Reaction mechanism of azoreductases suggests convergent evolution with quinone oxidoreductases

Azoreductases are involved in the bioremediation by bacteria of azo dyes found in waste water.In the gut flora,they activate azo pro-drugs,which are used for treatment of inflammatory bowel disease,releasing the active component 5-aminosalycilic acid.The bacterium P.aeruginosa has three azoreductase genes,paAzoR1,paAzoR2 and paAzoR3,which as recombinant enzymes have been shown to have different substrate specificities.The mechanism of azoreduction relies upon tautomerisation of the substrate to the hydrazone form.We report here the characterization of the P.aeruginosa azoreductase enzymes,including determining their thermostability,cofactor preference and kinetic constants against a range of their favoured substrates.The expression levels of these enzymes during growth of P.aeruginosa are altered by the presence of azo substrates.It is shown that enzymes that were originally described as azoreductases,are likely to act as NADH quinone oxidoreductases.The low sequence identities observed among NAD(P)H quinone oxidoreductase and azoreductase enzymes suggests convergent evolution.

P450-mediated dehydrotyrosine formation during WS9326 biosynthesis proceeds via dehydrogenation of a specific acylated dipeptide substrate

WS9326A is a peptide antibiotic containing a highly unusual N-methyl-E-2-3-dehydrotyrosine(NMet-Dht)residue that is incorporated during peptide assembly on a non-ribosomal peptide synthetase(NRPS).The cytochrome P450 encoded by sas16(P450Sas)has been shown to be essential for the formation of the alkene moiety in NMet-Dht,but the timing and mechanism of the P450Sas-mediatedα,β-dehydrogenation of Dht remained unclear.Here,we show that the substrate of P450Sas is the NRPS-associated peptidyl carrier protein(PCP)-bound dipeptide intermediate(Z)-2-pent-1′-enyl-cinnamoyl-Thr-N-Me-Tyr.We demonstrate that P450Sas-mediated incorporation of the double bond follows N-methylation of the Tyr by the N-methyl transferase domain found within the NRPS,and further that P450Sas appears to be specific for substrates containing the(Z)-2-pent-1’-enyl-cinnamoyl group.A crystal structure of P450Sas reveals differences between P450Sas and other P450s involved in the modification of NRPS-associated substrates,including the substitution of the canonical active site alcohol residue with a phenylalanine(F250),which in turn is critical to P450Sas activity and WS9326A biosynthesis.Together,our results suggest that P450Sas catalyses the direct dehydrogenation of the NRPS-bound dipeptide substrate,thus expanding the repertoire of P450 enzymes that can be used to produce biologically active peptides.

Mechanistic Study of Oxidoreductase AprQ Involved in Biosynthesis of Aminoglycoside Antibiotic Apramycin

Main observation and conclusion The aminoglycoside antibiotic apramycin contains a unique bicyclic octose moiety,and biosynthesis of this moiety involves an oxidoreductase AprQ.Unlike other known“Q”series proteins involved in aminoglycosides biosynthesis,AprQ does not work with an aminotransferase partner,and performs a four-electron oxidation that converts a CH2OH moiety to a carboxylate group.

Melatonin biosynthesis pathways in nature and its production in engineered microorganisms

Melatonin is a biogenic amine that can be found in plants,animals and microorganism.The metabolic pathway of melatonin is different in various organisms,and biosynthetic endogenous melatonin acts as a molecular signal and antioxidant protection against external stress.Microbial synthesis pathways of melatonin are similar to those of animals but different from those of plants.At present,the method of using microorganism fermentation to produce melatonin is gradually prevailing,and exploring the biosynthetic pathway of melatonin to modify microorganism is becoming the mainstream,which has more advantages than traditional chemical synthesis.Here,we review recent advances in the synthesis,optimization of melatonin pathway.L-tryptophan is one of the two crucial precursors for the synthesis of melatonin,which can be produced through a four-step reaction.Enzymes involved in melatonin synthesis have low specificity and catalytic efficiency.Site-directed mutation,directed evolution or promotion of cofactor synthesis can enhance enzyme activity and increase the metabolic flow to promote microbial melatonin production.On the whole,the status and bottleneck of melatonin biosynthesis can be improved to a higher level,providing an effective reference for future microbial modification.