Pyridoxal-5′-phosphate-dependent enzyme GenB3 Catalyzes C-3′,4′-dideoxygenation in gentamicin biosynthesis

Abstract

Background The C-3′,4′-dideoxygenation structure in gentamicin can prevent deactivation by aminoglycoside 3′-phosphotransferase (APH(3′)) in drug-resistant pathogens. However, the enzyme catalyzing the dideoxygenation step in the gentamicin biosynthesis pathway remains unknown. Results Here, we report that GenP catalyzes 3′ phosphorylation of the gentamicin biosynthesis intermediates JI-20A, JI-20Ba, and JI-20B. We further demonstrate that the pyridoxal-5′-phosphate (PLP)-dependent enzyme GenB3 uses these phosphorylated substrates to form 3′,4′-dideoxy-4′,5′-ene-6′-oxo products. The following C-6′-transamination and the GenB4-catalyzed reduction of 4′,5′-olefin lead to the formation of gentamicin C. To the best of our knowledge, GenB3 is the first PLP-dependent enzyme catalyzing dideoxygenation in aminoglycoside biosynthesis. Conclusions This discovery solves a long-standing puzzle in gentamicin biosynthesis and enriches our knowledge of the chemistry of PLP-dependent enzymes. Interestingly, these results demonstrate that to evade APH(3′) deactivation by pathogens, the gentamicin producers evolved a smart strategy, which utilized their own APH(3′) to activate hydroxyls as leaving groups for the 3′,4′-dideoxygenation in gentamicin biosynthesis.

Mechanistic Insights into Dideoxygenation in Gentamicin Biosynthesis

“…After incubation with a recombinant GenB3, 3 and 4, purified from in vitro phosphorylation of 1 and 2 using GenP and ATP, were converted into 6′-deamino-6′-oxo-sisomicin (oxo-sisomicin, 5; Figure S1E) and 6′-deamino-6′-oxoverdamicin (oxo-verdamicin, 6), respectively, based on LC-ESI-HRMS and NMR analyses (Figures 3A,B, S1F, and S3 and Table S1), providing direct evidence that GenB3 catalyzes the removal of the 3′-phosphate and 4′-hydroxyl groups of 3 and 4 (Figure 1). During the preparation of this manuscript, Zhou et al 27 published a paper on GenB3 with similar results to those described herein.…”

(Meta)Genomic Analysis Reveals Diverse Energy Conservation Strategies Employed by Globally Distributed Gemmatimonadota

“…The biologically active form of the vitamin B 6 group (pyridoxal, pyridoxamine, and pyridoxine), pyridoxal 5′-phosphate (PLP), is known as an essential cofactor for cellular function in all domains of life ( 25 ). PLP-dependent enzymes have been reported as being involved in diverse cellular processes, such as the biosynthesis of amino acids, sugars, lipids, and antibiotics ( 25 – 27 ). However, pyridoxal 5′-phosphate synthase ( pdx ST), which can catalyze D-ribose-5-phosphate, D-glyceraldehyde-3-phosphate, and l -glutamine into PLP, has been found in only two genomes, suggesting it is not the main route for vitamin B 6 biosynthesis.…”

Biosynthesis of cyclitols

“…The sugar moieties are further modied to produce matured kanamycins [105][106][107] and gentamicins. 103,[108][109][110][111][112][113][114][115][116][117] 3.4.3 Biosynthesis of hygromycin B. In the hygromycin B biosynthesis, the 5-OH group of 2DOS is glycosylated with UDPgalactose to yield galacamine (Fig.…”

“…The sugar moieties are further modified to produce matured kanamycins 105–107 and gentamicins. 103,108–117…”