期刊
ACS CENTRAL SCIENCE
卷 6, 期 5, 页码 760-770出版社
AMER CHEMICAL SOC
DOI: 10.1021/acscentsci.0c00111
关键词
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资金
- Australian Research Council [FT130100103, DP120101396, DP180101957]
- BBSRC
- European Research Council [ERC-2012-AdG-322942]
- Natural Sciences and Engineering Research Council of Canada [201704910]
- Spanish Ministry of Science, Innovation and Universities (MICINN/AEI/FEDER) [CTQ2017-85496-P]
- Spanish Structures of Excellence Maria de Maeztu [MDM-2017-0767]
- Agency for Management of University and Research Grants of Generalitat de Catalunya (AGAUR) [2017SGR-1189]
- Royal Society Ken Murray Professorship
- Agencia Estatal de Investigacion of Spain [RTI2018-101269-BI00, RTI2018-094751-B-C21]
- European Research Council (RECGLYCANMR, ERC AdG) [788143]
- Severo Ochoa Excellence accreditation [SEV2016-0644]
- European Research Council (ERC) [788143] Funding Source: European Research Council (ERC)
Retaining glycoside hydrolases cleave their substrates through stereochemical retention at the anomeric position. Typically, this involves two-step mechanisms using either an enzymatic nucleophile via a covalent glycosyl enzyme intermediate or neighboring-group participation by a substrate-borne 2-acetamido neighboring group via an oxazoline intermediate; no enzymatic mechanism with participation of the sugar 2-hydroxyl has been reported. Here, we detail structural, computational, and kinetic evidence for neighboring-group participation by a mannose 2-hydroxyl in glycoside hydrolase family 99 endo-alpha-1,2-mannanases. We present a series of crystallographic snapshots of key species along the reaction coordinate: a Michaelis complex with a tetrasaccharide substrate; complexes with intermediate mimics, a sugar-shaped cyclitol beta-1,2-aziridine and beta-1,2-epoxide; and a product complex. The 1,2-epoxide intermediate mimic displayed hydrolytic and transfer reactivity analogous to that expected for the 1,2-anhydro sugar intermediate supporting its catalytic equivalence. Quantum mechanics/molecular mechanics modeling of the reaction coordinate predicted a reaction pathway through a 1,2-anhydro sugar via a transition state in an unusual flattened, envelope (E-3) conformation. Kinetic isotope effects (k(cat)/K-M) for anomeric-H-2 and anomeric-C-1(3) support an oxocarbenium ion-like transition state, and that for C2-O-18 (1.052 +/- 0.006) directly implicates nucleophilic participation by the C2-hydroxyl. Collectively, these data substantiate this unprecedented and long-imagined enzymatic mechanism.
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