Medline/PubMed Citation | Related Articles in PubMed | Download to Citation Manager

Biochem. J. (2007) 404 (121–130) (Printed in Great Britain)

Enhanced Full Text

PDF

Legacy HTML

Other papers of interest

Send to a friend

Add a comment

Functional domains and interdomain communication in Candida albicans glucosamine-6-phosphate synthase

Jarosaw OLCHOWY, Iwona GABRIEL and Sawomir MILEWSKI¹

Department of Pharmaceutical Technology and Biochemistry, Gdask University of Technology, 11/12 Narutowicza St., 80-952 Gdask, Poland

Functional and structural properties of several truncated or mutated variants of Candida albicans Gfa1p (glucosamine-6-phosphate synthase) were compared with those of the wild-type enzyme. Fragments encompassing residues 1–345 and 346–712 of Gfa1p, expressed heterogeneously in bacterial host as His₆ fusions, were identified as the functional GAH (glutamine amidehydrolysing) and ISOM (hexose phosphate-isomerizing) domains respectively. It was found that the native GAH domain is monomeric, whereas the native ISOM domain forms tetramers, as does the whole enzyme. Spectrofluorimetric and kinetic studies of the isolated domains, the D218–283Gfa1p mutein and the wild-type enzyme revealed that the binding site for the feedback inhibitor, uridine 5´-diphospho-N-acetyl-D-glucosamine, is located in the ISOM domain. Inhibitor binding affects amidohydrolysing activity of the GAH domain and, as a consequence, the GlcN-6-P (D-glucosamine-6-phosphate)-synthetic activity of the whole enzyme. The fragment containing residues 218–283 is neither involved in ligand binding nor in protein oligomerization. Comparison of the catalytic activities of Gfa1p^V711F, D709–712Gfa1p, Gfa1p^W97F and Gfa1p^W97G with those of the native Gfa1p and the isolated domains provided evidence for an intramolecular channel connecting the GAH and ISOM domains of Gfa1p. The channel becomes leaky upon deletion of amino acids 709–712 and in the W97F and W97G mutants. The Trp⁹⁷ residue was found to function as a molecular gate, opening and closing the channel. The W97G and V711F mutations resulted in an almost complete elimination of the GlcN-6-P-synthetic activity, with the retention of the amidohydrolase and sugar phosphate-isomerizing activities.

Key words: amidotransferase, glucosamine-6-phosphate synthase (Gfa1p), glutamine amide hydrolysing (GAH) domain, hexosephosphate-isomerizing (ISOM) domain, site-directed mutagenesis, intramolecular channel.

Abbreviations used: CPS, carbamoyl phosphate synthetase; DTT, dithiothreitol; Fru-6-P, D-fructose-6-phosphate; GAH, glutamine amide hydrolysing; GAHp–His₆, His-tagged C. albicans GAH domain; Gfa1p, glucosamine-6-phosphate synthase; GlcN-6-P, D-glucosamine-6-phosphate; Gln, glutamine; Glu, glutamate; GLUPA, g-glutamyl-p-nitroanilide; His₆–ISOMp, His-tagged C. albicans ISOM domain; IDA, ion-dependent adhesion; ISOM, hexosephosphate-isomerizing; LB, Luria–Bertani; PKA, protein kinase A; UDP-GlcNAc, uridine 5´-diphospho-N-acetyl-D-glucosamine.

¹To whom correspondence should be addressed (email milewski@chem.pg.gda.pl).

Received 3 October 2006/7 February 2007; accepted 19 February 2007

Published as BJ Immediate Publication 19 February 2007, doi:10.1042/BJ20061502

© 2007 Biochemical Society