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Lysine relay mechanism coordinates intermediate transfer in vitamin B6 biosynthesis.

ABSTRACT: Substrate channeling has emerged as a common mechanism for enzymatic intermediate transfer. A conspicuous gap in knowledge concerns the use of covalent lysine imines in the transfer of carbonyl-group-containing intermediates, despite their wideuse in enzymatic catalysis. Here we show how imine chemistry operates in the transfer of covalent intermediates in pyridoxal 5'-phosphate biosynthesis by the Arabidopsis thaliana enzyme Pdx1. An initial ribose 5-phosphate lysine imine is converted to the chromophoric I320 intermediate, simultaneously bound to two lysine residues and partially vacating the active site, which creates space for glyceraldehyde 3-phosphate to bind. Crystal structures show how substrate binding, catalysis and shuttling are coupled to conformational changes around strand ?6 of the Pdx1 (??)8-barrel. The dual-specificity active site and imine relay mechanism for migration of carbonyl intermediates provide elegant solutions to the challenge of coordinating a complex sequence of reactions that follow a path of over 20 Å between substrate- and product-binding sites.

SUBMITTER: Rodrigues MJ

PROVIDER: S-EPMC6078385 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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Lysine relay mechanism coordinates intermediate transfer in vitamin B6 biosynthesis.

Rodrigues Matthew J MJ Windeisen Volker V Zhang Yang Y Guédez Gabriela G Weber Stefan S Strohmeier Marco M Hanes Jeremiah W JW Royant Antoine A Evans Gwyndaf G Sinning Irmgard I Ealick Steven E SE Begley Tadhg P TP Tews Ivo I

Nature chemical biology 20170116 3

Substrate channeling has emerged as a common mechanism for enzymatic intermediate transfer. A conspicuous gap in knowledge concerns the use of covalent lysine imines in the transfer of carbonyl-group-containing intermediates, despite their wideuse in enzymatic catalysis. Here we show how imine chemistry operates in the transfer of covalent intermediates in pyridoxal 5'-phosphate biosynthesis by the Arabidopsis thaliana enzyme Pdx1. An initial ribose 5-phosphate lysine imine is converted to the c ...[more]

PMID: 28092359

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Project description:UnlabelledPhotorhabdus luminescens is a Gram-negative entomopathogenic bacterium which symbiotically associates with the entomopathogenic nematode Heterorhabditis bacteriophora P. luminescens is highly virulent to many insects and nonsymbiotic nematodes, including Caenorhabditis elegans To understand the virulence mechanisms of P. luminescens, we obtained virulence-deficient and -attenuated mutants against C. elegans through a transposon-mutagenized library. From the genetic screening, we identified the pdxB gene, encoding erythronate-4-phosphate dehydrogenase, as required for de novo vitamin B6 biosynthesis. Mutation in pdxB caused growth deficiency of P. luminescens in nutrient-poor medium, which was restored under nutrient-rich conditions or by supplementation with pyridoxal 5'-phosphate (PLP), an active form of vitamin B6 Supplementation with three other B6 vitamers (pyridoxal, pyridoxine, and pyridoxamine) also restored the growth of the pdxB mutant, suggesting the existence of a salvage pathway for vitamin B6 biosynthesis in P. luminescens Moreover, supplementation with PLP restored the virulence-deficient phenotype against C. elegans Combining these results with the fact that pdxB mutation also caused attenuation of insecticidal activity, we concluded that the production of appropriate amounts of vitamin B6 is critical for P. luminescens pathogenicity.ImportanceThe Gram-negative entomopathogenic bacterium Photorhabdus luminescens symbiotically associates with the entomopathogenic nematode Heterorhabditis bacteriophora P. luminescens is highly virulent to many insects and nonsymbiotic nematodes, including Caenorhabditis elegans We have obtained several virulence-deficient and -attenuated P. luminescens mutants against C. elegans through genetic screening. From the genetic analysis, we present the vitamin B6 biosynthetic pathways in P. luminescens that are important for its insecticidal activity. Mutation in pdxB, encoding erythronate-4-phosphate dehydrogenase and required for the de novo vitamin B6 biosynthesis pathway, caused virulence deficiency against C. elegans and growth deficiency of P. luminescens in nutrient-poor medium. Because such phenotypes were restored under nutrient-rich conditions or by supplementation with B6 vitamers, we showed the presence of the two vitamin B6 synthetic pathways (de novo and salvage) in P. luminescens and also showed that the ability to produce an appropriate amount of vitamin B6 is critical for P. luminescens pathogenicity.

Dataset Information

Lysine relay mechanism coordinates intermediate transfer in vitamin B6 biosynthesis.

Publications

Lysine relay mechanism coordinates intermediate transfer in vitamin B6 biosynthesis.

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