Adenylate‐Forming Enzymes of Rubradirin Biosynthesis: RubC1 Is a Bifunctional Enzyme with Aminocoumarin Acyl Ligase and Tyrosine‐Activating Domains

Abstract

The biosynthesis of aminocoumarin antibiotics requires two acyladenylate-forming enzymes: one for the activation of L-tyrosine as a precursor of the aminocoumarin moiety and another for the linkage of an acyl moiety to the aminocoumarin moiety. Unexpectedly, the biosynthetic gene cluster of the aminocoumarin antibiotic rubradirin was found to contain three genes for putative acyladenylate-forming enzymes of aminocoumarin biosynthesis and conjugation. We expressed, purified, and investigated these three proteins. Orf4 (55 kDa) was shown to be an active aminocoumarin acyl ligase. RubF6 (56 kDa) was inactive, but could be converted into an active enzyme by site-directed mutagenesis. RubC1 (138 kDa) was shown to be a unique bifunctional enzyme, comprising an aminocoumarin acyl ligase, and tyrosine-adenylation and peptidyl-carrier domains. This natural hybrid enzyme is unique among known proteins. A hypothesis is proposed as to how such an enzyme could offer a particularly effective machinery for aminocoumarin antibiotic biosynthesis.

Biosynthesis of 3,5-AHBA-derived natural products

“…The C-terminal part of the dual functional RubC1 is homologous to NovH, adenylating tyrosine in novobiocin biosynthesis, whereas the N-terminal part of RubC1, homologous to NovL, is an amide synthase catalyzing the connection of the AMC and DHDP moieties. 30,77 Together with the RubC1, homologs of NovI and NovJ in novobiocin biosynthesis, are involved in the biosynthesis of the AMC moiety and the amide bond formation (Fig. 7B).…”

A Domain of RubC1 of Rubradirin Biosynthesis Can Functionally Replace MbtH‐Like Proteins in Tyrosine Adenylation

“…We previously reported a biochemical study of RubC1, a unique bifunctional enzyme that catalyzes two different key reactions in the biosynthesis of the aminocoumarin antibiotic rubradirin 3. The C‐terminal region of RubC1, hereafter termed RubC1b (Figure 1), contains an adenylation domain and a peptidyl carrier protein (PCP) domain, and activates tyrosine as a precursor of the aminocoumarin moiety.…”

“…Recently, we proved that these three tyrosine‐adenylating enzymes are dependent on the presence of MbtH‐like proteins for their activity 2a. However, the rubradirin gene cluster4 does not contain an mbtH ‐like gene, and tyrosine‐adenylating activity of RubC1 has been observed in the absence of MbtH‐like proteins 3. This discrepancy in the MbtH requirement of closely related adenylating enzymes prompted us to reinvestigate tyrosine adenylation by RubC1.…”

“…In our previous study on RubC1,3 we had expressed the investigated proteins in Escherichia coli BL21(DE3). However, the genome of E. coli contains a gene for the MbtH‐like protein YbdZ, and in a subsequent study2a we found that YbdZ binds to adenylating enzymes expressed in E. coli , thereby obscuring their true biochemical properties.…”

“…RubC1b alone was found to be completely inactive ( k cat <0.5×10 −3 s −1 ), in contrast to our previous study where we had detected low activity ( k cat =3×10 −3 s −1 ) 3. It appears likely that this activity resulted from activation of RubC1b by YbdZ, similarly to findings with other tyrosine‐adenylating enzymes 2a.…”