skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: A High-Affinity Adenosine Kinase from Anopheles Gambiae

Abstract

Genome analysis revealed a mosquito orthologue of adenosine kinase in Anopheles gambiae (AgAK; the most important vector for the transmission of Plasmodium falciparum in Africa). P. falciparum are purine auxotrophs and do not express an adenosine kinase but rely on their hosts for purines. AgAK was kinetically characterized and found to have the highest affinity for adenosine (K{sub m} = 8.1 nM) of any known adenosine kinase. AgAK is specific for adenosine at the nucleoside site, but several nucleotide triphosphate phosphoryl donors are tolerated. The AgAK crystal structure with a bound bisubstrate analogue Ap{sub 4}A (2.0 {angstrom} resolution) reveals interactions for adenosine and ATP and the geometry for phosphoryl transfer. The polyphosphate charge is partly neutralized by a bound Mg{sup 2+} ion and an ion pair to a catalytic site Arg. The AgAK structure consists of a large catalytic core in a three-layer {alpha}/{beta}/{alpha} sandwich, and a small cap domain in contact with adenosine. The specificity and tight binding for adenosine arise from hydrogen bond interactions of Asn14, Leu16, Leu40, Leu133, Leu168, Phe168, and Thr171 and the backbone of Ile39 and Phe168 with the adenine ring as well as through hydrogen bond interactions between Asp18, Gly64, and Asn68 and themore » ribosyl 2'- and 3'-hydroxyl groups. The structure is more similar to that of human adenosine kinase (48% identical) than to that of AK from Toxoplasma gondii (31% identical). With this extraordinary affinity for AgAK, adenosine is efficiently captured and converted to AMP at near the diffusion limit, suggesting an important role for this enzyme in the maintenance of the adenine nucleotide pool. mRNA analysis verifies that AgAK transcripts are produced in the adult insects.« less

Authors:
; ; ; ; ; ;
Publication Date:
Research Org.:
BROOKHAVEN NATIONAL LABORATORY (BNL)
Sponsoring Org.:
USDOE SC OFFICE OF SCIENCE (SC)
OSTI Identifier:
1041940
Report Number(s):
BNL-97618-2012-JA
Journal ID: ISSN 0006-2960; BICHAW; TRN: US201212%%351
DOE Contract Number:
DE-AC02-98CH10886
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemistry (Eaton); Journal Volume: 50; Journal Issue: 11
Country of Publication:
United States
Language:
English
Subject:
08 HYDROGEN; ADENINES; ADENOSINE; ADULTS; AFFINITY; CRYSTAL STRUCTURE; DIFFUSION; ENZYMES; GEOMETRY; HYDROGEN; INSECTS; ION PAIRS; MOSQUITOES; NUCLEOSIDES; NUCLEOTIDES; PHOSPHOTRANSFERASES; PLASMODIUM; PURINES; RESOLUTION; SPECIFICITY; VECTORS

Citation Formats

M Cassera, M Ho, E Merino, E Burgos, A Rinaldo-Matthis, S Almo, and V Schramm. A High-Affinity Adenosine Kinase from Anopheles Gambiae. United States: N. p., 2011. Web. doi:10.1021/bi101921w.
M Cassera, M Ho, E Merino, E Burgos, A Rinaldo-Matthis, S Almo, & V Schramm. A High-Affinity Adenosine Kinase from Anopheles Gambiae. United States. doi:10.1021/bi101921w.
M Cassera, M Ho, E Merino, E Burgos, A Rinaldo-Matthis, S Almo, and V Schramm. Sat . "A High-Affinity Adenosine Kinase from Anopheles Gambiae". United States. doi:10.1021/bi101921w.
@article{osti_1041940,
title = {A High-Affinity Adenosine Kinase from Anopheles Gambiae},
author = {M Cassera and M Ho and E Merino and E Burgos and A Rinaldo-Matthis and S Almo and V Schramm},
abstractNote = {Genome analysis revealed a mosquito orthologue of adenosine kinase in Anopheles gambiae (AgAK; the most important vector for the transmission of Plasmodium falciparum in Africa). P. falciparum are purine auxotrophs and do not express an adenosine kinase but rely on their hosts for purines. AgAK was kinetically characterized and found to have the highest affinity for adenosine (K{sub m} = 8.1 nM) of any known adenosine kinase. AgAK is specific for adenosine at the nucleoside site, but several nucleotide triphosphate phosphoryl donors are tolerated. The AgAK crystal structure with a bound bisubstrate analogue Ap{sub 4}A (2.0 {angstrom} resolution) reveals interactions for adenosine and ATP and the geometry for phosphoryl transfer. The polyphosphate charge is partly neutralized by a bound Mg{sup 2+} ion and an ion pair to a catalytic site Arg. The AgAK structure consists of a large catalytic core in a three-layer {alpha}/{beta}/{alpha} sandwich, and a small cap domain in contact with adenosine. The specificity and tight binding for adenosine arise from hydrogen bond interactions of Asn14, Leu16, Leu40, Leu133, Leu168, Phe168, and Thr171 and the backbone of Ile39 and Phe168 with the adenine ring as well as through hydrogen bond interactions between Asp18, Gly64, and Asn68 and the ribosyl 2'- and 3'-hydroxyl groups. The structure is more similar to that of human adenosine kinase (48% identical) than to that of AK from Toxoplasma gondii (31% identical). With this extraordinary affinity for AgAK, adenosine is efficiently captured and converted to AMP at near the diffusion limit, suggesting an important role for this enzyme in the maintenance of the adenine nucleotide pool. mRNA analysis verifies that AgAK transcripts are produced in the adult insects.},
doi = {10.1021/bi101921w},
journal = {Biochemistry (Eaton)},
number = 11,
volume = 50,
place = {United States},
year = {Sat Dec 31 00:00:00 EST 2011},
month = {Sat Dec 31 00:00:00 EST 2011}
}
  • No abstract prepared.
  • Serine protease inhibitors (serpins) in insects function within development, wound healing and immunity. The genome of the African malaria vector,Anopheles gambiae, encodes 23 distinct serpin proteins, several of which are implicated in disease-relevant physiological responses.A. gambiaeserpin 18 (SRPN18) was previously categorized as non-inhibitory based on the sequence of its reactive-center loop (RCL), a region responsible for targeting and initiating protease inhibition. The crystal structure ofA. gambiaeSRPN18 was determined to a resolution of 1.45 Å, including nearly the entire RCL in one of the two molecules in the asymmetric unit. The structure reveals that the SRPN18 RCL is extremely short andmore » constricted, a feature associated with noncanonical inhibitors or non-inhibitory serpin superfamily members. Furthermore, the SRPN18 RCL does not contain a suitable protease target site and contains a large number of prolines. The SRPN18 structure therefore reveals a unique RCL architecture among the highly conserved serpin fold.« less
  • The purine salvage pathway of Anopheles gambiae, a mosquito that transmits malaria, has been identified in genome searches on the basis of sequence homology with characterized enzymes. Purine nucleoside phosphorylase (PNP) is a target for the development of therapeutic agents in humans and purine auxotrophs, including malarial parasites. The PNP from Anopheles gambiae (AgPNP) was expressed in Escherichia coli and compared to the PNPs from Homo sapiens (HsPNP) and Plasmodium falciparum (PfPNP). AgPNP has kcat values of 54 and 41 s-1 for 2'-deoxyinosine and inosine, its preferred substrates, and 1.0 s-1 for guanosine. However, the chemical step is fast formore » AgPNP at 226 s-1 for guanosine in pre-steady-state studies. 5'-Deaza-1'-aza-2'-deoxy-1'-(9-methylene)-Immucillin-H (DADMe-ImmH) is a transition-state mimic for a 2'-deoxyinosine ribocation with a fully dissociated N-ribosidic bond and is a slow-onset, tight-binding inhibitor with a dissociation constant of 3.5 pM. This is the tightest-binding inhibitor known for any PNP, with a remarkable Km/Ki* of 5.4 x 107, and is consistent with enzymatic transition state predictions of enhanced transition-state analogue binding in enzymes with enhanced catalytic efficiency. Deoxyguanosine is a weaker substrate than deoxyinosine, and DADMe-Immucillin-G is less tightly bound than DADMe-ImmH, with a dissociation constant of 23 pM for AgPNP as compared to 7 pM for HsPNP. The crystal structure of AgPNP was determined in complex with DADMe-ImmH and phosphate to a resolution of 2.2 Angstroms to reveal the differences in substrate and inhibitor specificity. The distance from the N1' cation to the phosphate O4 anion is shorter in the AgPNP{center_dot}DADMe-ImmH{center_dot}PO4 complex than in HsPNP{center_dot}DADMe-ImmH{center_dot}SO4, offering one explanation for the stronger inhibitory effect of DADMe-ImmH for AgPNP.« less
  • The leucine-rich repeat (LRR) proteins LRIM1 and APL1C control the function of the complement-like protein TEP1 in Anopheles mosquitoes. The molecular structure of LRIM1 and APL1C and the basis of their interaction with TEP1 represent a new type of innate immune complex. The LRIM1/APL1C complex specifically binds and solubilizes a cleaved form of TEP1 without an intact thioester bond. The LRIM1 and APL1C LRR domains have a large radius of curvature, glycosylated concave face, and a novel C-terminal capping motif. The LRIM1/APL1C complex is a heterodimer with a single intermolecular disulfide bond. The structure of the LRIM1/APL1C heterodimer reveals anmore » interface between the two LRR domains and an extensive C-terminal coiled-coil domain. We propose that a cleaved form of TEP1 may act as a convertase for activation of other TEP1 molecules and that the LRIM1/APL1C heterodimer regulates formation of this TEP1 convertase.« less