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A set of C-terminal deletion mutants of the RecA protein of Escherichia coli, progressively removing 6, 13, 17, and 25 amino acid residues, has been generated, expressed, and purified. In vivo, the deletion of 13 to 17 C-terminal residues results in increased sensitivity to mitomycin C. In vitro, the deletions enhance binding to duplex DNA as previously observed. We demonstrate that much of this enhancement involves the deletion of residues between positions 339 and 346. In addition, the C-terminal deletions cause a substantial upward shift in the pH-reaction profile of DNA strand exchange reactions. The C-terminal deletions of more than 13 amino acid residues result in strong inhibition of DNA strand exchange below pH 7, where the wild-type protein promotes a proficient reaction. However, at the same time, the deletion of 13-17 C-terminal residues eliminates the reduction in DNA strand exchange seen with the wild-type protein at pH values between 7.5 and 9. The results suggest the existence of extensive interactions, possibly involving multiple salt bridges, between the C terminus and other parts of the protein. These interactions affect the pK a of key groups involved in DNA strand exchange as well as the direct binding of RecA protein to duplex DNA.

Abstract4-Oxalocrotonate tautomerase (4-OT) isozymes play prominent roles in the bacterial utilization of aromatic hydrocarbons as sole carbon sources. These enzymes catalyze the conversion of 2-hydroxy-2,4-hexadienedioate (or 2-hydroxymuconate) to 2-oxo-3-hexenedioate, where Pro-1 functions as a general base and shuttles a proton from the 2-hydroxyl group of substrate to the C-5 position of product. 4-OT, a homohexamer from Pseudomonas putida mt-2, is the most extensively studied 4-OT isozyme and the founding member of the tautomerase superfamily. A search of five thermophilic bacterial genomes identified a coded amino acid sequence in each that had been annotated as a tautomerase-like protein but lacked Pro-1. However, a nearby sequence has Pro-1, but the sequence is not annotated as a tautomerase-like protein. In order to characterize † This research was supported by the National Institutes of Health Grants GM-41239 (CPW) and AI-60915 (SDP), the Robert A.Welch Foundation grant F-1334 (CPW), and the Department of Defense Grant W81XWH0710445 USAMRAA (SDP). Use of the Advanced Photon Source is supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract N. W-31-109-Eng-38. The Analytical Instrumentation Facility Core (College of Pharmacy, The University of Texas at Austin) is supported by an NIH Center grant ES07784. * Corresponding authors (CPW) Tel: 512-471-6198; Fax: 512-232-2606; whitman@mail.utexas.edu. (SDP) Tel: 303-871-2533; Fax: 303-871-2254; scott.pegan@du.edu. The atomic coordinates and structure factors have been deposited with the Brookhaven Protein Data Bank (PDB codes 3MB2). 1 Abbreviations: Ap, ampicillin; BSA, bovine serum albumin; dNTPs, deoxyribose nucleotide triphosphates; CHMI, 5-(carboxymethyl)-2-hydroxymuconate isomerase; CaaD, trans-3-chloroacrylic acid dehalogenase; cis-CaaD, cis-3-chloroacrylic acid dehalogenase; DSC, differential scanning calorimetry; HEPES, N-2-hydroxyethylpiperazine-N′-2-ethane sulfonate; hh4-OT, heterohexamer 4-oxalocrotonate tautomerase; IPTG, isopropyl-β-D-thiogalactoside; Kn, kanamycin; LB, Luria-Bertani; MIF, macrophage migration inhibitory factor; MSAD, malonate semialdehyde decarboxylase; NCBI, National Center for Biotechnology Information; NMR, nuclear magnetic resonance; 4-OT, 4-oxalocrotonate tautomerase; PEG, polyethylene glycol; PPT, phenylpyruvate tautomerase; PCR, polymerase chain reaction; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis. SUPPORTING INFORMATION AVAILABLEThe expression, overproduction, and purification protocols for the hh4-OT are provided in the Supporting Information. The experimental procedures used for the construction, expression, overproduction, purification, and mass spectral analysis of the hh4-OT mutants and the construction and expression of the separate subunits of the hh4-OT are also provided in the Supporting Information. Finally, the molecular modeling studies are described. This material is available free of charge via the Internet at http://pubs.acs.org....

The organophosphorus nerve agents sarin, soman, tabun, and VX exert their toxic effects by inhibiting the action of human acetylcholinesterase, a member of the serine hydrolase superfamily of enzymes. The current treatments for nerve agent exposure must be administered quickly to be effective and they often do not eliminate long-term toxic side effects associated with organophosphate poisoning. Thus, there is significant need for effective prophylactic methods to protect at-risk personnel from nerve agent exposure, and protein-based approaches have emerged as promising candidates. We present the 2.7 Å resolution crystal structures of the serine hydrolase human carboxylesterase 1 (hCE1), a broad-spectrum drug metabolism enzyme, in covalent acylenzyme intermediate complexes with the chemical weapons soman and tabun. The structures reveal that hCE1 binds stereoselectively to these nerve agents; for example, hCE1 appears to react preferentially with the 10 4 -fold more lethal P S stereoisomer of soman relative to the P R form. In addition, structural features of the hCE1 active site indicate that the enzyme may be resistant to deadend organophosphate aging reactions that permanently inactivate other serine hydrolases. Taken together, these data provide important structural details toward the goal of engineering hCE1 into an organophosphate hydrolase and protein-based therapeutic for nerve agent exposure.The organophosphorus (OP) nerve agents sarin, soman, tabun, and VX are among the deadliest man-made chemicals (1). While the military use of OP nerve agents is widely banned, these compounds have been employed in recent decades by rogue states and terrorist groups. In 1988, Iraq employed weaponized sarin against its own Kurdish citizens in Halabja, a town adjacent to the Iranian border, killing an estimated 5,000 (2). Coordinated attacks on the Tokyo subway system by the Japanese Aum Shinrikyo cult in 1995 also employed sarin, killing 12 and injuring † This work was supported, in part, by NIH grants CA98468 and NS58089, and the American Lebanese Syrian Associated Charities. ‡ Protein Data Bank codes are 2HRQ for the hCE1-soman structure and 2HRR for the hCE1-tabun structure. *Corresponding Author: Matthew R. Redinbo, Ph.D., Department of Chemistry, CB #3290, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-3290, (919)843-8910; Fax: (919)962-2388, redinbo@unc.edu. 1 Abbreviations: AcChE, Human acetylcholinesterase; BuChE, Human butyrylcholinesterase; CE, carboxylesterase; hCE1, Human carboxylesterase 1; HI-6, 1-(2-hydroxy-iminomethylpyridinium)-1-(4-carboxyamino)-pyridinium dimethylether dichloride; MuAcChE, Mus musculus acetylcholinesterase; OP, organophosphate; Ortho-7, 1,7-heptylene-bis-N,N′-2-pyridiniumaldoxime dichloride; PON1, human serum paraoxonase 1; rmsd, root mean square deviation; Sarin, methylethyl methylphosphonofluoridate; Soman, Tabun, ethyl N, TcAcChE, Torpedo californica acetylcholinesterase; VX, OP toxicity is thought to be mediated through the inhibition of human acety...