Mutational analysis of mycobacterium UvrD1 identifies functional groups required for ATP hydrolysis, DNA unwinding, and chemomechanical coupling Journal Article
| Authors: | Sinha, K. M.; Glickman, M. S.; Shuman, S. |
| Article Title: | Mutational analysis of mycobacterium UvrD1 identifies functional groups required for ATP hydrolysis, DNA unwinding, and chemomechanical coupling |
| Abstract: | Mycobacterial UvrD1 is a DNA-dependent ATPase and a Ku-dependent 3′ to 5′ DNA helicase. The UvrD1 motor domain resembles that of the prototypal superfamily I helicases UvrD and PcrA. Here we performed a mutational analysis of UvrD1 guided by the crystal structure of a DNA-bound Escherichia coli UvrD-ADP-MgF<sub>3</sub> transition state mimetic. Alanine scanning and conservative substitutions identified amino acids essential for both ATP hydrolysis and duplex unwinding, including those implicated in phosphohydrolase chemistry via transition state stabilization (Arg308, Arg648, Gln275), divalent cation coordination (Glu236), or activation of the nucleophilic water (Glu236, Gln275). Other residues important for ATPase/helicase activity include Phe280 and Phe72, which interact with the DNA 3′ single strand tail. ATP hydrolysis was uncoupled from duplex unwinding by mutations at Glu609 (in helicase motif V), which contacts the ATP ribose sugar. Introducing alanine in lieu of the adenine-binding "Q motif" glutamine (Gln24) relaxed the substrate specificity in NTP hydrolysis, e.g., eliciting a gain of function as a UTPase/TTPase, although the Q24A mutant still relied on ATP/dATP for duplex unwinding. Our studies highlight the role of the Q motif as a substrate filter and the contributions of adenosine-binding residues as couplers of NTP hydrolysis to motor activity. The Ku-binding function of UvrD1 lies within its C-terminal 270 amino acid segment. Here we found that deleting the 90 amino acid C-terminal domain, which is structurally uncharacterized, diminished DNA unwinding, without affecting ATP hydrolysis or binding to the DNA helicase substrate, apparently by affecting the strength of the UvrD1-Ku interaction. © 2009 American Chemical Society. |
| Keywords: | unclassified drug; gene deletion; nonhuman; dna repair; genes; amino acid substitution; phosphatase; carboxy terminal sequence; protein protein interaction; protein binding; enzyme activity; mutational analysis; bacterial protein; amines; bacterial proteins; dna; amino acid sequence; conserved sequence; molecular sequence data; sequence homology, amino acid; kinetics; nucleotide sequence; escherichia coli; nucleic acids; substrate specificity; crystal structure; models, molecular; protein structure, tertiary; binding sites; sugars; dna mutational analysis; helicase; alanine; amination; amino acids; organic acids; adenosine triphosphate; dna, single-stranded; motor activity; enzyme specificity; adenosine triphosphatase; mycobacterium; adenosine triphosphatases; hydrolysis; protein structure, secondary; glutamine; dna helicases; atp hydrolysis; dna helicase; substrates; amino acid motifs; escherichia coli proteins; enzyme active site; binding residues; c-terminal domains; chemo-mechanical couplings; divalent cation; dna unwinding; gain-of function; helicases; motor domain; mycobacterial; transition state; transition state stabilization; functional groups; motors; plants (botany); sugar (sucrose); functional group; nucleophile; protein uvrd1; dna binding motif; dna denaturation; enzyme chemistry; mycobacterium smegmatis; protein hydrolysis |
| Journal Title: | Biochemistry |
| Volume: | 48 |
| Issue: | 19 |
| ISSN: | 0006-2960 |
| Publisher: | American Chemical Society |
| Date Published: | 2009-05-19 |
| Start Page: | 4019 |
| End Page: | 4030 |
| Language: | English |
| DOI: | 10.1021/bi900103d |
| PUBMED: | 19317511 |
| PROVIDER: | scopus |
| PMCID: | PMC2761027 |
| DOI/URL: | |
| Notes: | --- - "Cited By (since 1996): 4" - "Export Date: 30 November 2010" - "CODEN: BICHA" - "Source: Scopus" |
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