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. 2007 Jan 9;46(1):78-86.
doi: 10.1021/bi061551q.

Mutational analysis of Escherichia coli MoeA: two functional activities map to the active site cleft

Jason D Nichols  1 , Song Xiang, Hermann Schindelin, K V Rajagopalan
Affiliations

Mutational analysis of Escherichia coli MoeA: two functional activities map to the active site cleft

Jason D Nichols et al. Biochemistry. .

Abstract

The molybdenum cofactor is ubiquitous in nature, and the pathway for Moco biosynthesis is conserved in all three domains of life. Recent work has helped to illuminate one of the most enigmatic steps in Moco biosynthesis, ligation of metal to molybdopterin (the organic component of the cofactor) to form the active cofactor. In Escherichia coli, the MoeA protein mediates ligation of Mo to molybdopterin while the MogA protein enhances this process in an ATP-dependent manner. The X-ray crystal structures for both proteins have been previously described as well as two essential MogA residues, Asp49 and Asp82. Here we describe a detailed mutational analysis of the MoeA protein. Variants of conserved residues at the putative active site of MoeA were analyzed for a loss of function in two different, previously described assays, one employing moeA- crude extracts and the other utilizing a defined system. Oddly, no correlation was observed between the activity in the two assays. In fact, our results showed a general trend toward an inverse relationship between the activity in each assay. Moco binding studies indicated a strong correlation between a variant's ability to bind Moco and its activity in the purified component assay. Crystal structures of the functionally characterized MoeA variants revealed no major structural changes, indicating that the functional differences observed are not due to disruption of the protein structure. On the basis of these results, two different functional areas were assigned to regions at or near the MoeA active site cleft.

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Figures

Figure 1
Figure 1. X-ray crystal structure of E. coli MoeA
The MoeA monomer (A) and dimer (B) are both shown. The structures are colored according to domain as follows: yellow (I), blue (II), red (III), and green (IV). Arrows indicate the putative MoeA active site clefts. Figures were generated using PyMol.
Figure 2
Figure 2
Location of mutated MoeA residues
Figure 3
Figure 3. Analysis of MoeA variants using the moeAβˆ’ crude extract assay
MoeA mutants were assayed for their ability to reconstitute recombinant SO activity in moeAβˆ’crude extract in the presence of 50 ΞΌM molybdate at various concentrations of each variant using the method previously described (9). To aide the viewer, data for variants that were only minimally effected have not been shown. Shown are the data for wild type MoeA (●), D59A (Γ—), D59N (β– ), T100W (β—‡), E188A (β—†), E188Q (β–‘), D228A (β–Ό), D228N (β–²), D259A (β–½), S371W (Ξ”), and Ξ”-MoeA (β—‹). Results for each variant were normalized to activity acheived with 20 uM wild type MoeA, plotted vs [protein] and fitted with the logarithmic equation y = m log10(uM protein) + b. The slope and y-intercept (listed in Table I) were utilized for comparisons of the variants. Each variant was assayed in at least two separate experiments, in duplicate for each experiment. Error bars represent the standard deviation of the data.
Figure 4
Figure 4. In vitro analysis of MoeA variants using purified components
MoeA variants were assayed for the ability to mediate apo-SO reconstitution vs. [molybdate] in the presence of 20 ΞΌM MoeA as described for wild-type MoeA (14). To aide the viewer, only those variants that were significantly attenuated relative to wild type MoeA have been included in the plot. Shown are the data for wild type MoeA (●), T100A (β–²), T100W (β—‡), R137Q (β—†), K275Q (β–‘), K279Q (β–Ό), R350A (+), S371W (β– ), Ξ”-MoeA (Ξ”), and no protein (Γ—). For data analysis, results for each variant were first normalized to the maximum level of apo-SO activation acheived with wild type MoeA in that experiment (up to 420 U/mg SO in the presence of 1 mM molybdate). Each variant was assayed in at least two separate experiments, in duplicate for each experiment. Normalized data were averaged and fitted to a standard michaelis-menton equation. A β€œVmax” (labeled ΞΈmax: the maximum percentage of wild type MoeA activity) and Km (the [molybdate] required to reached Β½ ΞΈmax) were extracted for all variants and are listed in Table II. Error bars represent the standard deviation of the data.
Figure 5
Figure 5. Moco binding by MoeA variants
Moco binding assays were performed as described in β€œMaterials and Methods”. The level of bound Moco was normalized to the level obtained with wild type MoeA following each experiment. Experiments were performed in triplicate for each variant. Error bars represent the standard deviation of the normalized data.
Figure 6
Figure 6. Model of MPT bound to MoeA
MPT was modeled into the MoeA 3-D structure based upon the superposition of the Cnx1 G domain/MPT complex structure (15) and subdomain III of MoeA as determined with the program O. An initial transformation was generated by matching residues 131–156 of the Cnx1 G domain to corresponding regions of MoeA (residues 291–318 of monomer A in the NatI crystal). A refinement of this transformation matched all the corresponding secondary structure elements in the two proteins, resulting in an RMS deviation of 1.92 Γ… between the CΞ± atoms.
Figure 7
Figure 7. Structural diversity of MoeA variants
Stereo representation of all available variant monomers superimposed onto monomer A of the wild type Nat I crystal structure. Monomers were superimposed by alignment of domains I/III/IV, with all RMS deviations below 1 Γ…. Domain II from monomers C and D of R137Q and monomer A of D142N are highlighted in red, green, and blue, respectively.
Figure 8
Figure 8. Two different functions map to the MoeA active site cleft
MoeA is shown in a surface representation viewed into the active site cleft. Residues that were attenuated in the crude extract assay are colored blue while those deficient in the purified component system and Moco binding are colored yellow. Ser371 was attenuated in both and is colored green.
Figure 9
Figure 9. Graphical comparison of MoeA variant activity and Moco binding ability
A graphic illustration of the data listed in Table III.
See this image and copyright information in PMC

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