scholarly journals The organophosphate-degrading enzyme from Agrobacterium radiobacter displays mechanistic flexibility for catalysis

2010 ◽  
Vol 432 (3) ◽  
pp. 565-573 ◽  
Author(s):  
Fernanda Ely ◽  
Kieran S. Hadler ◽  
Lawrence R. Gahan ◽  
Luke W. Guddat ◽  
David L. Ollis ◽  
...  
Keyword(s):  
Metal Ion ◽  
Binding Pocket ◽  
Ion Composition ◽  
Agrobacterium Radiobacter ◽  
Substrate Binding Pocket ◽  
Environmental Pressures ◽  
Degrading Enzyme ◽  
Protonation Equilibrium ◽  
Reaction Models ◽  
Bond Network

The OP (organophosphate)-degrading enzyme from Agrobacterium radiobacter (OpdA) is a binuclear metallohydrolase able to degrade highly toxic OP pesticides and nerve agents into less or non-toxic compounds. In the present study, the effect of metal ion substitutions and site-directed mutations on the catalytic properties of OpdA are investigated. The study shows the importance of both the metal ion composition and a hydrogen-bond network that connects the metal ion centre with the substrate-binding pocket using residues Arg254 and Tyr257 in the mechanism and substrate specificity of this enzyme. For the Co(II) derivative of OpdA two protonation equilibria (pKa1 ~5; pKa2 ~10) have been identified as relevant for catalysis, and a terminal hydroxide acts as the likely hydrolysis-initiating nucleophile. In contrast, the Zn(II) and Cd(II) derivatives only have one relevant protonation equilibrium (pKa ~4–5), and the μOH is the proposed nucleophile. The observed mechanistic flexibility may reconcile contrasting reaction models that have been published previously and may be beneficial for the rapid adaptation of OP-degrading enzymes to changing environmental pressures.

scholarly journals Structural and Genetic Determinants of Convergence in the Drosophila tRNA Structure–Function Map

2021 ◽  
Vol 89 (1-2) ◽  
pp. 103-116
Author(s):  
Julie Baker Phillips ◽  
David H. Ardell
Keyword(s):  
Structure Function ◽  
Metal Ion ◽  
Binding Pocket ◽  
Trna Synthetase ◽  
Heterologous Gene ◽  
Multigene Families ◽  
Ion Binding ◽  
Trna Genes ◽  
Structural Factors ◽  
Trna Structure

AbstractThe evolution of tRNA multigene families remains poorly understood, exhibiting unusual phenomena such as functional conversions of tRNA genes through anticodon shift substitutions. We improved FlyBase tRNA gene annotations from twelve Drosophila species, incorporating previously identified ortholog sets to compare substitution rates across tRNA bodies at single-site and base-pair resolution. All rapidly evolving sites fell within the same metal ion-binding pocket that lies at the interface of the two major stacked helical domains. We applied our tRNA Structure–Function Mapper (tSFM) method independently to each Drosophila species and one outgroup species Musca domestica and found that, although predicted tRNA structure–function maps are generally highly conserved in flies, one tRNA Class-Informative Feature (CIF) within the rapidly evolving ion-binding pocket—Cytosine 17 (C17), ancestrally informative for lysylation identity—independently gained asparaginylation identity and substituted in parallel across tRNAAsn paralogs at least once, possibly multiple times, during evolution of the genus. In D. melanogaster, most tRNALys and tRNAAsn genes are co-arrayed in one large heterologous gene cluster, suggesting that heterologous gene conversion as well as structural similarities of tRNA-binding interfaces in the closely related asparaginyl-tRNA synthetase (AsnRS) and lysyl-tRNA synthetase (LysRS) proteins may have played a role in these changes. A previously identified Asn-to-Lys anticodon shift substitution in D. ananassae may have arisen to compensate for the convergent and parallel gains of C17 in tRNAAsn paralogs in that lineage. Our results underscore the functional and evolutionary relevance of our tRNA structure–function map predictions and illuminate multiple genomic and structural factors contributing to rapid, parallel and compensatory evolution of tRNA multigene families.

scholarly journals Crystal structure of steroid reductase SRD5A reveals conserved steroid reduction mechanism

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Yufei Han ◽  
Qian Zhuang ◽  
Bo Sun ◽  
Wenping Lv ◽  
Sheng Wang ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Biochemical Characterization ◽  
Substrate Binding ◽  
Binding Pocket ◽  
Substrate Binding Pocket ◽  
Transmembrane Segments ◽  
Therapeutic Molecules ◽  
Binding Cavity ◽  
Immune System Regulation ◽  
Mediated Reduction

AbstractSteroid hormones are essential in stress response, immune system regulation, and reproduction in mammals. Steroids with 3-oxo-Δ4 structure, such as testosterone or progesterone, are catalyzed by steroid 5α-reductases (SRD5As) to generate their corresponding 3-oxo-5α steroids, which are essential for multiple physiological and pathological processes. SRD5A2 is already a target of clinically relevant drugs. However, the detailed mechanism of SRD5A-mediated reduction remains elusive. Here we report the crystal structure of PbSRD5A from Proteobacteria bacterium, a homolog of both SRD5A1 and SRD5A2, in complex with the cofactor NADPH at 2.0 Å resolution. PbSRD5A exists as a monomer comprised of seven transmembrane segments (TMs). The TM1-4 enclose a hydrophobic substrate binding cavity, whereas TM5-7 coordinate cofactor NADPH through extensive hydrogen bonds network. Homology-based structural models of HsSRD5A1 and -2, together with biochemical characterization, define the substrate binding pocket of SRD5As, explain the properties of disease-related mutants and provide an important framework for further understanding of the mechanism of NADPH mediated steroids 3-oxo-Δ4 reduction. Based on these analyses, the design of therapeutic molecules targeting SRD5As with improved specificity and therapeutic efficacy would be possible.

Phosphate-bound structure of an organophosphate-degrading enzyme from Agrobacterium radiobacter

2012 ◽  
Vol 106 (1) ◽  
pp. 19-22 ◽  
Author(s):  
Fernanda Ely ◽  
Marcelo M. Pedroso ◽  
Lawrence R. Gahan ◽  
David L. Ollis ◽  
Luke W. Guddat ◽  
...  
Keyword(s):  
Agrobacterium Radiobacter ◽  
Degrading Enzyme

Characterization of the Acyl Substrate Binding Pocket of Acetyl-CoA Synthetase†

Biochemistry ◽  
2006 ◽  
Vol 45 (38) ◽  
pp. 11482-11490 ◽  
Author(s):  
Cheryl Ingram-Smith ◽  
Barrett I. Woods ◽  
Kerry S. Smith
Keyword(s):  
Substrate Binding ◽  
Binding Pocket ◽  
Substrate Binding Pocket ◽  
Acetyl Coa

scholarly journals The Hydrophobic Hinge Region of Rat DNA Polymerase β Is Critical for Substrate Binding Pocket Geometry

2005 ◽  
Vol 280 (31) ◽  
pp. 28388-28393 ◽  
Author(s):  
Daniela Starcevic ◽  
Shibani Dalal ◽  
Joachim Jaeger ◽  
Joann B. Sweasy
Keyword(s):  
Dna Polymerase ◽  
Substrate Binding ◽  
Binding Pocket ◽  
Hinge Region ◽  
Dna Polymerase Β ◽  
Substrate Binding Pocket
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