Montelione GT

References (8)

Title : Structural Basis by Which the N-Terminal Polypeptide Segment of Rhizopus chinensis Lipase Regulates Its Substrate Binding Affinity - Zhang_2019_Biochemistry_58_3943
Author(s) : Zhang M , Yu XW , Xu Y , Guo RT , Swapna GVT , Szyperski T , Hunt JF , Montelione GT
Ref : Biochemistry , 58 :3943 , 2019
Abstract : Members of an important group of industrial enzymes, Rhizopus lipases, exhibit valuable hydrolytic features that underlie their biological functions. Particularly important is their N-terminal polypeptide segment (NTPS), which is required for secretion and proper folding but is removed in the process of enzyme maturation. A second common feature of this class of lipases is the alpha-helical "lid", which regulates the accessibility of the substrate to the enzyme active site. Some Rhizopus lipases also exhibit "interfacial activation" by micelle and/or aggregate surfaces. While it has long been recognized that the NTPS is critical for function, its dynamic features have frustrated efforts to characterize its structure by X-ray crystallography. Here, we combine nuclear magnetic resonance spectroscopy and X-ray crystallography to determine the structure and dynamics of Rhizopus chinensis lipase (RCL) with its 27-residue NTPS prosequence (r27RCL). Both r27RCL and the truncated mature form of RCL (mRCL) exhibit biphasic interfacial activation kinetics with p-nitrophenyl butyrate (pNPB). r27RCL exhibits a substrate binding affinity significantly lower than that of mRCL due to stabilization of the closed lid conformation by the NTPS. In contrast to previous predictions, the NTPS does not enhance lipase activity by increasing surface hydrophobicity but rather inhibits activity by forming conserved interactions with both the closed lid and the core protein structure. Single-site mutations and kinetic studies were used to confirm that the NTPS serves as internal competitive inhibitor and to develop a model of the associated process of interfacial activation. These structure-function studies provide the basis for engineering RCL lipases with enhanced catalytic activities.
ESTHER : Zhang_2019_Biochemistry_58_3943
PubMedSearch : Zhang_2019_Biochemistry_58_3943
PubMedID: 31436959
Gene_locus related to this paper: rhich-a3fm73

Title : Backbone and Ile-delta1, Leu, Val methyl (1)H, (15)N, and (13)C, chemical shift assignments for Rhizopus chinensis lipase - Zhang_2018_Biomol.NMR.Assign_12_63
Author(s) : Zhang M , Yu XW , Swapna GVT , Liu G , Xiao R , Xu Y , Montelione GT
Ref : Biomol NMR Assign , 12 :63 , 2018
Abstract : Lipase r27RCL is a 296-residue, 33 kDa monomeric enzyme with high ester hydrolysis activity, which has significant applications in the baking, paper and leather industries. The lipase gene proRCL from Rhizopus microsporus var. chinensis (also Rhizopus chinensis) CCTCC M201021 was cloned as a fusion construct C-terminal to a maltose-binding protein (MBP) tag, and expressed as MBP-proRCL in an Escherichia coli BL21 trxB (DE3) expression system with uniform (2)H,(13)C,(15)N-enrichment and Ile-delta1, Leu, and Val (13)CH3 methyl labeling. The fusion protein was hydrolyzed by Kex2 protease at the recognition site Lys-Arg between residues -29 and -28 of the prosequence, producing the enzyme form called r27RCL. Here we report extensive backbone (1)H, (15)N, and (13)C, as well as Ile-delta1, Leu, and Val side chain methyl, NMR resonance assignments for r27RCL.
ESTHER : Zhang_2018_Biomol.NMR.Assign_12_63
PubMedSearch : Zhang_2018_Biomol.NMR.Assign_12_63
PubMedID: 28929427
Gene_locus related to this paper: rhich-a3fm73

Title : Efficient production of (2)H, (13)C, (15)N-enriched industrial enzyme Rhizopus chinensis lipase with native disulfide bonds - Zhang_2016_Microb.Cell.Fact_15_123
Author(s) : Zhang M , Yu XW , Swapna GV , Xiao R , Zheng H , Sha C , Xu Y , Montelione GT
Ref : Microb Cell Fact , 15 :123 , 2016
Abstract : BACKGROUND: In order to use most modern methods of NMR spectroscopy to study protein structure and dynamics, isotope-enriched protein samples are essential. Especially for larger proteins (>20 kDa), perdeuterated and Ile (delta1), Leu, and Val methyl-protonated protein samples are required for suppressing nuclear relaxation to provide improved spectral quality, allowing key backbone and side chain resonance assignments needed for protein structure and dynamics studies. Escherichia coli and Pichia pastoris are two of the most popular expression systems for producing isotope-enriched, recombinant protein samples for NMR investigations. The P. pastoris system can be used to produce (13)C, (15)N-enriched and even (2)H,(13)C, (15)N-enriched protein samples, but efficient methods for producing perdeuterated proteins with Ile (delta1), Leu and Val methyl-protonated groups in P. pastoris are still unavailable. Glycosylation heterogeneity also provides challenges to NMR studies. E. coli expression systems are efficient for overexpressing perdeuterated and Ile (delta1), Leu, Val methyl-protonated protein samples, but are generally not successful for producing secreted eukaryotic proteins with native disulfide bonds.
RESULTS: The 33 kDa protein-Rhizopus chinensis lipase (RCL), an important industrial enzyme, was produced using both P. pastoris and E. coli BL21 trxB (DE3) systems. Samples produced from both systems exhibit identical native disulfide bond formation and similar 2D NMR spectra, indicating similar native protein folding. The yield of (13)C, (15)N-enriched r27RCL produced using P. pastoris was 1.7 times higher that obtained using E. coli, while the isotope-labeling efficiency was ~15 % lower. Protein samples produced in P. pastoris exhibit O-glycosylation, while the protein samples produced in E. coli were not glycosylated. The specific activity of r27RCL from P. pastoris was ~1.4 times higher than that produced in E. coli.
CONCLUSIONS: These data demonstrate efficient production of (2)H, (13)C, (15)N-enriched, Ile (delta1), Leu, Val methyl-protonated eukaryotic protein r27RCL with native disulfides using the E. coli BL21 trxB (DE3) system. For certain NMR studies, particularly efforts for resonance assignments, structural studies, and dynamic studies, E. coli provides a cost-effective system for producing isotope-enriched RCL. It should also be potential for producing other (2)H, (13)C, (15)N-enriched, Ile (delta1), Leu, Val methyl-protonated eukaryotic proteins with native disulfide bonds.
ESTHER : Zhang_2016_Microb.Cell.Fact_15_123
PubMedSearch : Zhang_2016_Microb.Cell.Fact_15_123
PubMedID: 27411547

Title : A bidirectional system for the dynamic small molecule control of intracellular fusion proteins - Neklesa_2013_ACS.Chem.Biol_8_2293
Author(s) : Neklesa TK , Noblin DJ , Kuzin AP , Lew S , Seetharaman J , Acton TB , Kornhaber GJ , Xiao R , Montelione GT , Tong L , Crews CM
Ref : ACS Chemical Biology , 8 :2293 , 2013
Abstract : Small molecule control of intracellular protein levels allows temporal and dose-dependent regulation of protein function. Recently, we developed a method to degrade proteins fused to a mutant dehalogenase (HaloTag2) using small molecule hydrophobic tags (HyTs). Here, we introduce a complementary method to stabilize the same HaloTag2 fusion proteins, resulting in a unified system allowing bidirectional control of cellular protein levels in a temporal and dose-dependent manner. From a small molecule screen, we identified N-(3,5-dichloro-2-ethoxybenzyl)-2H-tetrazol-5-amine as a nanomolar HALoTag2 Stabilizer (HALTS1) that reduces the Hsp70:HaloTag2 interaction, thereby preventing HaloTag2 ubiquitination. Finally, we demonstrate the utility of the HyT/HALTS system in probing the physiological role of therapeutic targets by modulating HaloTag2-fused oncogenic H-Ras, which resulted in either the cessation (HyT) or acceleration (HALTS) of cellular transformation. In sum, we present a general platform to study protein function, whereby any protein of interest fused to HaloTag2 can be either degraded 10-fold or stabilized 5-fold using two corresponding compounds.
ESTHER : Neklesa_2013_ACS.Chem.Biol_8_2293
PubMedSearch : Neklesa_2013_ACS.Chem.Biol_8_2293
PubMedID: 23978068
Gene_locus related to this paper: rhoso-halo1

Title : Computational design of catalytic dyads and oxyanion holes for ester hydrolysis - Richter_2012_J.Am.Chem.Soc_134_16197
Author(s) : Richter F , Blomberg R , Khare SD , Kiss G , Kuzin AP , Smith AJ , Gallaher J , Pianowski Z , Helgeson RC , Grjasnow A , Xiao R , Seetharaman J , Su M , Vorobiev S , Lew S , Forouhar F , Kornhaber GJ , Hunt JF , Montelione GT , Tong L , Houk KN , Hilvert D , Baker D
Ref : Journal of the American Chemical Society , 134 :16197 , 2012
Abstract : Nucleophilic catalysis is a general strategy for accelerating ester and amide hydrolysis. In natural active sites, nucleophilic elements such as catalytic dyads and triads are usually paired with oxyanion holes for substrate activation, but it is difficult to parse out the independent contributions of these elements or to understand how they emerged in the course of evolution. Here we explore the minimal requirements for esterase activity by computationally designing artificial catalysts using catalytic dyads and oxyanion holes. We found much higher success rates using designed oxyanion holes formed by backbone NH groups rather than by side chains or bridging water molecules and obtained four active designs in different scaffolds by combining this motif with a Cys-His dyad. Following active site optimization, the most active of the variants exhibited a catalytic efficiency (k(cat)/K(M)) of 400 M(-1) s(-1) for the cleavage of a p-nitrophenyl ester. Kinetic experiments indicate that the active site cysteines are rapidly acylated as programmed by design, but the subsequent slow hydrolysis of the acyl-enzyme intermediate limits overall catalytic efficiency. Moreover, the Cys-His dyads are not properly formed in crystal structures of the designed enzymes. These results highlight the challenges that computational design must overcome to achieve high levels of activity.
ESTHER : Richter_2012_J.Am.Chem.Soc_134_16197
PubMedSearch : Richter_2012_J.Am.Chem.Soc_134_16197
PubMedID: 22871159

Title : Human retinoblastoma binding protein 9, a serine hydrolase implicated in pancreatic cancers - Vorobiev_2012_Protein.Pept.Lett_19_194
Author(s) : Vorobiev SM , Huang YJ , Seetharaman J , Xiao R , Acton TB , Montelione GT , Tong L
Ref : Protein Pept Lett , 19 :194 , 2012
Abstract : Human retinoblastoma binding protein 9 (RBBP9) is an interacting partner of the retinoblastoma susceptibility protein (Rb). RBBP9 is a tumor-associated protein required for pancreatic neoplasia, affects cell cycle control, and is involved in the TGF-beta signalling pathway. Sequence analysis suggests that RBBP9 belongs to the alpha/beta hydrolase superfamily of enzymes. The serine hydrolase activity of RBBP9 is required for development of pancreatic carcinomas in part by inhibiting TGF-beta antiproliferative signaling through suppressing Smad2/3 phosphorylation. The crystal structure of human RBBP9 confirms the alpha/beta hydrolase fold, with a six-stranded parallel beta-sheet flanked by alpha helixes. The structure of RBBP9 resembles that of the YdeN protein from Bacillus subtilis, which is suggested to have carboxylesterase activity. RBBP9 contains a Ser75-His165-Asp138 catalytic triad, situated in a prominent pocket on the surface of the protein. The side chains of the LxCxE sequence motif that is important for interaction with Rb is mostly buried in the structure. Structure- function studies of RBBP9 suggest possible routes for novel cancer drug discovery programs.
ESTHER : Vorobiev_2012_Protein.Pept.Lett_19_194
PubMedSearch : Vorobiev_2012_Protein.Pept.Lett_19_194
PubMedID: 21933118
Gene_locus related to this paper: human-RBBP9

Title : Crystal structure of human retinoblastoma binding protein 9 -
Author(s) : Vorobiev SM , Su M , Seetharaman J , Huang YJ , Chen CX , Maglaqui M , Janjua H , Proudfoot M , Yakunin A , Xiao R , Acton TB , Montelione GT , Tong L
Ref : Proteins , 74 :526 , 2009
PubMedID: 19004028
Gene_locus related to this paper: human-RBBP9

Title : Structural and biochemical studies identify tobacco SABP2 as a methyl salicylate esterase and implicate it in plant innate immunity - Forouhar_2005_Proc.Natl.Acad.Sci.U.S.A_102_1773
Author(s) : Forouhar F , Yang Y , Kumar D , Chen Y , Fridman E , Park SW , Chiang Y , Acton TB , Montelione GT , Pichersky E , Klessig DF , Tong L
Ref : Proc Natl Acad Sci U S A , 102 :1773 , 2005
Abstract : Salicylic acid (SA) is a critical signal for the activation of plant defense responses against pathogen infections. We recently identified SA-binding protein 2 (SABP2) from tobacco as a protein that displays high affinity for SA and plays a crucial role in the activation of systemic acquired resistance to plant pathogens. Here we report the crystal structures of SABP2, alone and in complex with SA at up to 2.1-A resolution. The structures confirm that SABP2 is a member of the alpha/beta hydrolase superfamily of enzymes, with Ser-81, His-238, and Asp-210 as the catalytic triad. SA is bound in the active site and is completely shielded from the solvent, consistent with the high affinity of this compound for SABP2. Our biochemical studies reveal that SABP2 has strong esterase activity with methyl salicylate as the substrate, and that SA is a potent product inhibitor of this catalysis. Modeling of SABP2 with MeSA in the active site is consistent with all these biochemical observations. Our results suggest that SABP2 may be required to convert MeSA to SA as part of the signal transduction pathways that activate systemic acquired resistance and perhaps local defense responses as well.
ESTHER : Forouhar_2005_Proc.Natl.Acad.Sci.U.S.A_102_1773
PubMedSearch : Forouhar_2005_Proc.Natl.Acad.Sci.U.S.A_102_1773
PubMedID: 15668381
Gene_locus related to this paper: nicta-SABP2