Enzyme discovery beyond homology: a unique hydroxynitrile lyase in the Bet v1 superfamily

Abstract Background Nowadays there is a strong trend towards a circular economy using lignocellulosic biowaste for the production of biofuels and other bio-based products. The use of enzymes at several stages of the production process (e.g., saccharification) can offer a sustainable route due to avoidance of harsh chemicals and high temperatures. For novel enzyme discovery, physically linked gene clusters targeting carbohydrate degradation in bacteria, polysaccharide utilization loci (PULs), are recognized ‘treasure troves’ in the era of exponentially growing numbers of sequenced genomes. Results We determined the biochemical properties and structure of a protein of unknown function (PUF) encoded within PULs of metagenomes from beaver droppings and moose rumen enriched on poplar hydrolysate. The corresponding novel bifunctional carbohydrate esterase (CE), now named BD-FAE, displayed feruloyl esterase (FAE) and acetyl esterase activity on simple, synthetic substrates. Whereas acetyl xylan esterase (AcXE) activity was detected on acetylated glucuronoxylan from birchwood, only FAE activity was observed on acetylated and feruloylated xylooligosaccharides from corn fiber. The genomic contexts of 200 homologs of BD-FAE revealed that the 33 closest homologs appear in PULs likely involved in xylan breakdown, while the more distant homologs were found either in alginate-targeting PULs or else outside PUL contexts. Although the BD-FAE structure adopts a typical α/β-hydrolase fold with a catalytic triad (Ser-Asp-His), it is distinct from other biochemically characterized CEs. Conclusions The bifunctional CE, BD-FAE, represents a new candidate for biomass processing given its capacity to remove ferulic acid and acetic acid from natural corn and birchwood xylan substrates, respectively. Its detailed biochemical characterization and solved crystal structure add to the toolbox of enzymes for biomass valorization as well as structural information to inform the classification of new CEs.

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Apd1 and Aim32 Are Prototypes of Bis-Histidinyl-Coordinated Non-Rieske [2Fe-2S] Proteins

10.26434/chemrxiv.7443971.v1 ◽

2018 ◽

Author(s):

Kathrin Stegmaier ◽

Catharina M. Blinn ◽

Dominique F. Bechtel ◽

Carina Greth ◽

Hendrik Auerbach ◽

...

Keyword(s):

Convergent Evolution ◽

Yeast Cells ◽

Ferric Ion ◽

Mitochondrial Matrix ◽

Protein Fold ◽

Wild Type ◽

Iron Ion ◽

Yeast Protein ◽

Flexible System ◽

Proton Coupled Electron Transfer

Apd1, a cytosolic yeast protein, and Aim32, its counterpart in the mitochondrial matrix, have a C-terminal thioredoxinlike ferredoxin domain and a widely divergent N-terminal domain. These proteins are found in bacteria, plants, fungi and unicellular pathogenic eukaryotes, but not in Metazoa. Our chemogenetic experiments demonstrate that the highly conserved cysteine and histidine residues within the C-X8-C-X24-75-H-X-G-G-H motif of the TLF domain of Apd1 and Aim32 proteins are essential for viability upon treatment of yeast cells with the redox potentiators gallobenzophenone or pyrogallol, respectively. UV-Vis, EPR and Mössbauer spectroscopy of purified wild type Apd1 and three His to Cys variants demonstrated that Cys207 and Cys216 are the ligands of the ferric ion and His255 and His259 are the ligands of the reducible iron ion of the [2Fe-2S]2+/1+ cluster. The [2Fe-2S] center of Apd1 (Em,7 = -164±5 mV, pKox1,2=7.9±0.1 and 9.7±0.1) differs from both dioxygenase (Em,7 ≈ -150 mV, pKox1,2=9.8 and 11.5) and cytochrome bc1/b6f Rieske clusters (Em,7 ≈ +300 mV, pKox1,2= 7.7 and 9.8). Apd1 and its engineered variants represent an unprecedented flexible system for which a stable [2Fe-2S] cluster with two histidine ligands, (two different) single histidine ligands or only cysteinyl ligands is possible in the same protein fold. Our results define a remarkable example of convergent evolution of [2Fe-2S] cluster containing proteins with bis-histidinyl coordination and proton-coupled electron transfer.<br>

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Convergent evolution of the Hedgehog/Intein fold in protein splicing

10.1101/2020.03.19.998260 ◽

2020 ◽

Author(s):

Hannes M. Beyer ◽

Salla I. Virtanen ◽

A. Sesilja Aranko ◽

Kornelia M. Mikula ◽

George T. Lountos ◽

...

Keyword(s):

Convergent Evolution ◽

Divergent Evolution ◽

Structural Basis ◽

Protein Splicing ◽

Protein Fold ◽

Class 1 ◽

Molecular Evolutionary Clock ◽

Evolutionary Clock ◽

Esterification Reactions ◽

Evolution Of Proteins

AbstractThe widely used molecular evolutionary clock assumes the divergent evolution of proteins. Convergent evolution has been proposed only for small protein elements but not for an entire protein fold. We investigated the structural basis of the protein splicing mechanism by class 3 inteins, which is distinct from class 1 and 2 inteins. We gathered structural and mechanistic evidence supporting the notion that the Hedgehog/INTein (HINT) superfamily fold, commonly found in protein splicing and related phenomena, could be an example of convergent evolution of an entire protein fold. We propose that the HINT fold is a structural and biochemical solution for trans-peptidyl and trans-esterification reactions.

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Apd1 and Aim32 Are Prototypes of Bis-Histidinyl-Coordinated Non-Rieske [2Fe-2S] Proteins

10.26434/chemrxiv.7443971 ◽

2018 ◽

Author(s):

Kathrin Stegmaier ◽

Catharina M. Blinn ◽

Dominique F. Bechtel ◽

Carina Greth ◽

Hendrik Auerbach ◽

...

Keyword(s):

Convergent Evolution ◽

Yeast Cells ◽

Ferric Ion ◽

Mitochondrial Matrix ◽

Protein Fold ◽

Wild Type ◽

Iron Ion ◽

Yeast Protein ◽

Flexible System ◽

Proton Coupled Electron Transfer

Apd1, a cytosolic yeast protein, and Aim32, its counterpart in the mitochondrial matrix, have a C-terminal thioredoxinlike ferredoxin domain and a widely divergent N-terminal domain. These proteins are found in bacteria, plants, fungi and unicellular pathogenic eukaryotes, but not in Metazoa. Our chemogenetic experiments demonstrate that the highly conserved cysteine and histidine residues within the C-X8-C-X24-75-H-X-G-G-H motif of the TLF domain of Apd1 and Aim32 proteins are essential for viability upon treatment of yeast cells with the redox potentiators gallobenzophenone or pyrogallol, respectively. UV-Vis, EPR and Mössbauer spectroscopy of purified wild type Apd1 and three His to Cys variants demonstrated that Cys207 and Cys216 are the ligands of the ferric ion and His255 and His259 are the ligands of the reducible iron ion of the [2Fe-2S]2+/1+ cluster. The [2Fe-2S] center of Apd1 (Em,7 = -164±5 mV, pKox1,2=7.9±0.1 and 9.7±0.1) differs from both dioxygenase (Em,7 ≈ -150 mV, pKox1,2=9.8 and 11.5) and cytochrome bc1/b6f Rieske clusters (Em,7 ≈ +300 mV, pKox1,2= 7.7 and 9.8). Apd1 and its engineered variants represent an unprecedented flexible system for which a stable [2Fe-2S] cluster with two histidine ligands, (two different) single histidine ligands or only cysteinyl ligands is possible in the same protein fold. Our results define a remarkable example of convergent evolution of [2Fe-2S] cluster containing proteins with bis-histidinyl coordination and proton-coupled electron transfer.<br>

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Major reorientation of tRNA substrates defines specificity of dihydrouridine synthases

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1500161112 ◽

2015 ◽

Vol 112 (19) ◽

pp. 6033-6037 ◽

Cited By ~ 23

Author(s):

Robert T. Byrne ◽

Huw T. Jenkins ◽

Daniel T. Peters ◽

Fiona Whelan ◽

James Stowell ◽

...

Keyword(s):

Escherichia Coli ◽

Amino Acids ◽

Substrate Specificity ◽

Crystal Structures ◽

Active Site ◽

Protein Fold ◽

Catalytic Center ◽

Trna Recognition ◽

Trna Binding ◽

Positively Charged

The reduction of specific uridines to dihydrouridine is one of the most common modifications in tRNA. Increased levels of the dihydrouridine modification are associated with cancer. Dihydrouridine synthases (Dus) from different subfamilies selectively reduce distinct uridines, located at spatially unique positions of folded tRNA, into dihydrouridine. Because the catalytic center of all Dus enzymes is conserved, it is unclear how the same protein fold can be reprogrammed to ensure that nucleotides exposed at spatially distinct faces of tRNA can be accommodated in the same active site. We show that the Escherichia coli DusC is specific toward U16 of tRNA. Unexpectedly, crystal structures of DusC complexes with tRNAPhe and tRNATrp show that Dus subfamilies that selectively modify U16 or U20 in tRNA adopt identical folds but bind their respective tRNA substrates in an almost reverse orientation that differs by a 160° rotation. The tRNA docking orientation appears to be guided by subfamily-specific clusters of amino acids (“binding signatures”) together with differences in the shape of the positively charged tRNA-binding surfaces. tRNA orientations are further constrained by positional differences between the C-terminal “recognition” domains. The exquisite substrate specificity of Dus enzymes is therefore controlled by a relatively simple mechanism involving major reorientation of the whole tRNA molecule. Such reprogramming of the enzymatic specificity appears to be a unique evolutionary solution for altering tRNA recognition by the same protein fold.

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