scholarly journals Disruption of the crossover helix impairs dihydrofolate reductase activity in the bifunctional enzyme TS–DHFR from Cryptosporidium hominis

2009 ◽  
Vol 417 (3) ◽  
pp. 757-764 ◽  
Author(s):  
Melissa A. Vargo ◽  
W. Edward Martucci ◽  
Karen S. Anderson
Keyword(s):  
Dihydrofolate Reductase ◽  
Active Site ◽  
Reductase Activity ◽  
The Other ◽  
Site Directed Mutagenesis ◽  
Mutant Enzyme ◽  
Bifunctional Enzyme ◽  
Cryptosporidium Hominis ◽  
Single Polypeptide Chain ◽  
Species Specific

In contrast with most species, including humans, which have monofunctional forms of the folate biosynthetic enzymes TS (thymidylate synthase) and DHFR (dihydrofolate reductase), several pathogenic protozoal parasites, including Cryptosporidium hominis, contain a bifunctional form of the enzymes on a single polypeptide chain having both catalytic activities. The crystal structure of the bifunctional enzyme TS–DHFR C. hominis reveals a dimer with a ‘crossover helix’, a swap domain between DHFR domains, unique in that this helical region from one monomer makes extensive contacts with the DHFR active site of the other monomer. In the present study, we used site-directed mutagenesis to probe the role of this crossover helix in DHFR catalysis. Mutations were made to the crossover helix: an ‘alanine-face’ enzyme in which the residues on the face of the helix close to the DHFR active site of the other subunit were mutated to alanine, a ‘glycine-face’ enzyme in which the same residues were mutated to glycine, and an ‘all-alanine’ helix in which all residues of the helix were mutated to alanine. These mutant enzymes were studied using a rapid transient kinetic approach. The mutations caused a dramatic decrease in the DHFR activity. The DHFR catalytic activity of the alanine-face mutant enzyme was 30 s−1, the glycine-face mutant enzyme was 17 s−1, and the all-alanine helix enzyme was 16 s−1, all substantially impaired from the wild-type DHFR activity of 152 s−1. It is clear that loss of helix interactions results in a marked decrease in DHFR activity, supporting a role for this swap domain in DHFR catalysis. The crossover helix provides a unique structural feature of C. hominis bifunctional TS–DHFR that could be exploited as a target for species-specific non-active site inhibitors.

R67, the Other Dihydrofolate Reductase:  Rational Design of an Alternate Active Site Configuration†

Biochemistry ◽  
2008 ◽  
Vol 47 (2) ◽  
pp. 555-565 ◽  
Author(s):  
Jian Feng ◽  
Sumit Goswami ◽  
Elizabeth E. Howell
Keyword(s):  
Dihydrofolate Reductase ◽  
Active Site ◽  
Rational Design ◽  
The Other

scholarly journals Exploring novel strategies for AIDS protozoal pathogens: α-helix mimetics targeting a key allosteric protein–protein interaction in C. hominis thymidylate synthase-dihydrofolate reductase (TS-DHFR)

MedChemComm ◽  
2013 ◽  
Vol 4 (9) ◽  
pp. 1247-1256 ◽  
Author(s):  
W. Edward Martucci ◽  
Johanna M. Rodriguez ◽  
Melissa A. Vargo ◽  
Matthew Marr ◽  
Andrew D. Hamilton ◽  
...  
Keyword(s):  
Dihydrofolate Reductase ◽  
Protein Interaction ◽  
Thymidylate Synthase ◽  
Opportunistic Infections ◽  
Molecular Target ◽  
Bifunctional Enzyme ◽  
Cryptosporidium Hominis ◽  
Protein Protein Interaction ◽  
Helix Mimetics ◽  
Α Helix

The bifunctional enzyme TS–DHFR from Cryptosporidium hominis is a molecular target for design of antiparasitic therapies for AIDS-related opportunistic infections.

scholarly journals Dipeptidyl Aminopeptidase IV from Stenotrophomonas maltophilia Exhibits Activity against a Substrate Containing a 4-Hydroxyproline Residue

2008 ◽  
Vol 190 (23) ◽  
pp. 7819-7829 ◽  
Author(s):  
Yoshitaka Nakajima ◽  
Kiyoshi Ito ◽  
Tsubasa Toshima ◽  
Takashi Egawa ◽  
Heng Zheng ◽  
...  
Keyword(s):  
Active Site ◽  
Stenotrophomonas Maltophilia ◽  
Site Directed Mutagenesis ◽  
Mutant Enzyme ◽  
Subunit Structure ◽  
Hydrophobic Pocket ◽  
Cell Parameters ◽  
Replacement Method ◽  
Major Factors ◽  
Dipeptidyl Aminopeptidase

ABSTRACT The crystal structure of dipeptidyl aminopeptidase IV from Stenotrophomonas maltophilia was determined at 2.8-Å resolution by the multiple isomorphous replacement method, using platinum and selenomethionine derivatives. The crystals belong to space group P43212, with unit cell parameters a = b = 105.9 Å and c = 161.9 Å. Dipeptidyl aminopeptidase IV is a homodimer, and the subunit structure is composed of two domains, namely, N-terminal β-propeller and C-terminal catalytic domains. At the active site, a hydrophobic pocket to accommodate a proline residue of the substrate is conserved as well as those of mammalian enzymes. Stenotrophomonas dipeptidyl aminopeptidase IV exhibited activity toward a substrate containing a 4-hydroxyproline residue at the second position from the N terminus. In the Stenotrophomonas enzyme, one of the residues composing the hydrophobic pocket at the active site is changed to Asn611 from the corresponding residue of Tyr631 in the porcine enzyme, which showed very low activity against the substrate containing 4-hydroxyproline. The N611Y mutant enzyme was generated by site-directed mutagenesis. The activity of this mutant enzyme toward a substrate containing 4-hydroxyproline decreased to 30.6% of that of the wild-type enzyme. Accordingly, it was considered that Asn611 would be one of the major factors involved in the recognition of substrates containing 4-hydroxyproline.

scholarly journals Conversion of citrate synthase into citryl-CoA lyase as a result of mutation of the active-site aspartic acid residue to glutamic acid

1991 ◽  
Vol 280 (2) ◽  
pp. 521-526 ◽  
Author(s):  
W J Man ◽  
Y Li ◽  
C D O'Connor ◽  
D C Wilton
Keyword(s):  
Catalytic Activity ◽  
Aspartic Acid ◽  
Active Site ◽  
Citrate Synthase ◽  
Condensation Reaction ◽  
Site Directed Mutagenesis ◽  
Mutant Enzyme ◽  
Reverse Direction ◽  
Acetyl Coa ◽  
Aspartic Acid Residue

The active-site aspartic acid residue, Asp-362, of Escherichia coli citrate synthase was changed by site-directed mutagenesis to Glu-362, Asn-362 or Gly-362. Only very low catalytic activity could be detected with the Asp→Asn and Asp→Gly mutations. The Asp→Glu mutation produced an enzyme that expressed about 0.8% of the overall catalytic rate, and the hydrolysis step in the reaction, monitored as citryl-CoA hydrolysis, was inhibited to a similar extent. However, the condensation reaction, measured in the reverse direction as citryl-CoA cleavage to oxaloacetate and acetyl-CoA, was not affected by the mutation, and this citryl-CoA lyase activity was the major catalytic activity of the mutant enzyme. This high condensation activity in an enzyme in which the subsequent hydrolysis step was about 98% inhibited permitted considerable exchange of the methyl protons of acetyl-CoA during catalysis by the mutant enzyme. The Km for oxaloacetate was not significantly altered in the D362E mutant enzyme, whereas the Km for acetyl-CoA was about 5 times lower. A mechanism is proposed in which Asp-362 is involved in the hydrolysis reaction of this enzyme, and not as a base in the deprotonation of acetyl-CoA as recently suggested by others. [Karpusas, Branchaud & Remington (1990) Biochemistry 29, 2213-2219; Alter, Casazza, Zhi, Nemeth, Srere & Evans, (1990) Biochemistry 29, 7557-7563].

scholarly journals Activation-Independent Cyclization of Monoterpenoids

2011 ◽  
Vol 78 (4) ◽  
pp. 1055-1062 ◽  
Author(s):  
Gabriele Siedenburg ◽  
Dieter Jendrossek ◽  
Michael Breuer ◽  
Benjamin Juhl ◽  
Jürgen Pleiss ◽  
...  
Keyword(s):  
Active Site ◽  
Zymomonas Mobilis ◽  
The Other ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Combinatorial Approach ◽  
Content Type ◽  
Oxidosqualene Cyclases ◽  
Terpene Cyclase ◽  
Unique Capability

ABSTRACTThe biosynthesis of cyclic monoterpenes (C10) generally requires the cyclization of an activated linear precursor (geranyldiphosphate) by specific terpene cyclases. Cyclic triterpenes (C30), on the other hand, originate from the linear precursor squalene by the action of squalene-hopene cyclases (SHCs) or oxidosqualene cyclases (OSCs). Here, we report a novel terpene cyclase fromZymomonas mobilis(ZMO1548-Shc) with the unique capability to cyclize citronellal to isopulegol. To our knowledge, ZMO1548-Shc is the first biocatalyst with diphosphate-independent monoterpenoid cyclase activity. A combinatorial approach using site-directed mutagenesis and modeling of the active site with a bound substrate revealed that the cyclization of citronellal proceeds via a different mechanism than that of the cyclization of squalene.

scholarly journals Tuning PFKFB3 Bisphosphatase Activity Through Allosteric Interference

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Helena Macut ◽  
Xiao Hu ◽  
Delia Tarantino ◽  
Ettore Gilardoni ◽  
Francesca Clerici ◽  
...  
Keyword(s):  
Small Molecules ◽  
Active Site ◽  
Analytical Method ◽  
Kinase Activity ◽  
The Other ◽  
Bifunctional Enzyme ◽  
Allosteric Site ◽  
Small Peptides ◽  
Microscale Thermophoresis ◽  
Binding Peptides

AbstractThe human inducible phospho-fructokinase bisphosphatase isoform 3, PFKFB3, is a crucial regulatory node in the cellular metabolism. The enzyme is an important modulator regulating the intracellular fructose-2,6-bisphosphate level. PFKFB3 is a bifunctional enzyme with an exceptionally high kinase to phosphatase ratio around 740:1. Its kinase activity can be directly inhibited by small molecules acting directly on the kinase active site. On the other hand, here we propose an innovative and indirect strategy for the modulation of PFKFB3 activity, achieved through allosteric bisphosphatase activation. A library of small peptides targeting an allosteric site was discovered and synthesized. The binding affinity was evaluated by microscale thermophoresis (MST). Furthermore, a LC-MS/MS analytical method for assessing the bisphosphatase activity of PFKFB3 was developed. The new method was applied for measuring the activation on bisphosphatase activity with the PFKFB3-binding peptides. The molecular mechanical connection between the newly discovered allosteric site to the bisphosphatase activity was also investigated using both experimental and computational methods.

scholarly journals Structural and Functional Adaptation of Vancomycin Resistance VanT Serine Racemases

mBio ◽  
2015 ◽  
Vol 6 (4) ◽  
Author(s):  
Djalal Meziane-Cherif ◽  
Peter J. Stogios ◽  
Elena Evdokimova ◽  
Olga Egorova ◽  
Alexei Savchenko ◽  
...  
Keyword(s):  
Active Site ◽  
Catalytic Domain ◽  
Vancomycin Resistance ◽  
Functional Adaptation ◽  
Site Directed Mutagenesis ◽  
Resistant Bacteria ◽  
Bifunctional Enzyme ◽  
Gram Positive ◽  
Antibiotic Resistant ◽  
Membrane Bound

ABSTRACTVancomycin resistance in Gram-positive bacteria results from the replacement of thed-alanyl–d-alanine target of peptidoglycan precursors withd-alanyl–d-lactate ord-alanyl–d-serine (d-Ala-d-Ser), to which vancomycin has low binding affinity. VanT is one of the proteins required for the production ofd-Ala-d-Ser-terminating precursors by convertingl-Ser tod-Ser. VanT is composed of two domains, an N-terminal membrane-bound domain, likely involved inl-Ser uptake, and a C-terminal cytoplasmic catalytic domain which is related to bacterial alanine racemases. To gain insight into the molecular function of VanT, the crystal structure of the catalytic domain of VanTGfrom VanG-type resistantEnterococcus faecalisBM4518 was determined. The structure showed significant similarity to type III pyridoxal 5′-phosphate (PLP)-dependent alanine racemases, which are essential for peptidoglycan synthesis. Comparative structural analysis between VanTGand alanine racemases as well as site-directed mutagenesis identified three specific active site positions centered around Asn696which are responsible for thel-amino acid specificity. This analysis also suggested that VanT racemases evolved from regular alanine racemases by acquiring additional selectivity toward serine while preserving that for alanine. The 4-fold-lower relative catalytic efficiency of VanTGagainstl-Ser versusl-Ala implied that this enzyme relies on its membrane-bound domain forl-Ser transport to increase the overall rate ofd-Ser production. These findings illustrate how vancomycin pressure selected for molecular adaptation of a housekeeping enzyme to a bifunctional enzyme to allow for peptidoglycan remodeling, a strategy increasingly observed in antibiotic-resistant bacteria.IMPORTANCEVancomycin is one of the drugs of last resort against Gram-positive antibiotic-resistant pathogens. However, bacteria have evolved a sophisticated mechanism which remodels the drug target, thed-alanine ending precursors in cell wall synthesis, into precursors terminating withd-lactate ord-serine, to which vancomycin has less affinity.d-Ser is synthesized by VanT serine racemase, which has two unusual characteristics: (i) it is one of the few serine racemases identified in bacteria and (ii) it contains a membrane-bound domain involved inl-Ser uptake. The structure of the catalytic domain of VanTGshowed high similarity to alanine racemases, and we identified three specific active site substitutions responsible forl-Ser specificity. The data provide the molecular basis for VanT evolution to a bifunctional enzyme coordinating both transport and racemization. Our findings also illustrate the evolution of the essential alanine racemase into a vancomycin resistance enzyme in response to antibiotic pressure.

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