Abstract
The periplasmic aldehyde oxidoreductase PaoABC from Escherichia coli is a molybdenum enzyme involved in detoxification of aldehydes in the cell. It is an example of an αβγ heterotrimeric enzyme of the xanthine oxidase family of enzymes which does not dimerize via its molybdenum cofactor binding domain. In order to structurally characterize PaoABC, X-ray crystallography and small angle X-ray scattering (SAXS) have been carried out. The protein crystallizes in the presence of 20% (w/v) polyethylene glycol 3350 using the hanging-drop vapour diffusion method. Although crystals were initially twinned, several experiments were done to overcome twinning and lowering the crystallization temperature (293 K to 277 K) was the solution to the problem. The non-twinned crystals used to solve the structure diffract X-rays to beyond 1.80 Å and belong to the C2 space group, with cell parameters a = 109.42 Å, b = 78.08 Å, c = 151.77 Å, β = 99.77°, and one molecule in the asymmetric unit. A molecular replacement solution was found for each subunit separately, using several proteins as search models. SAXS data of PaoABC were also collected showing that, in solution, the protein is also an αβγ heterotrimer.
Highlights
Molybdenum is a transition metal that is incorporated, as a biologically active cofactor, in a class of widely distributed proteins collectively known as molybdoenzymes [1]
With the exception of R. capsulatus and Pseudomonas aeruginosa xanthine dehydrogenases (XDH) [20,21] which binds molybdenum containing molybdopterin (Mo-MPT), all bacterial XDHs characterized to date bind the molybdopterin cytosine dinucleotide (MCD) form of molybdenum cofactor (Moco)
While all the enzymes of the xanthine oxidase (XO) family that have so far been analysed by crystallography exhibit dimeric structures (homodimers or dimers of heterotrimers [38]), biochemical analysis suggested that PaoABC does not dimerize via its Moco-binding domain and remains an αβγ heterotrimer in solution
Summary
Molybdenum is a transition metal that is incorporated, as a biologically active cofactor (molybdenum cofactor, Moco), in a class of widely distributed proteins collectively known as molybdoenzymes [1]. With the exception of R. capsulatus and Pseudomonas aeruginosa XDH [20,21] which binds Mo-MPT, all bacterial XDHs characterized to date bind the molybdopterin cytosine dinucleotide (MCD) form of Moco. In these XDHs, molecular masses range from 140 to 300 kDa and different subunit structures were observed, like α2 in Steptomyces cyanogenus [22], αβγ in V. atypica [19], α3 in P. putida [23], α2β2 in R. capsulatus [15], α2β2 in Comamonas acidovorans [24,25], and α4β4 in P. putida 86 [17]. Additional SAXS experiments confirmed the αβγ heterotrimeric structure of the enzyme
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