Single Amino Acid Substitutions Disrupt Tetramer Formation in the Dihydroneopterin Aldolase Enzyme of Pneumocystis carinii

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• 作者：M. Carmen Thomas ; Stuart P. Ballantine ; Susanne S. Bethell ; Satty Bains ; Paul Kellam ; and Chris J. Delves
• 刊名：Biochemistry
• 出版年：1998
• 出版时间：August 18, 1998
• 年：1998
• 卷：37
• 期：33
• 页码：11629 - 11636
• 全文大小：141K
• 年卷期：v.37,no.33(August 18, 1998)
• ISSN：1520-4995

文摘

In the opportunistic pathogen Pneumocystis carinii, dihydroneopterin aldolase function isexpressed as the N-terminal portion of the multifunctional folic acid synthesis protein (Fas). This regionencompasses two domains, FasA and FasB, which are 27% amino acid identical. FasA and FasB alsoshare significant amino acid sequence similarity with bacterial dihydroneopterin aldolases. In the presentstudy, this enzyme function has been overproduced as an independent monofunctional activity in Escherichiacoli. Recombinant FasAB-Met23 (amino acids 23-290 of the predicted open reading frame) was purifiedand shown to contain dihydroneopterin aldolase activity. The native FasAB-Met23 is a tetramer of the30-kDa subunit, demonstrating characteristics of an associating-dissociating equilibrium system in whichonly the multimeric form of the enzyme is active. Multiple sequence alignment of FasA and FasB withother dihydroneopterin aldolases highlights only three positions where the amino acid is invariable betweenall the predicted proteins. The role of these conserved amino acid residues in enzyme function wasinvestigated using site-directed mutagenesis. Mutant FasAB-Met23 species were overproduced and purifiedto near homogeneity. Three FasA domain mutants and two FasB domain mutants had little or no detectabledihydroneopterin aldolase activity, implicating both FasA and FasB in the catalytic mechanism. We showthat each mutant protein containing an inactivating amino acid substitution has lost its ability to formstable tetramers.