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J Mol Biol. 2009 Feb 20;386(2):486-503. doi: 10.1016/j.jmb.2008.12.037. Epub 2008 Dec 24.

Structure and function of 2,3-dimethylmalate lyase, a PEP mutase/isocitrate lyase superfamily member.

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  • 1Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute, Rockville, 20850, USA.

Abstract

The Aspergillus niger genome contains four genes that encode proteins exhibiting greater than 30% amino acid sequence identity to the confirmed oxaloacetate acetyl hydrolase (OAH), an enzyme that belongs to the phosphoenolpyruvate mutase/isocitrate lyase superfamily. Previous studies have shown that a mutant A. niger strain lacking the OAH gene does not produce oxalate. To identify the function of the protein sharing the highest amino acid sequence identity with the OAH (An07g08390, Swiss-Prot entry Q2L887, 57% identity), we produced the protein in Escherichia coli and purified it for structural and functional studies. A focused substrate screen was used to determine the catalytic function of An07g08390 as (2R,3S)-dimethylmalate lyase (DMML): k(cat)=19.2 s(-1) and K(m)=220 microM. DMML also possesses significant OAH activity (k(cat)=0.5 s(-1) and K(m) =220 microM). DNA array analysis showed that unlike the A. niger oah gene, the DMML encoding gene is subject to catabolite repression. DMML is a key enzyme in bacterial nicotinate catabolism, catalyzing the last of nine enzymatic steps. This pathway does not have a known fungal counterpart. BLAST analysis of the A. niger genome for the presence of a similar pathway revealed the presence of homologs to only some of the pathway enzymes. This and the finding that A. niger does not thrive on nicotinamide as a sole carbon source suggest that the fungal DMML functions in a presently unknown metabolic pathway. The crystal structure of A. niger DMML (in complex with Mg(2+) and in complex with Mg(2+) and a substrate analog: the gem-diol of 3,3-difluoro-oxaloacetate) was determined for the purpose of identifying structural determinants of substrate recognition and catalysis. Structure-guided site-directed mutants were prepared and evaluated to test the contributions made by key active-site residues. In this article, we report the results in the broader context of the lyase branch of the phosphoenolpyruvate mutase/isocitrate lyase superfamily to provide insight into the evolution of functional diversity.

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