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2004 Annual Science Report

Pennsylvania State University Reporting  |  JUL 2003 – JUN 2004

Enzymes of Ancient Metabolic Pathways

4 Institutions
3 Teams
0 Publications
0 Field Sites
Field Sites

Project Progress

Since 1933, carbonic anhydrase research has focused on enzymes from mammals (α class) and plants (β class); however, two additional classes (γ and δ) were discovered recently. Cam, from the procaryote Methanosarcina thermophila, is the prototype of the γ class and the first carbonic anhydrase to be characterized from either an anaerobic organism or the Archaea domain. All enzymes characterized from the four classes have been purified aerobically and are reported to contain a catalytic zinc. Herein, we report the anaerobic reconstitution of apo-Cam with Fe2+ which yielded Cam with an effective kcat which exceeded that for the Zn2+-reconstituted enzyme. Mössbauer spectroscopy showed that the Fe2+-reconstituted enzyme contained high-spin Fe2+ that when oxidized to Fe+3 inactivated the enzyme. Reconstitution with Fe3 was unsuccessful. Reconstitution with Cu2, Mn2+, Ni2+, or Cd2+ yielded enzymes with effective kcat values that were 10% or less than the value for the Zn2+-reconstituted Cam. Cam produced in Escherichia coli and purified anaerobically contained iron with effective kcat and kcat/Km values exceeding the values for Zn2+-reconstituted Cam. The results identify a previously unrecognized biological function for iron.

    Francisco Cruz
    Doctoral Student

    Caleb Bell III
    Undergraduate Student

    Objective 1.1
    Models of formation and evolution of habitable planets

    Objective 3.2
    Origins and evolution of functional biomolecules

    Objective 3.3
    Origins of energy transduction

    Objective 4.1
    Earth's early biosphere

    Objective 4.2
    Foundations of complex life

    Objective 5.1
    Environment-dependent, molecular evolution in microorganisms

    Objective 5.3
    Biochemical adaptation to extreme environments

    Objective 6.1
    Environmental changes and the cycling of elements by the biota, communities, and ecosystems

    Objective 6.2
    Adaptation and evolution of life beyond Earth