The Enzymological Mechanism Study Of Natural Product Biosynthesis Related Non-heme Iron Oxidases OvoA And PtlH

Mononuclear non-heme iron oxidases are a large group of enzymes in nature and are famous for the varieties of it catalyzed reactions,including Dehydrogenation reaction,hydroxylation reaction and epoxidation reaction.Non-heme iron enzymes played important roles in natural products biosynthesis,such as OvoA involved in Ovothiol biosynthesis and PtlH involved in Pentalenolactone biosynthesis.Ovothiol is a histidine thiol derivative,which showed anti-oxidation and anti-cancer activities.OvoA catalyzed one of the key steps in Ovothiol biosynthesis which is oxidative coupling between cysteine and histidine.Pentalenolactone is a sesquiterpenoid antibiotic that is active against a variety of microorganisms.Its carbon skeleton was extensively modified by the action of six redox enzymes,including a hydroxylation reaction catalyzed by PtlH.OvoA catalyzed the formation of sulfoxide and cysteine sulfinic acid with cysteine and histidine as substrates.To the wild-tyoe OvoA enzyme reaction,the ratio between coupling product and cysteine sulfinic acid is 9:1.If the OvoA Y417 was replaced with phenylalanine,cysteine sulfinic acid has become the dominant product.The OvoA Y417 was further replaced with unnatural amino acid(Methylthio tyrosine,MtTyr).Intriguingly,the ratio between coupling product and cysteine sulfinic acid changed into 7:3.It means that OvoA Y417 plays a key role in the formation of coupling product and we can modulate the ratio of these two products by unnatural amino acid incorporation.To reveal the enzymatic mechanism of OvoA,isotopically sensitive branching method was employed to study how the two products were partitioned in OvoA reaction.Based on high resolution mass spectrometry and 13C NMR analysis,substrate kinetic isotope effect(KIE)of wild-type OvoA reaction is 1.01� 0.02,and solvent KIE is 1.29�0.01.To the unnatural amino acid containing OvoA Y417MtTyr mutant protein reaction,the substrate KIE is 1.08 � 0.01 and solvent KIE is 2.09�0.02.It means that the coupling product and cysteine sulfinic acid was branched out from a common intermediate,which was proposed to be a Fe3+-superoxo species in this study.The reaction goes to the cysteine sulfinic acid formation pathway if Feru abstracts an electron from substrate cysteine sulfur atom to generate a cysteine based radical.Contrarily,the reaction goes to the coupling product formation pathway if a proton-coupled electron transfers from OvoA Y417 to the Fe3+-superoxo species to generate a Y417 based radical.PtlH is a non-heme iron catalyzed the hydroxylation of 1-deoxypentalenic acid to 11b-hydroxy-1-deoxypentalenic acid.The reported PtlH crystal structure showed that PtlH Y142 locates between the Fe2+ center and substrate.There were two different conformations were observed for this tyrosine,one of them is closed and one of them is open.In this study,PtlH Y142 was replaced with phenylalanine and alanine,respectively.To the PtlH Y142F mutant,it showed similar activity with wild-type PtlH,but the PtlH Y142A mutant was not active any more.Besides the normal hydroxylation product 11?-hydroxy-1-deoxypentalenic acid,a dihydroxylated by-product was detected from the PtlH reaction.In addition,the kcat of PtlH reaction increased around 7 time and the dihydroxylated by-product amount was decreased dramatically when ascorbic acid was present in the reaction.The ascorbic acid binding pocket was predicted based on molecular docking.If the small size amino acid S1 13 was replaced with a large size amino acid Trp,the PtlH S113W mutant protein did not show ascorbic acid effect any more.These results indicate that the PtlH Y142 hydroxyl group is unnecessary for the PtlH activity,while the benzene ring plays an important role in substrate binding and product releasing due to certain hydrophobic interactions.Distinct from our known knowledge,ascorbic acid is not only a reductant but also an allosteric effector,which effects product formation directly.It is probably that ascorbic acid promotes product releasing to prevent the formation of dihydroxylated by-product.We detected the ascorbic acid effect of PtlH and defined the ascorbic acid binding pocket for the first time.It expands our understanding about ascorbic acid function.