Abstract
Aspartate-β-semialdehyde dehydrogenase (ASADH) catalyzes the second reaction in the aspartate pathway, a pathway required for the biosynthesis of one fifth of the essential amino acids in plants and microorganisms. Microarray analysis of a fungal pathogen T. rubrum responsible for most human dermatophytoses identified the upregulation of ASADH (trASADH) expression when the fungus is exposed to human skin, underscoring its potential as a drug target. Here we report the crystal structure of trASADH, revealing a tetrameric ASADH with a GAPDH-like fold. The tetramerization of trASADH was confirmed by sedimentation and SAXS experiments. Native PAGE demonstrated that this ASADH tetramerization is apparently universal in fungal species, unlike the functional dimer that is observed in all bacterial ASADHs. The helical subdomain in dimeric bacteria ASADH is replaced by the cover loop in archaeal/fungal ASADHs, presenting the determinant for this altered oligomerization. Mutations that disrupt the tetramerization of trASADH also abolish the catalytic activity, suggesting that the tetrameric state is required to produce the active fungal enzyme form. Our findings provide a basis to categorize ASADHs into dimeric and tetrameric enzymes, adopting a different orientation for NADP binding and offer a structural framework for designing drugs that can specifically target the fungal pathogens.
Highlights
Trichophyton rubrum is the most prevalent fungal pathogen for human dermatophytoses, accounting for ~ 70% of the total dermatophyte infections[1]
The hinge residues are mostly conserved within the aspartate-β -semialdehyde dehydrogenase (ASADH) family, NADP induced conformational dynamics have been observed in bacterial ASADHs, but not in the archaeal/fungal branch[18]
Structure comparison demonstrates that the structure of the upregulation of ASADH (trASADH) is closely related to the ASADHs from Methanococcus jannaschii, denoted mjASADH and Candida albicans, denoted caASADH (Protein Data Bank ID: 1ys[4] and 3hsk) belonging to the fungal/archaeal ASADH branch
Summary
Trichophyton rubrum is the most prevalent fungal pathogen for human dermatophytoses, accounting for ~ 70% of the total dermatophyte infections[1]. The representative structures from mjASADH and caASADH shows a complete absence of the helical subdomain[14,18] Because of this missing helical subdomain, the archaeal/fungal ASADHs are more related to the fold found in an archaeal malonyl-coenzyme A reductase (MCR) and in glyceraldehyde-3-phosphate dehydrogenase (GAPDH) rather than in the bacterial ASADHs. MCR and GAPDH are enzymes that are known to be active as tetramers[20]. The conformational dynamics of the cover loop coupled to coenzyme binding suggest a different NADP recognition mechanism by tetrameric ASADHs. Our findings offer a novel criterion to classify ASADH into dimeric and tetrameric enzymes, and provide a novel structural framework for biocide design targeting the fungal pathogens
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