title: Delineating the reaction mechanism of reductase domains of Nonribosomal Peptide Synthetases from mycobacteria.
abstract: Substrate binding to enzymes often follows a precise order where catalysis is accomplished through programmed conformational changes. Short-chain dehydrogenase/reductase (SDR) enzymes follow sequential order 'bi-bi' reaction kinetics. The mechanistic study of a SDR homolog, reductase (R) domain, from multifunctional enzymes, e.g. Nonribosomal Peptide Synthetases (NRPSs) and Polyketide Synthases (PKSs) has revealed that it reductively releases 4'-phosphopantetheinyl arm-tethered peptidyl product. We report that the R-domains of NRPSs from Mycobacterium tuberculosis (RNRP) and Mycobacterium smegmatis (RGPL) do not strictly adhere to the obligatory mode of catalysis performed by SDRs, but instead can carry out reductive catalysis of substrate following random bi-bi reaction mechanism as deciphered by NMR and SAXS studies. The crucial conformational change associated with NADPH binding necessary to achieve catalytically competent conformation is also delineated by SAXS studies. Using ITC, we have demonstrated that mutation of catalytic tyrosine to phenylalanine in R-domains results in 3-4-fold decrease in affinity for NADPH and attribute this phenomenon to loss of the noncovalent cation-π interactions present between the tyrosine and nicotinamide ring. We propose that the adaptation to an alternative theme of bi-bi catalytic mechanism enables the R-domains to process the substrates transferred by upstream domains and maintain assembly-line enzymology.
authors.affiliation: CSIR-Centre for Cellular and Molecular Biology, Hyderabad 500007, India.
authors.forename: Asfarul S
authors.initials: AS
authors.lastname: Haque
authors.affiliation: CSIR-Centre for Cellular and Molecular Biology, Hyderabad 500007, India.
authors.forename: Ketan D
authors.initials: KD
authors.lastname: Patel
authors.affiliation: CSIR-Centre for Cellular and Molecular Biology, Hyderabad 500007, India.
authors.forename: Mandar V
authors.initials: MV
authors.lastname: Deshmukh
authors.affiliation: National Institute of Immunology, New Delhi 110067, India.
authors.forename: Arush
authors.initials: A
authors.lastname: Chhabra
authors.affiliation: National Institute of Immunology, New Delhi 110067, India; CSIR-Institute of Genomics and Integrative Biology, Delhi 110007, India; Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore 560064, India.
authors.forename: Rajesh S
authors.initials: RS
authors.lastname: Gokhale
authors.affiliation: CSIR-Centre for Cellular and Molecular Biology, Hyderabad 500007, India. Electronic address: sankar@ccmb.res.in.
authors.forename: Rajan
authors.initials: R
authors.lastname: Sankaranarayanan
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive; D002151:Calorimetry / Q000379:methods; D020134:Catalytic Domain; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D015394:Molecular Structure; D009154:Mutation; D020102:Mycobacterium smegmatis / Q000201:enzymology / Q000235:genetics; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics; D009249:NADP / Q000737:chemistry / Q000378:metabolism; D009536:Niacinamide / Q000737:chemistry / Q000378:metabolism; D010453:Peptide Synthases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010649:Phenylalanine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D053838:Scattering, Small Angle; D013816:Thermodynamics; D014443:Tyrosine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014961:X-Ray Diffraction
chemical_list: D001426:Bacterial Proteins; D009536:Niacinamide; D014443:Tyrosine; D010649:Phenylalanine; D009249:NADP; D010453:Peptide Synthases; C104385:non-ribosomal peptide synthase
keywords: Cation–π interaction; NRPS; Reductase domain; Sequential order bi–bi reaction mechanism; Short-chain Dehydrogenase/Reductase (SDR)