Site Directed Mutagenesis of the E coli Gcd Gene

Goldstein, Alan

Abstract

Quinoproteins have emerged as the third major class of dehydrogenases and are essential to the bioenergetics of many bacterial genera. Quinoproteins are also present in higher eukaryotes and probably participate in essential processes such as tissue development and differentiation in humans, animals and plants. Information about quinoprotein dehydrogenases is severely limited compared to NADP- dependent and flavin-containing dehydrogenases. Therefore, fundamental molecular genetic and biochemical studies of this class of enzymes are essential. The long term objective is to enhance basic knowledge of quinoprotein structure and function. Specifically, they will use the E. coli quinoprotein glucose dehydrogenase to study this class of proteins. Analysis of the deduced amino acid sequences of 9 cloned PQQ- dehydrogenases has revealed a 'highly conserved' region that includes both identical and similar amino acids. It was proposed that this domain plays an essential role in enzyme structure/function. Importantly, the highly conserved region is present in the soluble glucose dehydrogenase of A. calcoaceticus , an enzyme that shares no other homology with the 8 remaining PQQ-dehydrogenases. They have used site-directed mutagenesis to alanine-scan four amino acids from the highly conserved region that are identical in all nine proteins. They have assayed 3 of these mutants and shown that enzyme activity is greatly reduced. They have also replaced 3 of these sites with amino acids with drastically different R groups and obtained two knockouts and a near knockout (7%. of control). Preliminary data show that the knockouts are not the result of a trivial failure in folding or transport to the periplasm. Structural models of GDH have been developed in this laboratory and in Chris Anthony's laboratory using the crystal structure of the quinoprotein methanol dehydrogenase (MDH) published by Anthony and coworkers. In both GDH and NMH, the highly conserved region is part of a beta-propeller fold motif that has been reported in other proteins including the influenza virus neuraminidase. They expect to contribute to the basic understanding of protein structure as well as the unique mechanism(s) of quinoprotein dehydrogenases. A second specific aim of this project is to strengthen the research environment within the Division of Biology at Alfred University and promote the integration of research and education in this predominantly undergraduate institution.