scholarly journals Not Cleaving the His-tag of Thal Results in More Tightly Packed and Better-Diffracting Crystals

Crystals ◽  
2020 ◽  
Vol 10 (12) ◽  
pp. 1135
Author(s):  
Ann-Christin Moritzer ◽  
Tina Prior ◽  
Hartmut H. Niemann
Keyword(s):  
Active Site ◽  
Flavin Adenine Dinucleotide ◽  
Substrate Binding ◽  
Environmentally Friendly ◽  
Space Group ◽  
Cofactor Binding ◽  
Closed Conformation ◽  
His Tag ◽  
Halide Salts ◽  
Binding Loop

Flavin-dependent halogenases chlorinate or brominate their substrates in an environmentally friendly manner, only requiring the cofactor reduced flavin adenine dinucleotide (FADH2), oxygen, and halide salts. The tryptophan 6-halogenase Thal exhibits two flexible loops, which become ordered (substrate-binding loop) or adopt a closed conformation (FAD loop) upon substrate or cofactor binding. Here, we describe the structure of NHis-Thal-RebH5 containing an N-terminal His-tag from pET28a, which crystallized in a different space group (P21) and, surprisingly, diffracted to a higher resolution of 1.63 Å than previously deposited Thal structures (P64; ~2.2 Å) with cleaved His-tag. Interestingly, the binding of glycine in the active site can induce an ordered conformation of the substrate-binding loop.

scholarly journals Structural characterization of tartrate dehydrogenase: a versatile enzyme catalyzing multiple reactions

2010 ◽  
Vol 66 (6) ◽  
pp. 673-684 ◽  
Author(s):  
Radhika Malik ◽  
Ronald E. Viola
Keyword(s):  
Active Site ◽  
Metal Ion ◽  
Substrate Binding ◽  
Hydride Transfer ◽  
Divalent Metal ◽  
Ammonium Ion ◽  
Hydroxyl Group ◽  
Metal Ion Binding ◽  
Cofactor Binding ◽  
Divalent Metal Ion

The first structure of an NAD-dependent tartrate dehydrogenase (TDH) has been solved to 2 Å resolution by single anomalous diffraction (SAD) phasing as a complex with the intermediate analog oxalate, Mg2+and NADH. This TDH structure fromPseudomonas putidahas a similar overall fold and domain organization to other structurally characterized members of the hydroxy-acid dehydrogenase family. However, there are considerable differences between TDH and these functionally related enzymes in the regions connecting the core secondary structure and in the relative positioning of important loops and helices. The active site in these complexes is highly ordered, allowing the identification of the substrate-binding and cofactor-binding groups and the ligands to the metal ions. Residues from the adjacent subunit are involved in both the substrate and divalent metal ion binding sites, establishing a dimer as the functional unit and providing structural support for an alternating-site reaction mechanism. The divalent metal ion plays a prominent role in substrate binding and orientation, together with several active-site arginines. Functional groups from both subunits form the cofactor-binding site and the ammonium ion aids in the orientation of the nicotinamide ring of the cofactor. A lysyl amino group (Lys192) is the base responsible for the water-mediated proton abstraction from the C2 hydroxyl group of the substrate that begins the catalytic reaction, followed by hydride transfer to NAD. A tyrosyl hydroxyl group (Tyr141) functions as a general acid to protonate the enolate intermediate. Each substrate undergoes the initial hydride transfer, but differences in substrate orientation are proposed to account for the different reactions catalyzed by TDH.

scholarly journals Structural comparisons lead to the definition of a new superfamily of NAD(P)(H)-accepting oxidoreductases: the single-domain reductases/epimerases/dehydrogenases (the ‘RED’ family)

1994 ◽  
Vol 304 (1) ◽  
pp. 95-99 ◽  
Author(s):  
G Labesse ◽  
A Vidal-Cros ◽  
J Chomilier ◽  
M Gaudry ◽  
J P Mornon
Keyword(s):  
Amino Acids ◽  
Sequence Alignment ◽  
Active Site ◽  
Tertiary Structure ◽  
Binding Specificity ◽  
Single Domain ◽  
Divergent Evolution ◽  
Cofactor Binding ◽  
Sequence Identity ◽  
Definition Of

Using both primary- and tertiary-structure comparisons, we have established new structural similarities shared by reductases, epimerases and dehydrogenases not previously known to be related. Despite the low sequence identity (down to 10%), short consensus segments are identified. We show that the sequence, the active site and the supersecondary structure are well conserved in these proteins. New homologues (the protochlorophyllide reductases) are detected, and we define a new superfamily composed of single-domain dinucleotide-binding enzymes. Rules for the cofactor-binding specificity are deduced from our sequence alignment. The involvement of some amino acids in catalysis is discussed. Comparison with two-domain dehydrogenases allows us to distinguish two general mechanisms of divergent evolution.

Substrate Binding StabilizesS-Adenosylhomocysteine Hydrolase in a Closed Conformation†

Biochemistry ◽  
2000 ◽  
Vol 39 (32) ◽  
pp. 9811-9818 ◽  
Author(s):  
Dan Yin ◽  
Xiaoda Yang ◽  
Yongbo Hu ◽  
Krysztof Kuczera ◽  
Richard L. Schowen ◽  
...  
Keyword(s):  
Substrate Binding ◽  
Adenosylhomocysteine Hydrolase ◽  
Closed Conformation

Substrate-binding specificity of chitinase and chitosanase as revealed by active-site architecture analysis

2015 ◽  
Vol 418 ◽  
pp. 50-56 ◽  
Author(s):  
Shijia Liu ◽  
Shangjin Shao ◽  
Linlin Li ◽  
Zhi Cheng ◽  
Li Tian ◽  
...  
Keyword(s):  
Active Site ◽  
Substrate Binding ◽  
Binding Specificity ◽  
Architecture Analysis

scholarly journals 1P-121 Roles of Asp297 in the vicinity of the active site of cytochrome P450cam in substrate binding(The 46th Annual Meeting of the Biophysical Society of Japan)

2008 ◽  
Vol 48 (supplement) ◽  
pp. S40
Author(s):  
Keisuke Sakurai ◽  
Katsuyoshi Harada ◽  
Kunitoshi Shimokata ◽  
Takashi Hayashi ◽  
Hideo Shimada
Keyword(s):  
Annual Meeting ◽  
Active Site ◽  
Substrate Binding ◽  
Cytochrome P450cam ◽  
Biophysical Society

scholarly journals A crystallographic study of the Toho-1 β-lactamase acylation mechanism

2014 ◽  
Vol 70 (a1) ◽  
pp. C1207-C1207
Author(s):  
Leighton Coates
Keyword(s):  
Hydrogen Bonding ◽  
Transition State ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Substrate Binding ◽  
Ligand Complex ◽  
Transition State Analog ◽  
Hydrogen Bonding Interactions ◽  
Active Site Region ◽  
Neutron Crystallography

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].

scholarly journals Structure of the human heterotetrameric cis-prenyltransferase complex

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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