Cloning, characterization and anion inhibition studies of a new γ-carbonic anhydrase from the Antarctic bacterium Pseudoalteromonas haloplanktis

Abstract

A new γ-class carbonic anhydrase (CA, EC 4.2.1.1) was cloned, purified and characterized from the Antarctic bacterium Pseudoalteromonas haloplanktis, PhaCAγ. The enzyme has a medium-low catalytic activity for the physiologic reaction of CO2 hydration to bicarbonate and protons, with a kcat of 1.4×105s−1 and a kcat/Km of 1.9×106M−1s−1. An anion inhibition study of PhaCAγ with inorganic anions and small molecule inhibitors is also reported. Many anions present in sea water, such as chloride, fluoride, sulfate, iodide, but also others such as azide, perchlorate and tetrafluoroborate did not inhibit this enzyme. Pseudohalides such as cyanate, thiocyanate, cyanide, selenocyanide, and also bicarbonate, nitrate, nitrite and many complex inorganic anions showed inhibition in the millimolar range (KI in the range of 1.7–9.3mM). The best PhaCAγ inhibitors detected in this study were diethyldithiocarbamate (KI of 0.96mM) as well as sulfamide, sulfamate, phenylboronic acid and phenylarsonic acid (KI in the range of 82–91μM). Since γ-CAs are poorly understood at this moment, being present in carboxysomes and thus involved in photosynthesis, this study may be relevant for a better understanding of these processes in Antarctic bacteria/cyanobacteria.