Biochemical Characterization of Aspergillus fumigatus AroH, a Putative Aromatic Amino Acid Aminotransferase

Abstract

The rise in the frequency of nosocomial infections is becoming a major problem for public health, in particular in immunocompromised patients. Aspergillus fumigatus is an opportunistic fungus normally present in the environment directly responsible for lethal invasive infections. Recent results suggest that the metabolic pathways related to amino acid metabolism can regulate the fungus-host interaction and that an important role is played by enzymes involved in the catabolism of L-tryptophan. In particular, in A. fumigatus L-tryptophan regulates Aro genes. Among them, AroH encodes a putative pyridoxal 5'-phosphate-dependent aminotransferase. Here we analyzed the biochemical features of recombinant purified AroH by spectroscopic and kinetic analyses corroborated by in silico studies. We found that the protein is dimeric and tightly binds the coenzyme forming a deprotonated internal aldimine in equilibrium with a protonated ketoenamine form. By setting up a new rapid assay method, we measured the kinetic parameters for the overall transamination of substrates and we demonstrated that AroH behaves as an aromatic amino acid aminotransferase, but also accepts L-kynurenine and α-aminoadipate as amino donors. Interestingly, computational approaches showed that the predicted overall fold and active site topology of the protein are similar to those of its yeast ortholog, albeit with some differences in the regions at the entrance of the active site, which could possibly influence substrate specificity. Should targeting fungal metabolic adaptation be of therapeutic value, the results of the present study may pave the way to the design of specific AroH modulators as potential novel agents at the host/fungus interface.

Keywords: aromatic amino acid aminotransferase; enzymatic assay; enzyme kinetics; enzyme spectroscopy; fungal infection; homology modeling; pyridoxal 5'-phosphate.

Figure 1

SDS-PAGE, Western Blot and SEC analyses on recombinant AroH. (A) SDS-PAGE of recombinant AroH purification. Lane M: protein molecular weight markers; lane 1: 30 μg of total bacterial lysate upon induction with IPTG; lane 2: 20 μg of purified recombinant AroH after affinity chromatography; lane 3: 30 μg of column flow-through. (B) Western blot of purified AroH. Lane M: molecular weight marker of His-tagged proteins; lane 1: 10 μg of total lysate after induction with IPTG; lane 2: 5 μg of purified recombinant AroH. (C) Elution profile of 5μM AroH on a Superdex 200 SEC column equilibrated and run in 0.1M KP pH 7.4.

Figure 2

Spectroscopic features of holoAroH. (A) Absorbance and CD (inset) spectrum of 8 μM holoAroH in 0.1 M KP, pH 7.4. (B) Internal aldimine emission fluorescence of 1 μM holoAroH upon excitation at 350 nm (—) and 432 nm (- -). (C,D) pH-dependence of the visible absorbance spectrum of holoAroH (10 μM) and of the internal aldimine emission fluorescence at 500 nm (exc. at 432 nm; 1 μM enzyme concentration) in KP 0.1 M at pH 6.02, 6.43, 6.75, 7.03, 7.44, and 8.31. The insets of both panels show the fitting of data to obtain the pK_spec.

Figure 3

Secondary and tertiary structure of AroH in the apo and holo-form. (A) Far-UV CD spectrum registered at 1 μM enzyme concentration in KP 0.1 M pH 7.4. The inset shows the percentage of secondary structure obtained by spectrum deconvolution. (B) Intrinsic fluorescence emission (exc. at 280 nm) of apo- (—) and holo- (- -) AroH at 0.5 μM concentration in KP 0.1 M pH 7.4. The inset shows the quenching of intrinsic emission fluorescence at increasing PLP concentrations. (C) K_D(PLP) calculation. The graph shows the quenching of the AroH intrinsic fluorescence as a function of PLP concentration. The line shows the theoretical fit obtained using Equation 1.

Figure 4

External aldimine of AroH upon binding with different amino donors. AroH (10 μM) (- - - ) was incubated with 6 mM L-tyrosine, 20 mM L-tryptophan, 15 mM L-phenylalanine, or 100 mM L-glutamate, as indicated (—), in 0.1 M KP pH 7.4, at 25°C.

Figure 5

AroH L-phenylalanine transaminase activity assay. Time-dependent changes of the absorbance at 365 nm during the continuous assay of AroH (0.2 μM) transaminase activity in the presence of L-phenylalanine at the indicated concentrations and 1 mM α-ketoglutarate in 66 mM KP, pH 7.4 at 25°C.

Figure 6

Activity of AroH as a function of pH and temperature. (A) AroH activity as a function of pH. The activity measurements were performed in KP 0.1 M at 25°C. The amino donor used were L-tryptophan (o) or L-phenylalanine (•) while the amino acceptor was α -ketoglutarate. The reaction was stopped in 10% TCA (v/v) after 10 min. L-glutamate determination was performed as reported in Material and Methods. (B) The figure shows the residual enzymatic activity of AroH in the holo-(□) and apo-(■) form in KP 0.1 M pH 7.4 at 25°C, upon 10 min incubation in the temperature range 25–85°C. In both panels the continuous and dashed line indicate the holo- or apo-form, respectively.

Figure 7

AroH homology model. (A) Homology model of dimeric AroH as ribbon representation obtained using the SWISS-MODEL portal. The two monomers are colored orange and white. (B) Superimposition between the structure of Aro8 (light purple) and that modeled for AroH (light orange). Flexible regions are indicated with arrows. (C) Active site topology of superimposed AroH and Aro8 structures. Residues involved in PLP binding are represented as gray sticks. The PLP cofactor is represented as green stick. The arrow indicates the position of the main-chain of Ser185 and Asn186. Tyr149^* belongs to the neighboring subunit. Numbers under parentheses indicate residue position in Aro8.

Figure 8

Structural features of the AroH active site regions. (A) Ribbon representation of superimposition of the region surrounding the active site in AroH and Aro8. Residues involved in PLP or substrate binding are highlighted as gray or green sticks, respectively. The PLP cofactor is shown as light green stick. (B,C) Electrostatic surface potential of Aro8 (B) and AroH (C) calculated using non-linear Poisson Boltzmann equation [−4 (red) and +4 (blue) kT/e] at 25°C, pH 7.4. (D,E) Particular of the electrostatic map of the active site channel of Aro8 (D) and AroH (E). Residues involved in the formation of active site channel of Aro8 are highlighted as sticks. Around the sticks transparency (80%) of the surface representation has been applied. Potential contours are [−4 (red) and +4 (blue) kT/e] around both proteins.