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.

Modeling Approaches Reveal New Regulatory Networks in Aspergillus fumigatus Metabolism

“…Idos proteins in Aspergillus seem to degrade Trp in to kynurenines, with different affinity for Trp and velocity rate [ 8 ]. The Aro pathway in Aspergillus is less characterized, although we have recently characterized the protein function, and we have shown that the catabolic cascade induces the release of indole derivatives as indole acetate from Trp (manuscript submitted) [ 31 ].…”

“…The scheme of the experiment and the forward analysis based on expression data are shown in Figure 2 . Knowing that both pathways may potentially degrade Trp [ 4 , 31 ], we maximally induced the catabolism on the other axis by removing one axis, as shown by qPCR results ( Supplementary Materials Figure S2 ).…”

Tryptophan Co-Metabolism at the Host-Pathogen Interface

“…Trp metabolites produced by the fungus Candida, in particular 5-hydroxytryptophan metabolites, are also able to modulate Th17 response, similarly to kynurenines as shown for mammalians (34). The opportunistic fungal pathogen A. fumigatus also has three ido genes (idoA,B,C) in its genome (35)(36)(37). Enzymatic studies suggest that Idos of A. oryzae, participate in Trp degradation (38).…”

Aspergillus fumigatus tryptophan metabolic route differently affects host immunity

“…The opportunistic fungal pathogen Aspergillus fumigatus possesses three putative ido genes ( idoA , idoB , and idoC ) and two putative aro8 and aro9 genes ( aroH and aroI ) ( Yuasa and Ball, 2013 ; Wang et al., 2016 ; Dindo et al., 2018 ). Enzymatic studies suggest that Idos of A. oryzae participate in Trp degradation ( Yuasa and Ball, 2011 ).…”

“…Meanwhile, A. fumigatus grown on Trp upregulate ido gene expression ( Wang et al., 2016 ). Researchers recently purified a recombinant form of Aspergillus AroH and examined the biochemical features of the protein ( Dindo et al., 2018 ). The data indicated that AroH displays structural properties and a pyridoxal-5′-phosphate (PLP) binding capacity reminiscent of the yeast ortholog Aro8.…”