Functional and Phylogenetic Divergence of Fungal Adenylate-Forming Reductases

Abstract

A key step in fungal L-lysine biosynthesis is catalyzed by adenylate-forming L-␣-aminoadipic acid reductases, organized in domains for adenylation, thiolation, and the reduction step. However, the genomes of numerous ascomycetes and basidiomycetes contain an unexpectedly large number of additional genes encoding similar but functionally distinct enzymes. Here, we describe the functional in vitro characterization of four reductases which were heterologously produced in Escherichia coli. The Ceriporiopsis subvermispora serine reductase Nps1 features a terminal ferredoxin-NADP ؉ reductase (FNR) domain and thus belongs to a hitherto undescribed class of fungal multidomain enzymes. The second major class is characterized by the canonical terminal short-chain dehydrogenase/reductase domain and represented by Ceriporiopsis subvermispora Nps3 as the first biochemically characterized L-␣-aminoadipic acid reductase of basidiomycete origin. Aspergillus flavus L-tyrosine reductases LnaA and LnbA are members of a distinct phylogenetic clade. Phylogenetic analysis supports the view that fungal adenylate-forming reductases are more diverse than previously recognized and belong to four distinct classes.

Two separate key enzymes and two pathway‐specific transcription factors are involved in fusaric acid biosynthesis in Fusarium fujikuroi

“…However, the canonical condensation (C) domain, involved in amide bond formation between two cognate aminoacyl‐ S ‐PCPs is missing (Stack et al ., ; Du and Lou, ). Therefore, NRPS34 likely confers the activation of only one amino acid in a similar manner to the α‐aminoadipate reductases involved in lysine biosynthesis in fungi (Bushley and Turgeon, ; Kalb et al ., ). All have an identical structure consisting of an A‐T unit followed by a TR domain (IPR010080), a member of the NAD(P)‐binding Rossman fold superfamily.…”

Biosynthesis of LL‐Z1272β: Discovery of a New Member of NRPS‐like Enzymes for Aryl‐Aldehyde Formation

“…A recent phylogenetic analysis of fungal adenylate‐forming reductases revealed that these enzymes can be grouped into four classes . Although StbB and ATEG_03630 belong to class III (aryl acid reductases), they display clearly distinct substrate specificities, as ATEG_03630 is known to accept orsellinic acid ( 2 ) as well .…”

Activity of α‐Aminoadipate Reductase Depends on the N‐Terminally Extending Domain

“…Still, they follow mechanistically identical biochemistry: that is, aminoacyl adenylate formation, covalent substrate tethering through a thioester, and subsequent reductive product release. Given that the ADA domain is dispensable for the functionality of these enzymes, we investigated its role by using C. subvermispora NPS3, a model basidiomycete Lys2 α‐aminoadipate reductase that had been biochemically characterized previously 6b. It was hypothesized that the function of ADA domains has relevance for the catalytic functionality of the reductase 4.…”

“…These two truncated NPS3 versions were produced as N‐terminally tagged fusions. To produce the 159.6 kDa C‐terminally tagged hexahistidine‐fusion full‐length protein, plasmid pDK21 was used 6b…”

“…Construction of expression plasmids : Plasmid pDK216b is a pET21b‐based expression construct harboring the full‐length nps3 gene, which served as PCR template to produce truncated nps3 versions. The gene lacking the ADA‐domain‐encoding portion entirely was amplified by use of forward primer oDK15 (5′‐TATAT AGAAT TCGTT GGTGC AGTTC CG‐3′) and reverse primer oDK17 (5′‐ACGAT ACATA CGATC GCGCA CACC‐3′).…”