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Introduction.

  1. Top of page
  2. Introduction.
  3. Materials and Methods.
  4. Results and Discussion.
  5. Acknowledgements
  6. REFERENCES

The Tas gene of Escherichia coli encodes an uncharacterized protein of 38.5-kDa molecular weight. The amino acid sequence shows homology to the family of aldo-keto reductases that reduce aldehyde or ketone functional groups to primary or secondary alcohols.The basic chemical reaction is the stereospecific transfer of hydride between the nicotinamide ring of NADPHa and a carbon center on the substrate molecule. The aldo-keto reductases metabolize a wide range of small molecule substrates including sugars, aliphatic aldehydes, aromatic hydrocarbons, steroids, and prostaglandins.1 For many members of the family, however, the physiological substrates are not known.

Crystallographic studies of human aldose reductase2–4 have established the molecular architecture, geometry of cofactor binding, and the catalytic mechanism of aldo-keto reductases. All members of the family have a (β/α)8 TIM barrel fold. The catalytic site with the bound NADP(H) cofactor is located at the C-terminal edge of the β-strands. Loops surrounding the active site form the substrate recognition cylindrical surface. Crystal complexes with various inhibitors5–8 have indicated the ability of aldo-keto reductases to accommodate a wide range of substrates due to the conformational flexibility of the recognition loops.

Tas was identified as the gene able to complement the tyrosine requirement in E. coli tyrosine auxotroph strains under starvation conditions,9 hence the name Tas (tyrosine auxotrophy suppressor). Most likely, the enzymatic activity of the Tas protein directly substitutes for the lacking prephenate dehydrogenase activity of the auxotroph strain; however, the involvement of Tas in the mutation process resulting in slow-growing Tyr+ revertants cannot be ruled out. Tas was among the genes significantly induced in response to the DNA damage caused by mitomycin C10 and therefore may be part of a general protective response.

The crystal structure determination of Tas was undertaken as part of a structural genomics effort11 (http://s2f.carb.nist.gov) to assist with the functional assignment of the protein. The Tas protein from E. coli was cloned, expressed, and the crystal structure determined at 1.6-Å resolution.

Materials and Methods.

  1. Top of page
  2. Introduction.
  3. Materials and Methods.
  4. Results and Discussion.
  5. Acknowledgements
  6. REFERENCES

Cloning, expression, and purification. The Tas gene was polymerase chain reaction (PCR) amplified from E. coli MG1655 genomic DNA and subcloned into a pDONR201 plasmid using the Gateway technology (Invitrogen). For expression, the coding sequence was transferred into a pDEST14 plasmid using site-specific recombination (Invitrogen). The protein was produced in E. coli strain BL21 Star (DE3) (Invitrogen) that was transformed with pDEST14. Cells were grown on LB media containing 100 μg/μL ampicillin at 37°C to an A600 of 0.6 and induced with 1 mM isopropyl β-D-thiogalactoside for 3 h. The protein was purified by column chromatography in two steps using Source 30Q (Pharmacia) and Butyl-560M (Toyopearl).

Crystallization and structure determination. Tas crystals were grown by vapor diffusion in hanging drops at room temperature from 0.1 M Tris, pH 8.5, 15% polyethyleneglycol 8000, 50 mM ammonium sulfate, and 0.2 M magnesium chloride. The crystals belong to the space group P212121 with unit cell parameters: a = 58.3 Å, b = 81.5 Å, c = 145.0 Å. There are two protein molecules in the asymmetrical unit with 45% solvent content. A heavy atom derivative was obtained by soaking the crystal in 1 mM methylmercury chloride for 1 h. For X-ray data collection, 20% glycerol was added to the mother liquor and the crystal was flash-frozen in liquid propane.

The structure was solved by the multiwavelength anomalous diffraction method using 1 Hg derivative crystal. X-ray data to 1.6-Å resolution were measured at three wavelengths (Table I) using the IMCA-CAT beamline 17-ID at the APS (Argonne, IL) equipped with a ADSC Quantum-4 CCD detector, and processed with HKL2000.12 Two mercury sites (one per protein molecule) were located by the Shake-and-Bake method13 and were used for phasing with MLPHARE/DM.14 The atomic model was built using RESOLVE15 and O16 and refined with REFMAC.17 The model contains all 346 residues expected from the amino acid sequence. The atomic coordinates of the Tas protein were deposited in the Protein Data Bank under the accession code 1LQA.

Table I. X-Ray Data and Refinement Statistics
Data setPeakInflectionHigh-E
  • a

    Anomalous pairs not merged.

Wavelength (Å)1.00721.00910.9999
Resolution (Å)1.61.61.6
Number of unique reflectionsa160,862161,083160,940
Completeness (%)92.091.791.5
Redundancy3.33.33.1
Rsym (Σ|I − 〈I〉)/ΣI)0.0630.0620.112
II〉 (outer shell)2.91.51.4
Rcryst (Σ||Fo| − |Fc||)/Σ|Fo|)0.146  
Rfree (3% data)0.186  
Number of protein atoms5,432  
Number of NADPH atoms96  
Number of water molecules866  
RMSD in bonds (Å)0.017  
RMSD in angles (°)1.8  
RMSD in main-chain B factors (Å2)3.1  

Results and Discussion.

  1. Top of page
  2. Introduction.
  3. Materials and Methods.
  4. Results and Discussion.
  5. Acknowledgements
  6. REFERENCES

The Tas protein has a topology of a canonical (β/α)8 barrel (Fig. 1). In addition to the 16 structural elements of the barrel, there are 2 helices, H1 and H2, in the loops following β-strands 7 and 8 that form a helical subdomain at the periphery of the molecule. The N-terminal β-hairpin caps the barrel at the N-terminal side. The NADP(H) molecule is observed at the C-terminal side of the barrel, where the loops following β-strands form a deep cleft that is the putative active site of the enzyme.

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Figure 1. Ribbon presentation of the polypeptide fold of the Tas protein. The N-terminal β-hairpin is green. NADPH is shown as a ball-and-stick model.

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The overall structure, the cofactor binding mode, and the location of the active site pocket are similar to those of other aldo-keto reductases. The N-terminal β-hairpin and the α-helical subdomain are present in all aldo-keto reductases and may be considered as a fingerprint of the family with respect to other TIM barrel proteins. The structural similarity to the β subunit of the voltage-dependent K+ channel (KVB2)18 is in particular striking. Tas and KVB2 can be superimposed with a root mean square deviation (RMSD) of 1.9 Å over 305 common Cα atoms. Tas shares 32% sequence identity with KVB2. The 3D similarity to other aldo-keto reductases is significantly lower, which reflects their lower sequence similarity (less than 22% identity).

The crystal structure reveals NADP(H) bound to the protein, although no cofactor was present in the crystallization solution. NADP(H) is almost completely sequestered from the solvent by the loop following β7 so that only the nicotinamide portion remains accessible at the bottom of the active site (Fig. 2). The observed extended conformation of NADP(H) corresponds to its binding mode in other aldo-keto reductases. Numerous interactions with the protein involve both hydrogen bonds and hydrophobic contacts.

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Figure 2. Active site in the Tas protein. The C4 atom of nicotinamide is yellow and the water molecule at the substrate carbonyl site is magenta. Hydrogen bonds are shown as broken lines.

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The orientation of the nicotinamide ring, which is crucial for catalysis, is supported through a few key interactions that involve stacking with the phenol ring of Tyr233 and hydrogen bonds between the amide oxygen and Asn180 and between the amide nitrogen and Ser179 and Gln205. This orientation directs the 4-pro-R hydrogen of C4 (shown in yellow in Fig. 2) toward the opening where the substrate enters the catalytic site. The catalytic mechanism of aldo-keto reductases includes the hydride transfer from the nicotinamide to the carbonyl carbon of the substrate, followed by the proton transfer from a donor group on the protein to the carbonyl oxygen of the substrate.19–21 The proton donor is likely a tyrosine residue with the pK lowered by interaction with the lysine and aspartate residues. Such a charge relay system is also present in Tas and includes Tyr53[BOND]Lys87[BOND]Asp48. A water molecule bound to Tyr53 marks the postulated position of the carbonyl group of a substrate.

The residues lining the walls of the active site pocket determine the substrate specificity of an aldo-keto reductase. A high proportion of hydrophobic residues is typical for most members of the family. In the Tas protein, hydrophobic residues line the specificity pocket close to the catalytic site, whereas a number of basic residues (Arg93, Lys143, Arg261, Arg264) form the outer layer of the recognition site, suggesting the importance of electrostatic interactions. The substrate binding cleft is narrower and deeper in Tas when compared to other aldo-keto reductases. This is due to the longer loops 2, 3, and 4 in Tas. In other members of the family, two of these loops are usually shorter, which accounts for a more open recognition site.

While the crystal structure proves the molecular function of Tas as an aldo-keto reductase, the biologic role of the protein in the cell remains unclear. Analysis of the gene expression levels in E. coli under the conditions of genotoxic stress10 indicates that Tas may be part of a general protective response. A similar role may me ascribed to the yeast homolog of Tas, GCY1, which appeared to be induced in Zygosaccharomyces rouxii in response to salt stress.22 Further biochemical and biophysical studies will shed more light on the molecular and cellular functions of the Tas protein. These studies will be facilitated by the 3D structure of Tas.

Acknowledgements

  1. Top of page
  2. Introduction.
  3. Materials and Methods.
  4. Results and Discussion.
  5. Acknowledgements
  6. REFERENCES

Use of the Advanced Photon Source was supported by the U.S. Department of Energy, Basic Energy Sciences, Office of Science, under Contract W-31-109-Eng-38. This work was also supported in part by an award from the W.M. Keck Foundation.

REFERENCES

  1. Top of page
  2. Introduction.
  3. Materials and Methods.
  4. Results and Discussion.
  5. Acknowledgements
  6. REFERENCES