NAD⁺‐Dependent Dehydrogenase PctP and Pyridoxal 5′‐Phosphate Dependent Aminotransferase PctC Catalyze the First Postglycosylation Modification of the Sugar Intermediate in Pactamycin Biosynthesis

Abstract

The unique five-membered aminocyclitol core of the antitumor antibiotic pactamycin originates from d-glucose, so unprecedented enzymatic modifications of the sugar intermediate are involved in the biosynthesis. However, the order of the modification reactions remains elusive. Herein, we examined the timing of introduction of an amino group into certain sugar-derived intermediates by using recombinant enzymes that were encoded in the pactamycin biosynthesis gene cluster. We found that the NAD -dependent alcohol dehydrogenase PctP and pyridoxal 5'-phosphate dependent aminotransferase PctC converted N-acetyl-d-glucosaminyl-3-aminoacetophonone into 3'-amino-3'-deoxy-N-acetyl-d-glucosaminyl-3-aminoacetophenone. Further, N-acetyl-d-glucosaminyl-3-aminophenyl-β-oxopropanoic acid ethyl ester was converted into the corresponding 3'-amino derivative. However, PctP did not oxidize most of the tested d-glucose derivatives, including UDP-GlcNAc. Thus, modification of the GlcNAc moiety in pactamycin biosynthesis appears to occur after the glycosylation of aniline derivatives.

Glycosylation of acyl carrier protein-bound polyketides during pactamycin biosynthesis

“…These modifications may involve the oxidoreductase PtmN, the aminotransferase PtmA, the carbamoyltransferase PtmB, the deacetylase PtmG, the radical SAM-dependent C-methyltransferases PtmL and PtmM, and/or rearrangement and cyclization to an aminocyclopentitol intermediate by the radical SAM protein PtmC. 2,3,28 PtmJ is a putative GT-A type protein belonging to family 2 of the glycosyltransferase superfamily. Members of this family include chitin synthases, Nacetylglucosaminyltransferases, and hyaluronan synthases.…”

Biochemical and Structural Analysis of a Dehydrogenase, KanD2, and an Aminotransferase, KanS2, That Are Responsible for the Construction of the Kanosamine Moiety in Kanamycin Biosynthesis

“…These results suggest that the postglycosylation modification mechanism is likely used in the kanamycin producer strain to construct the kanosamine moiety of kanamycins as reported for the gentamicin biosynthetic pathway . This reaction order is also the same as that of the biosynthesis of the aminocyclitol antibiotic pactamycin in which one amino group of the aminocyclitol core is introduced into N -acetyl- d -glucosamine aniline derivatives by the dehydrogenase PctP and the aminotransferase PctC . UDP-Glc seems to be oxidized by the dehydrogenase RifL and subsequently transaminated by the aminotransferase RifK to yield UDP-kanosamine in the early stage of the biosynthetic pathway for 3-amino-5-hydroxybenzoic acid in rifamycin biosynthesis (Scheme S3).…”

“…6 This reaction order is also the same as that of the biosynthesis of the aminocyclitol antibiotic pactamycin in which one amino group of the aminocyclitol core is introduced into N-acetyl-D-glucosamine aniline derivatives by the dehydrogenase PctP and the aminotransferase PctC. 7 UDP-Glc seems to be oxidized by the dehydrogenase RifL and subsequently transaminated by the aminotransferase RifK to yield UDP-kanosamine in the early stage of the biosynthetic pathway for 3-amino-5-hydroxybenzoic acid in rifamycin biosynthesis (Scheme S3). 8 In the biosynthesis of kanosamine 6-phosphate in Bacillus subtilis, G6P is oxidized by the dehydrogenase NtdC and subsequently transaminated by the aminotransferase NtdA.…”

Functional Characterization of 3‐Aminobenzoic Acid Adenylation Enzyme PctU and UDP‐N‐Acetyl‐d‐Glucosamine: 3‐Aminobenzoyl‐ACP Glycosyltransferase PctL in Pactamycin Biosynthesis

“…An NAD + ‐dependent dehydrogenase, PctP, and a PLP‐dependent aminotransferase (PctC) are responsible for introducing the amino group into the C3 position of the GlcNAc‐aniline derivative during pactamycin biosynthesis . Thus, GlcNAc‐3ABA‐PctK is converted to 3‐amino‐3‐deoxy‐GlcNAc‐3ABA‐PctK by PctP and PctC.…”