Mechanistic Insights into Dideoxygenation in Gentamicin Biosynthesis

Abstract

Gentamicin is an important aminoglycoside antibiotic used for treatment of infections caused by Gram-negative bacteria. Although most of the biosynthetic pathways of gentamicin have been elucidated, a remaining intriguing question is how the intermediates JI-20A and JI-20B undergo a dideoxygenation to form gentamicin C complex. Here we show that the dideoxygenation process starts with GenP-catalyzed phosphorylation of JI-20A and JI-20Ba. The phosphorylated products are successively modified by concerted actions of two PLP (pyridoxal 5′-phosphate)-dependent enzymes: elimination of water and then phosphate by GenB3 and double bond migration by GenB4. Each of these reactions liberates an imine which hydrolyses to a ketone or aldehyde and is then reaminated by GenB3 using an amino donor. Importantly, crystal structures of GenB3 and GenB4 have guided site-directed mutagenesis to reveal crucial residues for the enzymes’ functions. We propose catalytic mechanisms for GenB3 and GenB4, which shed light on the already unrivalled catalytic versatility of PLP-dependent enzymes.

Facile and selective N-alkylation of gentamicin antibiotics via chemoenzymatic synthesis

“…S18). This different reactivity of GenB4 was consistent with prior studies which showed that the enzyme isomerizes the C-4'/C-5'-double bond of 1f 30,31 . ForB deaminated 1f in equal conversions to NeoB, despite being tested at almost half the enzyme concentration, however it had very little activity on all other substrates tested.…”

“…GenB4 converted 1f to 2e in near quantitative yield. Additionally, Li and co-workers demonstrated that GenB4 does not require an amine acceptor for the C-4'/C-5' double bond isomerisation 31 (Figure 1A), which is favourable as it reduces the number of components present in the reaction and could potentially ease subsequent product isolation. Therefore, the reaction conditions of the GenB4 mediated isomerisation of sisomicin (1f) were explored.…”

“…Interestingly, the C-6'-TAm, NeoB, from neomycin biosynthesis was also shown to deaminate the C-6''' position of neomycin C, generating an aldehyde in this position 21 [Scheme 1C(i)]. More recently, another biosynthetic TAm, GenB4 from gentamicin biosynthesis, was shown to naturally convert sisomicin to 6'-oxogentamicin C1a in quantitative conversions by isomerisation of the C-4'/C-5' double bond [Scheme 1C(ii)] 30,31 . These enzymatic reactions were of interest as they showed regioselective amine conversion to a more chemically versatile aldehyde group in a single step.…”

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…”