Defining critical residues for substrate binding to 1-deoxy-d-xylulose 5-phosphate synthase - active site substitutions stabilize the predecarboxylation intermediate C2α-lactylthiamin diphosphate

β-lactam antibiotics have been used effectively over several decades against many types of highly virulent bacteria. The predominant cause of resistance to these antibiotics in Gram-negative bacterial pathogens is the production of serine β-lactamase enzymes. A key aspect of the class A serine β-lactamase mechanism that remains unresolved and controversial is the identity of the residue acting as the catalytic base during the acylation reaction. Multiple mechanisms have been proposed for the formation of the acyl-enzyme intermediate that are predicated on understanding the protonation states and hydrogen-bonding interactions among the important residues involved in substrate binding and catalysis of these enzymes. For resolving a controversy of this nature surrounding the catalytic mechanism, neutron crystallography is a powerful complement to X-ray crystallography that can explicitly determine the location of deuterium atoms in proteins, thereby directly revealing the hydrogen-bonding interactions of important amino acid residues. Neutron crystallography was used to unambiguously reveal the ground-state active site protonation states and the resulting hydrogen-bonding network in two ligand-free Toho-1 β-lactamase mutants which provided remarkably clear pictures of the active site region prior to substrate binding and subsequent acylation [1,2] and an acylation transition-state analog, benzothiophene-2-boronic acid (BZB), which was also isotopically enriched with 11B. The neutron structure revealed the locations of all deuterium atoms in the active site region and clearly indicated that Glu166 is protonated in the BZB transition-state analog complex. As a result, the complete hydrogen-bonding pathway throughout the active site region could then deduced for this protein-ligand complex that mimics the acylation tetrahedral intermediate [3].

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Structure of the human heterotetrameric cis-prenyltransferase complex

10.1101/2020.06.02.095570 ◽

2020 ◽

Author(s):

Michal Lisnyansky Bar-El ◽

Pavla Vankova ◽

Petr Man ◽

Yoni Haitin ◽

Moshe Giladi

Keyword(s):

Crystal Structure ◽

Active Site ◽

Substrate Binding ◽

Synthesis Mechanism ◽

Allosteric Communication ◽

Pt Complex ◽

C Terminus ◽

Length Determination ◽

Enzymatic Complex

AbstractThe human cis-prenyltransferase (hcis-PT) is an enzymatic complex essential for protein N-glycosylation. Synthesizing the precursor of the glycosyl carrier dolichol-phosphate, we reveal here that hcis-PT exhibits a novel heterotetrameric assembly in solution, composed of two catalytic dehydrodolichyl diphosphate synthase (DHDDS) and two inactive Nogo-B receptor (NgBR) subunits. The 2.3 Å crystal structure of the complex exposes a dimer-of-heterodimers arrangement, with DHDDS C-termini serving as homotypic assembly domains. Furthermore, the structure elucidates the molecular details associated with substrate binding, catalysis, and product length determination. Importantly, the distal C-terminus of NgBR transverses across the heterodimeric interface, directly participating in substrate binding and underlying the allosteric communication between the subunits. Finally, mapping disease-associated hcis-PT mutations involved in blindness, neurological and glycosylation disorders onto the structure reveals their clustering around the active site. Together, our structure of the hcis-PT complex unveils the dolichol synthesis mechanism and its perturbation in disease.

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Functional analysis of box I mutations in yeast site-specific recombinases Flp and R: pairwise complementation with recombinase variants lacking the active-site tyrosine

Molecular and Cellular Biology ◽

10.1128/mcb.12.9.3757-3765.1992 ◽

1992 ◽

Vol 12 (9) ◽

pp. 3757-3765

Author(s):

J W Chen ◽

B R Evans ◽

S H Yang ◽

H Araki ◽

Y Oshima ◽

...

Keyword(s):

Active Site ◽

Dna Cleavage ◽

Substrate Binding ◽

Point Mutations ◽

Transfer Reactions ◽

Strand Transfer ◽

Site Specific ◽

Arginine Residues ◽

Active Site Tyrosine ◽

Half Site

The site-specific recombinases Flp and R from Saccharomyces cerevisiae and Zygosaccharomyces rouxii, respectively, are related proteins that belong to the yeast family of site-specific recombinases. They share approximately 30% amino acid matches and exhibit a common reaction mechanism that appears to be conserved within the larger integrase family of site-specific recombinases. Two regions of the proteins, designated box I and box II, also harbor a significantly high degree of homology at the nucleotide sequence level. We have analyzed the properties of Flp and R variants carrying point mutations within the box I segment in substrate-binding, DNA cleavage, and full-site and half-site strand transfer reactions. All mutations abolish or seriously diminish recombinase function either at the substrate-binding step or at the catalytic steps of strand cleavage or strand transfer. Of particular interest are mutations of Arg-191 of Flp and R, residues which correspond to one of the two invariant arginine residues of the integrase family. These variant proteins bind substrate with affinities comparable to those of the corresponding wild-type recombinases. Among the binding-competent variants, only Flp(R191K) is capable of efficient substrate cleavage in a full recombination target. However, this protein does not cleave a half recombination site and fails to complete strand exchange in a full site. Strikingly, the Arg-191 mutants of Flp and R can be rescued in half-site strand transfer reactions by a second point mutant of the corresponding recombinase that lacks its active-site tyrosine (Tyr-343). Similarly, Flp and R variants of Cys-189 and Flp variants at Asp-194 and Asp-199 can also be complemented by the corresponding Tyr-343-to-phenylalanine recombinase mutant.

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Revealing the origin of multiphasic dynamic behaviors in cyanobacteriochrome

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2001114117 ◽

2020 ◽

Vol 117 (33) ◽

pp. 19731-19736 ◽

Cited By ~ 2

Author(s):

Dihao Wang ◽

Xiankun Li ◽

Sheng Zhang ◽

Lijuan Wang ◽

Xiaojing Yang ◽

...

Keyword(s):

Active Site ◽

Transient Absorption ◽

Photophysical Properties ◽

Isomerization Reaction ◽

Solvation Dynamics ◽

Nonequilibrium Processes ◽

Dynamic Behaviors ◽

Local Structures ◽

Critical Residues ◽

Local Water

Cyanobacteriochromes are photoreceptors in cyanobacteria that exhibit a wide spectral coverage and unique photophysical properties from the photoinduced isomerization of a linear tetrapyrrole chromophore. Here, we integrate femtosecond-resolved fluorescence and transient-absorption methods and unambiguously showed the significant solvation dynamics occurring at the active site from a few to hundreds of picoseconds. These motions of local water molecules and polar side chains are continuously convoluted with the isomerization reaction, leading to a nonequilibrium processes with continuous active-site motions. By mutations of critical residues at the active site, the modified local structures become looser, resulting in faster solvation relaxations and isomerization reaction. The observation of solvation dynamics is significant and critical to the correct interpretation of often-observed multiphasic dynamic behaviors, and thus the previously invoked ground-state heterogeneity may not be relevant to the excited-state isomerization reaction.

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Domain sliding of two Staphylococcus aureus N-acetylglucosaminidases enables their substrate-binding prior to its catalysis

Communications Biology ◽

10.1038/s42003-020-0911-7 ◽

2020 ◽

Vol 3 (1) ◽

Cited By ~ 3

Author(s):

Sara Pintar ◽

Jure Borišek ◽

Aleksandra Usenik ◽

Andrej Perdih ◽

Dušan Turk

Keyword(s):

Crystal Structures ◽

Active Site ◽

Drug Targets ◽

Substrate Binding ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Human Pathogen ◽

Active Site Cleft ◽

Novel Drug ◽

Novel Drug Targets

AbstractTo achieve productive binding, enzymes and substrates must align their geometries to complement each other along an entire substrate binding site, which may require enzyme flexibility. In pursuit of novel drug targets for the human pathogen S. aureus, we studied peptidoglycan N-acetylglucosaminidases, whose structures are composed of two domains forming a V-shaped active site cleft. Combined insights from crystal structures supported by site-directed mutagenesis, modeling, and molecular dynamics enabled us to elucidate the substrate binding mechanism of SagB and AtlA-gl. This mechanism requires domain sliding from the open form observed in their crystal structures, leading to polysaccharide substrate binding in the closed form, which can enzymatically process the bound substrate. We suggest that these two hydrolases must exhibit unusual extents of flexibility to cleave the rigid structure of a bacterial cell wall.

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