Purification, characterization, and functional analysis of a truncated Klebsiella aerogenes UreE urease accessory protein lacking the histidine-rich carboxyl terminus.

The oomycete microorganism,Pythium insidiosum, causes the life-threatening infectious condition, pythiosis, in humans and animals worldwide. Affected individuals typically endure surgical removal of the infected organ(s). Detection ofP. insidiosumby the established microbiological, immunological, or molecular methods is not feasible in non-reference laboratories, resulting in delayed diagnosis. Biochemical assays have been used to characterizeP. insidiosum, some of which could aid in the clinical identification of this organism. Although hydrolysis of maltose and sucrose has been proposed as the key biochemical feature useful in discriminatingP. insidiosumfrom other oomycetes and fungi, this technique requires a more rigorous evaluation involving a wider selection ofP. insidiosumstrains. Here, we evaluated 10 routinely available biochemical assays for characterization of 26P. insidiosumstrains, isolated from different hosts and geographic origins. Initial assessment revealed diverse biochemical characteristics across theP. insidiosumstrains tested. Failure to hydrolyze sugars is observed, especially in slow-growing strains. Because hydrolysis of maltose and sucrose varied among different strains, use of the biochemical assays for identification ofP. insidiosumshould be cautioned. The ability ofP. insidiosumto hydrolyze urea is our focus, because this metabolic process relies on the enzyme urease, an important virulence factor of other pathogens. The ability to hydrolyze urea varied amongP. insidiosumstrains and was not associated with growth rates. Genome analyses demonstrated that urease- and urease accessory protein-encoding genes are present in both urea-hydrolyzing and non-urea-hydrolyzing strains ofP. insidiosum. Urease genes are phylogenetically conserved inP. insidiosumand related oomycetes, while the presence of urease accessory protein-encoding genes is markedly diverse in these organisms. In summary, we dissected biochemical characteristics and drew new insights into clinical identification and urease-related evolution ofP. insidiosum.

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

Journal of Bacteriology ◽

10.1128/jb.01426-09 ◽

2010 ◽

Vol 192 (9) ◽

pp. 2294-2304 ◽

Cited By ~ 22

Author(s):

Eric L. Carter ◽

Robert P. Hausinger

Keyword(s):

Binding Protein ◽

Equilibrium Dialysis ◽

Maltose Binding Protein ◽

Accessory Protein ◽

Klebsiella Aerogenes ◽

E Coli ◽

Cell Extracts ◽

Recombinant Cells

ABSTRACT Assembly of the Klebsiella aerogenes urease metallocenter requires four accessory proteins, UreD, UreE, UreF, and UreG, to effectively deliver and incorporate two Ni2+ 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 ΔureD-urease cluster in this host microorganism. MBP-UreD was purified as a large multimer (>670 kDa) that bound approximately 2.5 Ni2+ ions (Kd of ∼50 μM, where Kd is the dissociation constant) per UreD protomer according to equilibrium dialysis measurements. Zn2+ directly competes with 10-fold higher affinity (∼4 Zn2+ ions per protomer; Kd of 5 μM) for the Ni2+ binding sites. MBP pulldown experiments demonstrated that the UreD domain of MBP-UreD formed in vivo complexes with UreF, UreG, UreF plus UreG, or UreABC when these proteins were overproduced in the same E. coli cells. In addition, a UreABC-(MBP-UreD)-UreFG complex was observed in cells producing all urease components. Comparative in vitro binding experiments with purified proteins demonstrated an approximate 1:1 binding ratio between the UreD domain of MBP-UreD and the UreF domain of the UreEF fusion, only weak or transient interaction between MBP-UreD and UreG, and no binding with UreABC. These studies are the first to describe the properties of purified UreD, and they extend our understanding of its binding partners both in vitro and in the cell.

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