Analysis of Escherichia coli nicotinate mononucleotide adenylyltransferase mutants in vivo and in vitro

Investigation of the C-terminus

Based on the nadD72 mutation, we designed the mutant NadD72short with the same change of the ten C-terminal amino acid residues as nadD72, while the length of the protein is the same as in the wild type enzyme. We also constructed two mutants where either Tyr-205 or Ile-206 is changed to alanine.

We have earlier shown that the intracellular level of NAD⁺ correlates to growth ability on different media and at different temperatures [15]. Thus, we decided to investigate the effect on growth by the different mutant enzymes. Plasmids with either of the four mutant alleles, pZZNadD72, pZZNadD72short, pZZNadDY205A, pZZNadDI206A, or the wild type gene (pZZNadD) were transformed into strain MS10 with plasmid pKanNadD selecting for Ap^R. Transformants were restreaked on LB with ampicillin and arabinose to induce expression of the respective nadD alleles. The transformants were tested for loss of Km^R to ensure plasmid exchange. Thereafter, MS10 with each respective plasmid were streaked on LB plates with or without arabinose and incubated at 30°C. The diameter of the colonies was measured. The results are shown in Table 4. Addition of 0.1 mM arabinose resulted in growth of all strains. In the absence of arabinose leakage expression of the wild type enzyme is enough to support normal growth while neither of the two mutant proteins NadD72 or NadD72short are active enough to do so. This indicates that it is the change in the last 10 amino acids and not the elongated C-terminus that impairs the enzyme. The two mutants with either of the two conserved residues changed, supported growth like the wild type enzyme. This shows that to affect enzyme activity more than one amino acid has to be changed in the C-terminus.

The same test was performed on minimal medium and it was found that NadD72 and NadD72short could not support growth in the presence of 0.1 mM arabinose. However, at 0.2 mM arabinose enough enzyme was produced to allow growth (not shown).

A test was also performed on strain MS10 containing plasmids carrying the nadD alleles without a ZZ'-tag. We could not detect any difference in growth behavior whether the nadD alleles were tagged or not (not shown). This makes us confident to use the ZZ'-tagged enzymes in vitro.

The enzyme activity of the mutants changed in the C-terminus were measured in vitro. The result can be seen in Figure 2. The activity of the wild type enzyme was set to 1. The enzymes NadD72 and NadD72short have almost no activity in good agreement with the in vivo phenotype. The other two mutants, NadDY205A and NadDI206A are less efficient than the wild type enzyme but not enough to show as a change in the growth phenotype.

The results obtained both in vivo and in vitro for NadD72short could indicate that it is not the extension of NadD72 that causes the deficiency in the protein but rather the change in the C-terminus. However, this conclusion is complicated by the finding during purification of NadD72short that two bands were visible on the protein gel, indicating instability of the protein. Thus, the results obtained for NadD72short are inconclusive.

Another possibility should also be considered. Since the nadD72 allele causes temperature sensitivity it is possible that the enzyme activity is lowered at 37°C the temperature at which the in vitro experiments were performed. To test this, we grew all mutants at 30°C, 37°C, and 42°C, respectively. We found that the Ts phenotype not only disappears in the presence of arabinose but that the cells grow better at the higher temperatures than at 30°C. Therefore, we do not think that the changes in enzyme activity are caused by changes in reaction temperature optimum.

To understand the role of the elongated C-terminus of the NadD72 mutant, we consider the role of the corresponding region of the human NMNAT. Human NMNAT has a 24 amino acid residues longer C-terminus than E. coli NAMNAT. It has been suggested that the C-terminus in human NMNAT plays a role in substrate recognition [18, 19]. The NadD72 enzyme has 17 extra amino acid residues and it is possible that the extension interferes with substrate binding, which would lead to low enzyme activity. All we can say with certainty is that the C-terminus is important for stability of the protein and that the two conserved amino acid residues do not have a great influence on activity.

Investigation of the active site

The nadD74 mutation leads to an amino acid change in position 13 (Asp to Val). The mutated residue is two amino acids away from the ATP-binding motif, T/HXGH (position 16 to 19). Crystal structure information was used to decide which amino acid positions to mutagenize to learn more about the active site. Residues within 6 Å distance from the oxygens of the two adjacent phosphate-groups of the bound NAAD molecule are shown in Figure 3. Based on their close contact and H-bonding abilities Thr-11, His-19, Asn-40, His-45, Arg-46, Asp-109 and Ser-110 were selected for mutagenesis. All these residues were changed to alanine by site-directed mutagenesis. The recombinant proteins were cloned and analyzed, as were the C-terminal mutants.

Asp-109 and Ser-110 is located in the region connecting strand d and helix D of NAMNAT [10]. This region is one of three regions observed to undergo large conformational changes upon substrate binding. Interestingly, mutations of these two amino acids affect enzymatic activity very differently. On the one hand, binding of the substrate brings Ser-110 closer to the substrate. It is possible that the side chain of Ser-110 is H-bonded to the 2'-OH of AMP-ribose. However, mutation of Ser-110 to alanine resulted in an enzyme with 80% activity as compared to that of the wild type NadD. Our results indicate that the interaction between Ser-110 and the ribose is dispensable for substrate coordination. On the other hand, the change of the highly conserved Asp-109 had a severe effect on activity. Asp-109 has been proposed to form an H-bond to the 2'-OH group of AMP [10]. It was also suggested by the same authors that the carboxylate oxygen of the residue might be involved in the coordination of a Mg²⁺ ion shown to be important for the enzyme function [10, 12]. The location of Asp-109 is ideal to position an Mg²⁺ ion which could act as a Lewis acid stabilizing the transition state in the transesterification reaction [10]. However, the functional importance of this conserved residue has not been investigated earlier. The serious decrease in enzymatic activity (0.038%) that we observe supports the above mechanism and serves as an experimental evidence for the involvement of Asp-109 in the catalysis.

The Thr-11 main chain nitrogen is H-bonded to the AMP phosphate in the crystal structure of the NadD-NAAD complex. This H-bond is expected to be independent of the side-chain character of the amino acid. However, the NadDT11A mutant leads to decreased enzyme activity (2%, Figure 2). The explanation might be that the hydroxyl group of Thr-11 forms an H-bridge to Asn-40, which has been shown to interact with 2'-hydroxyl group of NAMN-ribose [10]. Such an interaction could contribute to substrate binding and coordination. The change of Asn-40 to alanine leads to a decreased enzyme activity (23%, Figure 2). We conclude that the disruption of this H-bonding network might lead to inefficient substrate coordination. Therefore, the effect on enzyme activity by the T11A and/or N40A mutation is possibly indirect.

The mutation D13V (NadD74) was found to lead to decreased activity of the enzyme. A similar mutation has been described in CTP:glycerol-3-phosphate cytidyltranferase that is a member of the same enzyme super family [20]. The residue Asp-11 (which corresponds to Asp-13 in NAMNAT) was changed to alanine and enzyme activity was severely reduced. Both valine and alanine are hydrophobic amino acids and it is possible that the change disturbs ATP binding; resulting in lower enzyme activity. It is not clear whether the role of Asp in this position is catalytical or structural.

The role of the second histidine (His-19) in the conserved T/HXGH motif has been tested in several studies and been shown to play a role in ATP-binding and stabilization, but the role for the two histidine residues can vary between enzymes [14, 21]. Our results confirm the previous observations. The mutation H19A leads to a decrease in enzyme activity to 0.62% of the wild type activity.

Amino acid residues His-45 and Arg-46 are part of a flexible loop in the NadD enzyme, which moves upon substrate binding. The two residues are conserved within NMNAT from Bacteria and Eukarya but not in the known archaeal enzymes. His-45 is involved in a hydrophobic stacking interaction with the pyridine ring in NAMN and is also likely to form an H-bond with the NAMN phosphate group. Histidines are often involved in acid base catalysis, and prone to activate nucleophiles by abstracting a proton. The H45A change led to an enzyme with 4% residual activity, while the R46A mutant was the most affected of all tested active site mutants. If the role of Arg-46 in the substrate binding loop was simply to protect the bound substrates, higher rates of ATP hydrolysis is expected in the case of the R46A mutant. Since the R46A mutant did not produce AMP as by-product in the in vitro experiment, the role of this arginine side chain must be more than simply protecting the bound substrates from water molecules. The guanidinium group of Arg-46 lies in an ideal position to serve as a positively charged moiety that stabilizes the juxtaposed and negatively charged phosphates of the substrate molecules, as well as of the product NAAD. The archaeal orthologues lack the precise sequence homology in the sequence aligned to the H45-R46, but they have a conserved arginine (Arg-8 in Methanococcus jannaschii) that occupies the same position with its side chain in the ATP-enzyme complex as that of Arg-46 in the E. coli NadD enzyme [10, 12]. On the other hand another hypothesis should be considered as well. Mutational studies on NMNAT from Methanobacterium thermoautotrophicum indicate that the archaeal enzyme is involved in the reaction merely by placing the substrates in an ideal position [14]. Combining the facts above, we conclude that Arg-46 in E. coli NadD plays an important role in stabilizing the two adjacent, negatively charged, phosphate moieties during catalysis. A similar role might be attributed to Arg-8 in the archaeal counterparts but this has to be tested.

Bacterial strains and media

Standard recombinant DNA techniques were used for cloning of DNA [23]. E. coli strain DH5α was used as a recipient for cloned DNA. Restriction and modification enzymes were purchased from New England Biolabs, Amersham Pharmacia Biotech or Life Technologies. DNA fragments were separated by agarose gel electrophoresis, excised and purified using the Qiaex II Gel Extraction Kit (Qiagen). Oligonucleotides were purchased from MWG Biotech. Plasmid DNA was purified with QiaPrep Kit (Qiagen). MWG Biotech did DNA sequencing. ATP and NAMN were purchased from Sigma.

Construction of a nadD strain and a plasmid-exchange system

To delete the nadD gene from the chromosome we used linear DNA transformation and the λ red recombination system [16]. Primers used for amplification of the chloramphenicol acetyltransferase gene with homologies to the ends of nadD were PNadko1b and PNadko2b. The amplified linear DNA was electroporated into strain MRA530/pKanNadD and chloramphenicol resistant (Cm^R) colonies were selected. Recombinants were checked for proper exchange by PCR amplification and sequence verification. A P1 phage lysate made on one such recombinant was transduced to RI10/pKanNadD selecting for Cm^R. Transductants were verified by PCR, one clone was kept and named MS10. The plasmid could now be exchanged for a pBADmyc-hisA (Ap^R) plasmid carrying different nadD alleles. The exchange relies on the incompatibility of pKanNadD and pBADmyc-hisA.

Construction of expression vectors with different nadD alleles

The nadD72 allele from RI8 was amplified with primers PARA1 and PD1, the nadD74 allele from RI12 with primers PARA1 and PARA2 and the nadD72short allele from RI8 with primers PARA1 and PD72short. The nadD72short allele has the same change at the C-terminus as the nadD72 allele but with the same length as the wild type allele. Site-directed mutagenesis was performed on the cloned wild type allele using QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA). Oligonucleotides used are listed in Table 3.

All allele variants were cloned in the expression vector pBADmyc-hisA under the control of an arabinose-promoter using the Nco I and Kpn I sites. The resulting plasmids are listed in Table 2. Thereafter all alleles were fused at their 5'-end to a fragment containing an IgG binding ZZ'-domain from the pEZZ18 vector. This set of plasmids has the same origin of replication as the pKanNadD but they confer ampicillin resistance (Table 2). This makes it possible to exchange plasmid pKanNadD in the MS10 strain for plasmids carrying mutant nadD alleles.

Protein expression and purification

E. coli TOP10 cells containing plasmids with respective nadD allele (pZZNadDXX) were grown in LB medium at 30°C. When the cultures reached OD₅₅₀ 0.5, arabinose was added to a final concentration of 0.4 mM. After ~4 h growth, the cultures were quickly chilled on ice and harvested by centrifugation. Pellets were stored at -20°C. The frozen pellets were thawed, resuspended in 10 × TST (0.5 M Tris pH 7.4, 1.5 M NaCl, 5% w/v Tween 20), lysozyme (1 mg/ml), DNase I (20 μg/ml) and RNase A (20 μg/ml). After incubation at 37°C and several freezing (in liquid nitrogen) and thawing cycles, samples were sonicated and the lysate was cleared by centrifugation. Proteins were purified as described [24]. The solvent was changed to reaction buffer [9] on a NAP5 desalting column (Amersham Biosciences, Uppsala, Sweden). Purification efficiency was monitored by SDS-polyacrylamide gel elecrophoresis (PAGE) and stained by Coomasie blue. The protein concentration was determined by UV spectrophotometry at 280 nm [26].

Assay of enzymatic activity

In vitro enzymatic activity of NAMNAT was determined as described [9]. The substrate concentrations were 2 mM ATP and 1 mM NAMN and the reaction was carried out at 37°C. The enzyme concentration was 1 μg/100 μl. Reactions were terminated by immersing the tubes in boiling water for 5 minutes. They were thereafter cooled on ice and filtered through Nanosep 10 K microcentrifugal devices to take away the enzyme (Pall, Ann Arbor, MI). 20 μl aliquots were analyzed by high-pressure liquid chromatography on a Gilson LC system by using Supelcosil LC-18-T 15 cm by 4.6 mm column (Supelco, Bellefonte, PA).

The product formation was monitored at 254 nm as a function of time and the initial reaction rate was calculated from the slope of the curve. The rate for the wild type enzyme that was set to 1, rates for the mutant enzymes were correlated to this.