Expression, purification, kinetic, and structural characterization of an alpha-class carbonic anhydrase from Aedes aegypti (AaCA1).
Carbonic anhydrases (CAs) are zinc-containing metalloenzymes that catalyze the interconversion of carbon dioxide and bicarbonate. The alpha-class CAs are found predominantly in vertebrates, but they are also expressed in insects like mosquitoes. Recently, an alpha-CA from the midgut of Aedes aegypti larvae (AaCA1) was identified, cloned, and subsequently shown to share high sequence homologous to human CA I (HCA I). This paper presents the bacterial expression, purification, and kinetic characterization of the soluble CA domain of AaCA1. The data show AaCA1 is a highly active CA that displays inhibition by methazolamide and ethoxzolamide with nM affinity. Additionally, a homology model of AaCA1, based on the crystal structure of HCA I, is presented and the overall structure, active site, and surface charge properties are compared to those of HCA I and II. Measurements of catalysis show that AaCA1 is more like HCA II in terms of proton transfer, but more similar to HCA I in terms of conversion of carbon dioxide to bicarbonate, and these differences are rationalized in terms of structure. These results also indicate that amino acid differences in the active site of AaCA1 compared to human CAs could be used to design specific CA inhibitors for the management of mosquito populations.
Fisher
S Zoë
SZ
Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL 32610, USA.
Tariku
Iyerus
I
Case
Nicolette M
NM
Tu
Chingkuang
C
Seron
Teri
T
Silverman
David N
DN
Linser
Paul J
PJ
McKenna
Robert
R
D000330:Aedes / Q000201:enzymology* / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D002256:Carbonic Anhydrases / Q000737:chemistry / Q000145:classification / Q000235:genetics* / Q000378:metabolism*; D020134:Catalytic Domain; D015870:Gene Expression; D017344:Genes, Insect; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D055672:Static Electricity
D011994:Recombinant Proteins; D002256:Carbonic Anhydrases