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Conformational gating mechanism for processive catalysis of beta(1,3) -glucans

ABSTRACT:

Processive catalysis is a fundamental molecular mechanism in nature to both build and dismantle complex biological polymers such as nucleic acids, proteins and carbohydrates, underpinning a broad spectrum of biotechnological applications. Here, we uncover a processive mechanism for the breakdown of β(1,3)-glucans, a carbohydrate class widespread across biological kingdoms from prokaryotes to eukaryotes. This mechanism involves a structurally dynamic active site, which adopts a tunnel-like conformation upon substrate binding, productively positioning the glycosidic bond for cleavage. Upon product release, the disruption of a pivotal salt bridge triggers the transition to an open conformation, establishing new polar interactions with the remnant substrate that are essential for translocation and the subsequent catalytic cycle. QM/MM calculations further reveal that this processive cleavage involves a non-canonical 4C1 reactive sugar puckering, a characteristic hitherto limited to certain exo-acting enzymes. Taken together, these findings establish the mechanistic basis for β(1,3)-glucan processive catalysis, demonstrating reaction steps from substrate recognition, tunnel formation, nucleophilic attack, intermediate state stabilization and conformational changes associated with product release and translocation. Ultimately, this work advances our understanding of microbial enzymatic systems for β(1,3)-glucan breakdown and metabolism, demonstrating that processive catalysis is a conserved evolutionary strategy across all major classes of β-glucans in nature.

INSTRUMENT(S): Liquid Chromatography MS - positive - hilic

PROVIDER: MTBLS13959 | MetaboLights | 2026-03-02

REPOSITORIES: MetaboLights

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			Action	DRS
	a_MTBLS13959_LC-MS_positive_hilic_metabolite_profiling.txt	Txt
	i_Investigation.txt	Txt
	m_MTBLS13959_LC-MS_positive_hilic_metabolite_profiling_maf.tsv	Tabular
	s_MTBLS13959.txt	Txt

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Project description:We developed native elongating transcript sequencing (NET-seq, Churchman and Weissman Nature 2011, PMID: 21248844) combined with RNase footprinting of nascent transcripts (RNET-seq) to visualize translocation dynamics and nascent transcript errors in paused RNA polymerases in E. coli. We employed RNET-seq to the wild-type (WT) E. coli strain and to an isogenic strain deficient in genes for GreA and GreB (ΔgreAB). Gre factors and their eukaryotic analog TFIIS rescue backtracked complexes of RNAP. Briefly, the cells were rapidly lysed via spheroplasting, and the transcribing RNAPs were released from the genomic DNA by digestion with DNase I. Any ribosomes involved in co-transcriptional translation were separated from RNAP by digestion with RNase A. All RNAPs including those associated with the fragmented double-stranded DNAs and their 5’-truncated nascent RNAs were immobilized on Ni2+-NTA beads through the hexa-histidine-tagged β’ subunit and then extensively washed with a high-salt buffer. The 5’ ends of the transcripts in ECs were trimmed with RNase T1/V1 to leave a minimal length of RNA protected by RNAP. The RNases were subsequently removed by further washing of the beads. Elution with imidazole generated ECs carrying ~6-30 nt long transcripts. The nascent RNAs isolated from the ΔgreAB strain were longer than those from the WT strain and peaked at 18 nt versus 16 nt suggesting an enrichment of backtracked ECs, which is expected to occur in the absence of Gre-dependent 3’ RNA cleavage. The RNET-seq method involves trimming of excess nascent RNA from the 5’ ends leaving only the nascent RNA that is protected by RNAP. A previous in vitro study showed that an RNAP forming an EC protects different lengths of the 3’-proximal transcript from trimming by RNases A and T1 depending on the EC translocation state. Post-translocated, pre-translocated, and backtracked complexes protect 14-nt, 15-nt and >15-nt segments of the RNA, respectively. Because the very 3’ end of the RNA is extruded to a narrow pore within front of the enzyme during backtracking, the extruded RNA remains inaccessible to RNases increasing in length as backtracking increases. Thus, paused RNAP in either the pre- and post-translocated states as well as in different backtracked distances were monitored over the entire genome. The unique properties of our RNET-seq approach provided an opportunity to dissect the core mechanisms of different types of pausing in living cells.

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Conformational gating mechanism for processive catalysis of beta(1,3) -glucans

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