Widespread Family of NAD<sup>+</sup>-Dependent Sulfoquinovosidases at the Gateway to Sulfoquinovose Catabolism

Kaur, Arashdeep; Pickles, Isabelle B.; Sharma, Mahima; Madeido Soler, Niccolay; Scott, Nichollas E.; Pidot, Sacha J.; Goddard-Borger, Ethan D.; Davies, Gideon J.; Williams, Spencer J.

doi:10.1021/jacs.3c11126

“…K-12 MG1655, the sqgA gene of Arthrobacter sp. strain AK01 27 , and the sulfo-EMP2 genes of Alkalicoccus urumqiensis BZ-SZ-XJ18 25 . To validate homology between gene products, we compared protein sequences using the Needleman-Wunsch algorithm with default settings 39 .…”

Section: Methodsmentioning

confidence: 99%

“…The yih locus in Arthrobacter sp. strain AK01 was reported to include the sqgA gene that codes for sulfoquinovosidase (SQase) as an alternative to YihQ 27 . However, the co-localization patterns of the csqR gene have not been fully described, and the distribution of its homologs among bacterial species remains unclear.…”

Section: Introductionmentioning

confidence: 99%

Phylogeny and structural modeling of the transcription factor CsqR (YihW) from Escherichia coli

Rybina,

Glushak,

Bessonova

et al. 2024

Sci Rep

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CsqR (YihW) is a local transcription factor that controls expression of yih genes involved in degradation of sulfoquinovose in Escherichia coli. We recently showed that expression of the respective gene cassette might be regulated by lactose. Here, we explore the phylogenetic and functional traits of CsqR. Phylogenetic analysis revealed that CsqR had a conserved Met25. Western blot demonstrated that CsqR was synthesized in the bacterial cell as two protein forms, 28.5 (CsqR-l) and 26 kDa (CsqR-s), the latter corresponding to start of translation at Met25. CsqR-s was dramatically activated during growth with sulfoquinovose as a sole carbon source, and displaced CsqR-l in the stationary phase during growth on rich medium. Molecular dynamic simulations revealed two possible states of the CsqR-s structure, with the interdomain linker being represented by either a disordered loop or an ɑ-helix. This helix allowed the hinge-like motion of the N-terminal domain resulting in a switch of CsqR-s between two conformational states, “open” and “compact”. We then modeled the interaction of both CsqR forms with putative effectors sulfoquinovose, sulforhamnose, sulfoquinovosyl glycerol, and lactose, and revealed that they all preferred the same pocket in CsqR-l, while in CsqR-s there were two possible options dependent on the linker structure.

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“…The majority of SQases are classical glycosidases that belong to glycoside hydrolase family 31 (GH31) of the carbohydrate active enzyme classification Scheme (although a new NAD + -dependent SQase family, GH188, has recently been reported), [7] and possess a conserved sulfonate binding motif that distinguishes them from other glycosidases within this family. [4,8] These SQases use a two-step mechanism that results in the stereochemically retaining hydrolysis of the glycosidic linkage, and which has been studied in detail for the E. coli SQase, YihQ (Figure 1b).…”

Section: Introductionmentioning

confidence: 99%

Detection of Sulfoquinovosidase Activity in Cell Lysates Using Activity‐Based Probes

Li,

Pickles,

Sharma

et al. 2024

Angew Chem Int Ed

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The sulfolipid sulfoquinovosyl diacylglycerol (SQDG), produced by plants, algae, and cyanobacteria, constitutes a major sulfur reserve in the biosphere. Microbial breakdown of SQDG is critical for the biological utilization of its sulfur. This commences through release of the parent sugar, sulfoquinovose (SQ), catalyzed by sulfoquinovosidases (SQases). These vanguard enzymes are encoded in gene clusters that code for diverse SQ catabolic pathways. To identify, visualize and isolate glycoside hydrolase CAZY‐family 31 (GH31) SQases in complex biological environments, we introduce SQ cyclophellitol‐aziridine activity‐based probes (ABPs). These ABPs label the active site nucleophile of this enzyme family, consistent with specific recognition of the SQ cyclophellitol‐aziridine in the active site, as evidenced in the 3D structure of Bacillus megaterium SQase. A fluorescent Cy5‐probe enables visualization of SQases in crude cell lysates from bacteria harbouring different SQ breakdown pathways, whilst a biotin‐probe enables SQase capture and identification by proteomics. The Cy5‐probe facilitates monitoring of active SQase levels during different stages of bacterial growth which show great contrast to more traditional mRNA analysis obtained by RT‐qPCR. Given the importance of SQases in global sulfur cycling and in human microbiota, these SQase ABPs provide a new tool with which to study SQase occurrence, activity and stability.

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Detection of Sulfoquinovosidase Activity in Cell Lysates Using Activity‐Based Probes

Li,

Pickles,

Sharma

et al. 2024

Angewandte Chemie

Self Cite

0

View full text Add to dashboard Cite

The sulfolipid sulfoquinovosyl diacylglycerol (SQDG), produced by plants, algae, and cyanobacteria, constitutes a major sulfur reserve in the biosphere. Microbial breakdown of SQDG is critical for the biological utilization of its sulfur. This commences through release of the parent sugar, sulfoquinovose (SQ), catalyzed by sulfoquinovosidases (SQases). These vanguard enzymes are encoded in gene clusters that code for diverse SQ catabolic pathways. To identify, visualize and isolate glycoside hydrolase CAZY‐family 31 (GH31) SQases in complex biological environments, we introduce SQ cyclophellitol‐aziridine activity‐based probes (ABPs). These ABPs label the active site nucleophile of this enzyme family, consistent with specific recognition of the SQ cyclophellitol‐aziridine in the active site, as evidenced in the 3D structure of Bacillus megaterium SQase. A fluorescent Cy5‐probe enables visualization of SQases in crude cell lysates from bacteria harbouring different SQ breakdown pathways, whilst a biotin‐probe enables SQase capture and identification by proteomics. The Cy5‐probe facilitates monitoring of active SQase levels during different stages of bacterial growth which show great contrast to more traditional mRNA analysis obtained by RT‐qPCR. Given the importance of SQases in global sulfur cycling and in human microbiota, these SQase ABPs provide a new tool with which to study SQase occurrence, activity and stability.

show abstract

Widespread Family of NAD⁺-Dependent Sulfoquinovosidases at the Gateway to Sulfoquinovose Catabolism

Cited by 7 publications

References 27 publications

Phylogeny and structural modeling of the transcription factor CsqR (YihW) from Escherichia coli

Phylogeny and structural modeling of the transcription factor CsqR (YihW) from Escherichia coli

Detection of Sulfoquinovosidase Activity in Cell Lysates Using Activity‐Based Probes

Detection of Sulfoquinovosidase Activity in Cell Lysates Using Activity‐Based Probes

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Widespread Family of NAD+-Dependent Sulfoquinovosidases at the Gateway to Sulfoquinovose Catabolism

Cited by 7 publications

References 27 publications

Phylogeny and structural modeling of the transcription factor CsqR (YihW) from Escherichia coli

Phylogeny and structural modeling of the transcription factor CsqR (YihW) from Escherichia coli

Detection of Sulfoquinovosidase Activity in Cell Lysates Using Activity‐Based Probes

Detection of Sulfoquinovosidase Activity in Cell Lysates Using Activity‐Based Probes

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Widespread Family of NAD⁺-Dependent Sulfoquinovosidases at the Gateway to Sulfoquinovose Catabolism