Biochemical and Structural Studies of N5-Carboxyaminoimidazole Ribonucleotide Mutase from the Acidophilic Bacterium Acetobacter aceti

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• 作者：Charles Z. Constantine ; Courtney M. Starks ; Christopher P. Mill ; Aaron E. Ransome ; Steven J. Karpowicz ; Julie A. Francois ; Rena A. Goodman ; and T. Joseph Kappock
• 刊名：Biochemistry
• 出版年：2006
• 出版时间：July 11, 2006
• 年：2006
• 卷：45
• 期：27
• 页码：8193 - 8208
• 全文大小：508K
• 年卷期：v.45,no.27(July 11, 2006)
• ISSN：1520-4995

文摘

N⁵-Carboxyaminoimidazole ribonucleotide (N⁵-CAIR) mutase (PurE) catalyzes the reversibleinterconversion of acid-labile compounds N⁵-CAIR and 4-carboxy-5-aminoimidazole ribonucleotide (CAIR).We have examined PurE from the acidophilic bacterium Acetobacter aceti (AaPurE), focusing on itsadaptation to acid pH and the roles of conserved residues His59 and His89. Both AaPurE and Escherichiacoli PurE showed quasi-reversible acid-mediated inactivation, but wt AaPurE was much more stable atpH 3.5, with a mages/entities/ge.gif">20 mages/entities/deg.gif">C higher thermal unfolding temperature at all pHs. His89 is not essential and doesnot function as part of a proton relay system. The k_cat pH-rate profile was consistent with the assignmentof pK₁ to unproductive protonation of bound nucleotide and pK₂ to deprotonation of His59. A 1.85 Åresolution crystal structure of the inactive mutant H59N-AaPurE soaked in CAIR showed that protonationof CAIR C4 can occur in the absence of His59. The resulting species, modeled as isoCAIR [4(R)-carboxy-5-iminoimidazoline ribonucleotide], is strongly stabilized by extensive interactions with the enzyme anda water molecule. The carboxylate moiety is positioned in a small pocket proposed to facilitate nucleotidedecarboxylation in the forward direction (N⁵-CAIR mages/entities/rarr.gif"> CAIR) [Meyer, E., Kappock, T. J., Osuji, C., andStubbe, J. (1999) Biochemistry 38, 3012-3018]. Comparisons with model studies suggest that in thereverse (nonbiosynthetic) direction PurE favors protonation of CAIR C4. We suggest that the essentialrole of protonated His59 is to lower the barrier to decarboxylation by stabilizing a CO₂-azaenolateintermediate.