Natural amino donation: A PLP-dependent aminotransferase PctV, encoded in the pactamycin biosynthetic gene cluster, was found to catalyze the formation of 3-aminobenzoate from 3-dehydroshikimate with L-glutamate as the amino donor. The PctV reaction comprises a transamination and two dehydration reactions. This is the first report of a simple 3-ABA synthase in nature.

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.

Pactamycin is an antibiotic produced by Streptomyces pactum with antitumor and antimalarial properties. Pactamycin has a unique aminocyclitol core that is decorated with 3‐aminoacetophenone, 6‐methylsaliciate, and an N,N‐dimethylcarbamoyl group. Herein, we show that the adenylation enzyme PctU activates 3‐aminobenzoic acid (3ABA) with adenosine triphosphate and ligates it to the holo form of the discrete acyl carrier protein PctK to yield 3ABA‐PctK. Then, 3ABA‐PctK is N‐glycosylated with uridine diphosphate‐N‐acetyl‐d‐glucosamine (UDP‐GlcNAc) by the glycosyltransferase PctL to yield GlcNAc‐3ABA‐PctK. Because 3ABA is known to be a precursor of the 3‐aminoacetophenone moiety, PctU appears to be a gatekeeper that selects the appropriate 3‐aminobenzoate starter unit. Overall, we propose that acyl carrier protein‐bound glycosylated 3ABA derivatives are biosynthetic intermediates of pactamycin biosynthesis.

Mutational analysis of the pyridoxal 5'-phosphate (PLP)-dependent enzyme PctV was carried out to elucidate the multi-step reaction mechanism for the formation of 3-aminobenzoate (3-ABA) from 3-dehydroshikimate (3-DSA). Introduction of mutation K276R led to the accumulation of a quinonoid intermediate with an absorption maximum at 580 nm after the reaction of pyridoxamine 5'-phosphate (PMP) with 3-DSA. The chemical structure of this intermediate was supported by X-ray crystallographic analysis of the complex formed between the K276R mutant and the quinonoid intermediate. These results clearly show that a quinonoid intermediate is involved in the formation of 3-ABA. They also indicate that Lys276 (in the active site of PctV) plays multiple roles, including acid/base catalysis during the dehydration reaction of the quinonoid intermediate.

Kanosamine (3-amino-3-deoxy-D-glucose) is a characteristic sugar unit found in kanamycins, a group of aminoglycoside antibiotics. The kanosamine moiety originates from D-glucose in kanamycin biosynthesis. However, the timing of the replacement of the 3-OH group of the D-glucose-derived biosynthetic intermediate with the amino group is elusive. Comparison of biosynthetic gene clusters for related aminoglycoside antibiotics suggests that the nicotinamide adenine dinucleotide (NAD + )dependent dehydrogenase KanD2 and the pyridoxal 5′-phosphate (PLP)-dependent aminotransferase KanS2 are responsible for the introduction of the amino group at the C3 position of kanosamine. In this study, we demonstrated that KanD2 and KanS2 convert kanamycin A, B, and C to the corresponding 3″-deamino-3″-hydroxykanamycins (3″-hks) in the presence of PLP, 2-oxoglutarate, and NADH via a reverse reaction in the pathway. Furthermore, we observed that all of the 3″-hks are oxidized by KanD2 with NAD + , but D-glucose, UDP-D-glucose, D-glucose 6-phosphate, and D-glucose 1-phosphate are not. Crystal structure analysis of KanD2 complexed with 3″-hkB and NADH illustrated the selective recognition of pseudotrisaccharides, especially the D-glucose moiety with 2-deoxystreptamine, by a combination of hydrogen bonds and CH−π interactions. Overall, it was clarified that the kanosamine moiety of kanamycins is constructed after the glucosylation of the pseudodisaccharide biosynthetic intermediates in kanamycin biosynthesis.