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Structure of the Month

Structure of the Month: June 2009 [see all]

Structure and Mechanism of a Eukaryotic FMN Adenylyltransferase

Carlos Huerta¹, Dominika Borek¹, Mischa Machius¹, Nick V. Grishin², and Hong Zhang1¹

¹Department of Biochemistry and ²Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas 75390

Our laboratory investigates enzymes involved in the biosynthesis of cofactors such as NAD, FAD and coenzyme A that are essential for the cell survival. Understanding the functional properties of these enzymes is facilitated by the use of X-ray crystallography and kinetic studies.

Recently, we have determined crystal structures of four different complexes of eukaryotic FMN adenylyltransferase (FMNAT) from the pathogenic yeast Candida glabrata (CgFMNAT). FMNAT catalyzes the formation of the essential flavocoenzyme FAD and appears to play an important role in flavocoenzyme homeostasis regulation in eukaryotes. The four structures [apo-form (PDB ID 3FWK), ATP binary complex (3G59), and substrate (3G5A) and product (3G6K) ternary complexes] determined at 1.20-1.95 Å resolutions provided details about the substrate binding and catalytic site configurations, which support the proposed ordered bi-bi kinetic mechanism of the enzyme. These structures revealed a novel flavin-binding mode and a unique enzyme bound FAD conformation that has not been seen in any other known FAD-binding proteins. Comparing the structures of bacterial and eukaryotic FMNATs helps to understand the structural basis for the convergent evolution of the same FMNAT activity from different protein ancestors. Lastly, these structures allowed a structure-based investigation of the kinetic properties of eukaryotic FMNAT which may offer insights into the mechanisms by which eukaryotic FMNAT modulates FAD biosynthesis to maintain cellular flavocoenzyme homeostasis.

Figure 1: High resolution diffraction image of apo-CgFMNAT and electron density of products. A) Diffraction image of apo-CgFMNAT recorded on an R-AXIS IV++ image plate detector to 1.66 Å at the edge and 1.46 Å at the corner, with X-rays generated from Rigaku FR-E+ SuperBright. The highest resolution obtainable on the detector is 1.32 Å at the corner. A 1.20 Å data set (3FWK) was collected at Argonne National Laboratory, Structural Biology Center at the Advanced Photon Source (APS) and was used for the structure determination of apo-CgFMNAT. Inset: The area defined by the black rectangle in A) is enlarged to show diffraction spots at the edge of the image plate. B) Simulated-annealing gray F_o-F_c omit map contoured at 3.0σ is shown for FAD, PP_i and Mg²⁺ in the product ternary complex (3G6K). Data set collected at 1.35 Å at the APS.

Figure 2: Cartoon representations of ATP binary (3G59) and substrate ternary (3G5A) complex structures of CgFMNAT. A) Simulated-annealing gold F_o-F_c omit map contoured at 3.0σ is shown for ATP. The ATP and selected residues are shown as sticks and hydrogen bonds are shown as black dashes. B) Substrate ternary complex is colored by B-factor scale with blue representing the lowest and red the highest B-value. The B-factor ranges from 7.9-43.2 Å². Substrate ATP and FMN are shown as sticks and a magnesium ion as a green sphere.

This work was performed in the laboratory of Hong Zhang at the University of Texas Southwestern Medical Center, Dallas, TX.

Data collection details

PDB ID	Apo-CgFMNAT	ATP binary complex (3G59)	Substrate ternary complex (3G5A)	Product ternary complex
Space group	P3₂21	P3₂21	C2	C2
Unit cell	a = b = 80.06 Å c = 78.05 Å	a = b = 79.79 Å c = 77.94 Å	a = 207.83 Å b = 81.75 Å c = 136.70 Å β = 129.67°	a = 206.56 Å b = 81.44 Å c = 136.65 Å β = 129.79°
Radiation	Cu Kα	Cu Kα	Cu Kα	Cu Kα
Generator	FR-E+ SuperBright	FR-E+ SuperBright	FR-E+ SuperBright	FR-E+ SuperBright
Optic	VariMax HR	VariMax HR	VariMax HR	VariMax HR
Detector	R-AXIS IV++	R-AXIS IV++	R-AXIS IV++	R-AXIS IV++
Crystal-to-detector distance	105 mm	130 mm	120 mm	120 mm
Exposure time per frame	2 min	3 min	3 min	3 min
Oscillation width	0.5°	0.5°	0.5°	0.5°
Number of frames	228	180	240	240
Data processing	HKL-2000®	HKL-2000	HKL-2000	HKL-2000
Resolution range	50.0-1.66 Å	50.0-1.87 Å	50.0-1.78 Å (truncated to 1.95 Å during scaling)	50.0-1.78 Å
R_sym1	0.037 (0.097)²	0.078 (0.549)	0.088 (0.447)³	0.026 (0.159)

R_sym = Σ_hkl [( Σ_j (|I_j - <I>|) / Σ_jI_j ]. <I> is the average for all j measurements of reflection hkl.
Values in parenthesis are for the highest resolution shell.
Values for 1.95 Å data set.

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