther,s ince our metagenomic libraries are expressed in the E. coli strain EPI300, b-glucuronidase from E. coli [17] (BGE) will always be present in the screening media, giving rise to ahigh background activity (although this could be avoided in future screens by using BGE deficient strains of E. coli).…”

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confidence: 99%

A Mechanism‐Based Approach to Screening Metagenomic Libraries for Discovery of Unconventional Glycosidases

et al. 2018

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Functional metagenomics has opened new opportunities for enzyme discovery. To exploit the full potential of this new tool, the design of selective screens is essential, especially when searching for rare enzymes. To identify novel glycosidases that employ cleavage strategies other than the conventional Koshland mechanisms, a suitable screen was needed. Focusing on the unsaturated glucuronidases (UGLs), it was found that use of simple aryl glycoside substrates did not allow sufficient discrimination against β-glucuronidases, which are widespread in bacteria. While conventional glycosidases cannot generally hydrolyze thioglycosides efficiently, UGLs follow a distinct mechanism that allows them to do so. Thus, fluorogenic thioglycoside substrates featuring thiol-based self-immolative linkers were synthesized and assessed as selective substrates. The generality of the approach was validated with another family of unconventional glycosidases, the GH4 enzymes. Finally, the utility of these substrates was tested by screening a small metagenomic library.

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“…Moreover, these studies suggest that this is the rate‐determining step of this mechanism . Interestingly, this makes UGLs relatively promiscuous enzymes that are capable of accepting a range of substrates with different aglycones, since the aglycone is not directly involved in the rate determining step of this mechanism …”

Section: Figurementioning

confidence: 99%

A Mechanism‐Based Approach to Screening Metagenomic Libraries for Discovery of Unconventional Glycosidases

et al. 2018

Self Cite

View full text Add to dashboard Cite

Functional metagenomics has opened new opportunities for enzyme discovery. To exploit the full potential of this new tool, the design of selective screens is essential, especially when searching for rare enzymes. To identify novel glycosidases that employ cleavage strategies other than the conventional Koshland mechanisms, a suitable screen was needed. Focusing on the unsaturated glucuronidases (UGLs), it was found that use of simple aryl glycoside substrates did not allow sufficient discrimination against β‐glucuronidases, which are widespread in bacteria. While conventional glycosidases cannot generally hydrolyze thioglycosides efficiently, UGLs follow a distinct mechanism that allows them to do so. Thus, fluorogenic thioglycoside substrates featuring thiol‐based self‐immolative linkers were synthesized and assessed as selective substrates. The generality of the approach was validated with another family of unconventional glycosidases, the GH4 enzymes. Finally, the utility of these substrates was tested by screening a small metagenomic library.

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Mechanistic Investigations of Unsaturated Glucuronyl Hydrolase from Clostridium perfringens

Cited by 8 publications

References 56 publications

Biochemical Reconstruction of a Metabolic Pathway from a Marine Bacterium Reveals Its Mechanism of Pectin Depolymerization

Biochemical Reconstruction of a Metabolic Pathway from a Marine Bacterium Reveals Its Mechanism of Pectin Depolymerization

A Mechanism‐Based Approach to Screening Metagenomic Libraries for Discovery of Unconventional Glycosidases

A Mechanism‐Based Approach to Screening Metagenomic Libraries for Discovery of Unconventional Glycosidases

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