Structure of a NADH-Insensitive Hexameric Citrate Synthase that Resists Acid Inactivation

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• 作者：Julie A. Francois ; Courtney M. Starks ; Sasitorn Sivanuntakorn ; Hong Jiang ; Aaron E. Ransome ; Jeong-Won Nam ; Charles Z. Constantine ; T. Joseph Kappock
• 刊名：Biochemistry
• 出版年：2006
• 出版时间：November 14, 2006
• 年：2006
• 卷：45
• 期：45
• 页码：13487 - 13499
• 全文大小：618K
• 年卷期：v.45,no.45(November 14, 2006)
• ISSN：1520-4995

文摘

Acetobacter aceti converts ethanol to acetic acid, and strains highly resistant to both are usedto make vinegar. A. aceti survives acetic acid exposure by tolerating cytoplasmic acidification, whichimplies an unusual adaptation of cytoplasmic components to acidic conditions. A. aceti citrate synthase(AaCS), a hexameric type II citrate synthase, is required for acetic acid resistance and, therefore, wouldbe expected to function at low pH. Recombinant AaCS has intrinsic acid stability that may be a consequenceof strong selective pressure to function at low pH, and unexpectedly high thermal stability for a proteinthat has evolved to function at ~30 mages/entities/deg.gif">C. The crystal structure of AaCS, complexed with oxaloacetate(OAA) and the inhibitor carboxymethyldethia-coenzyme A (CMX), was determined to 1.85 Å resolutionusing protein purified by a tandem affinity purification procedure. This is the first crystal structure of a"closed" type II CS, and its active site residues interact with OAA and CMX in the same manner observedin the corresponding type I chicken CS·OAA·CMX complex. While AaCS is not regulated by NADH, itretains many of the residues used by Escherichia coli CS (EcCS) for NADH binding. The surface ofAaCS is abundantly decorated with basic side chains and has many fewer uncompensated acidic chargesthan EcCS; this constellation of charged residues is stable in varied pH environments and may beadvantageous in the A. aceti cytoplasm.