The UreEF Fusion Protein Provides a Soluble and Functional Form of the UreF Urease Accessory Protein

2010

J Bacteriol

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Assembly of the Klebsiella aerogenes urease metallocenter requires four accessory proteins, UreD, UreE, UreF, and UreG, to effectively deliver and incorporate two Ni 2؉ ions into the nascent active site of the urease apoprotein (UreABC). Each accessory protein has been purified and characterized with the exception of UreD due to its insolubility when it is overproduced in recombinant cells. In this study, a translational fusion was made between the maltose binding protein (MBP) and UreD, with the resulting MBP-UreD found to be soluble in Escherichia coli cell extracts and able to complement a ⌬ureD-urease cluster in this host microorganism. MBP-UreD was purified as a large multimer (>670 kDa) that bound approximately 2.

mentioning

confidence: 86%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Characterization of the Klebsiella aerogenes Urease Accessory Protein UreD in Fusion with the Maltose Binding Protein

Carter¹,

2010

J Bacteriol

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“…3), a complex that can be directly isolated from cells expressing the corresponding genes (35). Alternatively, in vitro incubation of UreE-UreF with Ure-ABC:UreD provides UreABC:UreD:UreE-UreF (37). Native gel electrophoresis of UreABC:UreD:UreF revealed multiple species, and zero to three pairs of UreD:UreF are suggested to bind per (UreABC) 3 .…”

Section: Ured/ureh: Scaffold For Recruitment Of Other Accessory Protementioning

confidence: 99%

Biosynthesis of the Urease Metallocenter

Farrugia

Macomber

Journal of Biological Chemistry

2013

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110

101

Metalloenzymes often require elaborate metallocenter assembly systems to create functional active sites. The medically important dinuclear nickel enzyme urease provides an excellent model for studying metallocenter assembly. Nickel is inserted into the urease active site in a GTP-dependent process with the assistance of UreD/UreH, UreE, UreF, and UreG. These accessory proteins orchestrate apoprotein activation by delivering the appropriate metal, facilitating protein conformational changes, and possibly providing a requisite post-translational modification. The activation mechanism and roles of each accessory protein in urease maturation are the subject of ongoing studies, with the latest findings presented in this minireview.Metallocenters serve essential biological functions such as transferring electrons, stabilizing biomolecules, binding substrates, and catalyzing desirable reactions. Synthesis of these sites must be tightly controlled because simple competition between metals may lead to misincorporation with loss of function and because excess cytoplasmic concentrations of free metal ions can have toxic cellular effects. In many cases, cells have evolved elaborate metallocenter assembly systems that sequester metal cofactors from the cellular milieu, thus offering protection from adventitious reactions while ensuring the fidelity of metal insertion. In addition to maintaining metal homeostasis, these assembly systems facilitate protein conformational changes and active site modifications that are required for full enzymatic activity. Several metalloproteins have been investigated as models to understand the mechanisms and dynamics of active site assembly and the complex orchestrations of their metallocenter assembly systems. In this minireview, we discuss recent findings related to maturation of the nickel-containing enzyme urease.

“…Yeast 2-hybrid experiments carried out using the H. pylori and Proteus mirabilis proteins also support an interaction between UreF and UreD/H (16, 17). Although K. aerogenes UreF is insoluble when produced alone, the fusion proteins with UreF linked to MBP (18) or to UreE (creating UreE-F) (19) are soluble. The gene encoding the fusion protein complements a ureF deletion mutant and binds in vitro to UreABC—UreD or MBP-UreD (14, 19).…”

mentioning

confidence: 99%

Klebsiella aerogenes UreF: Identification of the UreG Binding Site and Role in Enhancing the Fidelity of Urease Activation

Boer

2012

Biochemistry

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The Ni-containing active site of Klebsiella aerogenes urease is assembled through the concerted action of the UreD, UreE, UreF, and UreG accessory proteins. UreE functions as a metallochaperone that delivers Ni to a UreD–UreF–UreG complex bound to urease apoprotein, with UreG serving as a GTPase during enzyme activation. This study focuses on the role of UreF, previously proposed to act as a GTPase activating protein (GAP) of UreG. Sixteen conserved UreF surface residues that may play roles in protein–protein interactions were independently changed to Ala. When produced in the context of the entire urease gene cluster, cell-free extracts of nine site-directed mutants had less than 10% of the wild-type urease activity. Enrichment of the variant forms of UreF, as the UreE-F fusion proteins, uniformly resulted in copurification of UreD and urease apoprotein, whereas UreG bound to only a subset of the species. Notably, weakened interaction with UreG correlated with the low-activity mutants. The affected residues in UreF map to a distinct surface on the crystal structure, defining the UreG binding site. In contrast to the hypothesis that UreF is a GAP, the UreD–UreF–UreG–urease apoprotein complex containing K165A UreF exhibited significantly greater levels of GTPase activity than that containing the wild-type protein. Additional studies demonstrated the UreG GTPase activity was largely uncoupled from urease activation for the complex containing this UreF variant. Further experiments with these complexes provided evidence that UreF gates the GTPase activity of UreG to enhance the fidelity of urease metallocenter assembly, especially in the presence of the noncognate metal Zn.