title: Nonprocessive [2 + 2]e- off-loading reductase domains from mycobacterial nonribosomal peptide synthetases.
abstract: In mycobacteria, polyketide synthases and nonribosomal peptide synthetases (NRPSs) produce complex lipidic metabolites by using a thio-template mechanism of catalysis. In this study, we demonstrate that off-loading reductase (R) domain of mycobacterial NRPSs performs two consecutive [2 + 2]e(-) reductions to release thioester-bound lipopeptides as corresponding alcohols, using a nonprocessive mechanism of catalysis. The first crystal structure of an R domain from Mycobacterium tuberculosis NRPS provides strong support to this mechanistic model and suggests that the displacement of intermediate would be required for cofactor recycling. We show that 4e(-) reductases produce alcohols through a committed aldehyde intermediate, and the reduction of this intermediate is at least 10 times more efficient than the thioester-substrate. Structural and biochemical studies also provide evidence for the conformational changes associated with the reductive cycle. Further, we show that the large substrate-binding pocket with a hydrophobic platform accounts for the remarkable substrate promiscuity of these domains. Our studies present an elegant example of the recruitment of a canonical short-chain dehydrogenase/reductase family member as an off-loading domain in the context of assembly-line enzymology.
authors.affiliation: National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India.
authors.forename: Arush
authors.initials: A
authors.lastname: Chhabra
authors.forename: Asfarul S
authors.initials: AS
authors.lastname: Haque
authors.forename: Ravi Kant
authors.initials: RK
authors.lastname: Pal
authors.forename: Aneesh
authors.initials: A
authors.lastname: Goyal
authors.forename: Rajkishore
authors.initials: R
authors.lastname: Rai
authors.forename: Seema
authors.initials: S
authors.lastname: Joshi
authors.forename: Santosh
authors.initials: S
authors.lastname: Panjikar
authors.forename: Santosh
authors.initials: S
authors.lastname: Pasha
authors.forename: Rajan
authors.initials: R
authors.lastname: Sankaranarayanan
authors.forename: Rajesh S
authors.initials: RS
authors.lastname: Gokhale
mesh_terms: D000438:Alcohols / Q000378:metabolism; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004583:Electrons; D006020:Glycopeptides / Q000737:chemistry / Q000378:metabolism; D055666:Lipopeptides / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D009249:NADP; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry / Q000378:metabolism; D010453:Peptide Synthases / Q000737:chemistry* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity
chemical_list: D000438:Alcohols; D006020:Glycopeptides; D055666:Lipopeptides; D009249:NADP; D010088:Oxidoreductases; D010453:Peptide Synthases; C104385:non-ribosomal peptide synthase