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1.
bioRxiv ; 2024 Jun 19.
Article in English | MEDLINE | ID: mdl-38895306

ABSTRACT

How can a single protein domain encode a conformational landscape with multiple stably-folded states, and how do those states interconvert? Here, we use real-time and relaxation-dispersion NMR to characterize the conformational landscape of the circadian rhythm protein KaiB from Rhodobacter sphaeroides. Unique among known natural metamorphic proteins, this KaiB variant spontaneously interconverts between two monomeric states: the "Ground" and "Fold-switched" (FS) state. KaiB in its FS state interacts with multiple binding partners, including the central KaiC protein, to regulate circadian rhythms. We find that KaiB itself takes hours to interconvert between the Ground and FS state, underscoring the ability of a single sequence to encode the slow process needed for function. We reveal the rate-limiting step between the Ground and FS state is the cis-trans isomerization of three prolines in the fold-switching region by demonstrating interconversion acceleration by the prolyl isomerase CypA. The interconversion proceeds through a "partially disordered" (PD) state, where the C-terminal half becomes disordered while the N-terminal half remains stably folded. We discovered two additional properties of KaiB's landscape. Firstly, the Ground state experiences cold denaturation: at 4°C, the PD state becomes the majorly populated state. Secondly, the Ground state exchanges with a fourth state, the "Enigma" state, on the millisecond timescale. We combine AlphaFold2-based predictions and NMR chemical shift predictions to predict this "Enigma" state is a beta-strand register shift that eases buried charged residues, and support this structure experimentally. These results provide mechanistic insight in how evolution can design a single sequence that achieves specific timing needed for its function.

2.
Nature ; 625(7996): 832-839, 2024 Jan.
Article in English | MEDLINE | ID: mdl-37956700

ABSTRACT

AlphaFold2 (ref. 1) has revolutionized structural biology by accurately predicting single structures of proteins. However, a protein's biological function often depends on multiple conformational substates2, and disease-causing point mutations often cause population changes within these substates3,4. We demonstrate that clustering a multiple-sequence alignment by sequence similarity enables AlphaFold2 to sample alternative states of known metamorphic proteins with high confidence. Using this method, named AF-Cluster, we investigated the evolutionary distribution of predicted structures for the metamorphic protein KaiB5 and found that predictions of both conformations were distributed in clusters across the KaiB family. We used nuclear magnetic resonance spectroscopy to confirm an AF-Cluster prediction: a cyanobacteria KaiB variant is stabilized in the opposite state compared with the more widely studied variant. To test AF-Cluster's sensitivity to point mutations, we designed and experimentally verified a set of three mutations predicted to flip KaiB from Rhodobacter sphaeroides from the ground to the fold-switched state. Finally, screening for alternative states in protein families without known fold switching identified a putative alternative state for the oxidoreductase Mpt53 in Mycobacterium tuberculosis. Further development of such bioinformatic methods in tandem with experiments will probably have a considerable impact on predicting protein energy landscapes, essential for illuminating biological function.


Subject(s)
Cluster Analysis , Machine Learning , Protein Conformation , Protein Folding , Proteins , Sequence Alignment , Mutation , Proteins/chemistry , Proteins/genetics , Proteins/metabolism , Rhodobacter sphaeroides , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism
3.
Nature ; 616(7955): 183-189, 2023 04.
Article in English | MEDLINE | ID: mdl-36949197

ABSTRACT

Circadian rhythms play an essential part in many biological processes, and only three prokaryotic proteins are required to constitute a true post-translational circadian oscillator1. The evolutionary history of the three Kai proteins indicates that KaiC is the oldest member and a central component of the clock2. Subsequent additions of KaiB and KaiA regulate the phosphorylation state of KaiC for time synchronization. The canonical KaiABC system in cyanobacteria is well understood3-6, but little is known about more ancient systems that only possess KaiBC. However, there are reports that they might exhibit a basic, hourglass-like timekeeping mechanism7-9. Here we investigate the primordial circadian clock in Rhodobacter sphaeroides, which contains only KaiBC, to elucidate its inner workings despite missing KaiA. Using a combination of X-ray crystallography and cryogenic electron microscopy, we find a new dodecameric fold for KaiC, in which two hexamers are held together by a coiled-coil bundle of 12 helices. This interaction is formed by the carboxy-terminal extension of KaiC and serves as an ancient regulatory moiety that is later superseded by KaiA. A coiled-coil register shift between daytime and night-time conformations is connected to phosphorylation sites through a long-range allosteric network that spans over 140 Å. Our kinetic data identify the difference in the ATP-to-ADP ratio between day and night as the environmental cue that drives the clock. They also unravel mechanistic details that shed light on the evolution of self-sustained oscillators.


Subject(s)
Bacterial Proteins , Circadian Clocks , Circadian Rhythm , Rhodobacter sphaeroides , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Bacterial Proteins/ultrastructure , Phosphorylation , Rhodobacter sphaeroides/chemistry , Rhodobacter sphaeroides/metabolism , Crystallography, X-Ray , Cryoelectron Microscopy , Adenosine Triphosphate/metabolism , Adenosine Diphosphate/metabolism , Kinetics , Protein Folding , Protein Conformation , Allosteric Regulation
4.
Science ; 370(6523): 1442-1446, 2020 12 18.
Article in English | MEDLINE | ID: mdl-33214289

ABSTRACT

The advent of biocatalysts designed computationally and optimized by laboratory evolution provides an opportunity to explore molecular strategies for augmenting catalytic function. Applying a suite of nuclear magnetic resonance, crystallography, and stopped-flow techniques to an enzyme designed for an elementary proton transfer reaction, we show how directed evolution gradually altered the conformational ensemble of the protein scaffold to populate a narrow, highly active conformational ensemble and accelerate this transformation by nearly nine orders of magnitude. Mutations acquired during optimization enabled global conformational changes, including high-energy backbone rearrangements, that cooperatively organized the catalytic base and oxyanion stabilizer, thus perfecting transition-state stabilization. The development of protein catalysts for many chemical transformations could be facilitated by explicitly sampling conformational substates during design and specifically stabilizing productive substates over all unproductive conformations.


Subject(s)
Biocatalysis , Computer-Aided Design , Directed Molecular Evolution , Enzymes/chemistry , Enzymes/genetics , Proteins/chemistry , Proteins/genetics , Catalytic Domain , Nuclear Magnetic Resonance, Biomolecular , Protein Conformation
5.
Proc Natl Acad Sci U S A ; 117(32): 19221-19227, 2020 08 11.
Article in English | MEDLINE | ID: mdl-32719139

ABSTRACT

Despite the outstanding success of the cancer drug imatinib, one obstacle in prolonged treatment is the emergence of resistance mutations within the kinase domain of its target, Abl. We noticed that many patient-resistance mutations occur in the dynamic hot spots recently identified to be responsible for imatinib's high selectivity toward Abl. In this study, we provide an experimental analysis of the mechanism underlying drug resistance for three major resistance mutations (G250E, Y253F, and F317L). Our data settle controversies, revealing unexpected resistance mechanisms. The mutations alter the energy landscape of Abl in complex ways: increased kinase activity, altered affinity, and cooperativity for the substrates, and, surprisingly, only a modestly decreased imatinib affinity. Only under cellular adenosine triphosphate (ATP) concentrations, these changes cumulate in an order of magnitude increase in imatinib's half-maximal inhibitory concentration (IC50). These results highlight the importance of characterizing energy landscapes of targets and its changes by drug binding and by resistance mutations developed by patients.


Subject(s)
Antineoplastic Agents/pharmacology , Imatinib Mesylate/pharmacology , Neoplasms/enzymology , Oncogene Proteins v-abl/genetics , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Drug Resistance, Neoplasm , Humans , Neoplasms/drug therapy , Neoplasms/genetics , Oncogene Proteins v-abl/chemistry , Oncogene Proteins v-abl/metabolism
6.
Science ; 367(6480): 912-917, 2020 02 21.
Article in English | MEDLINE | ID: mdl-32079772

ABSTRACT

A myriad of cellular events are regulated by allostery; therefore, evolution of this process is of fundamental interest. Here, we use ancestral sequence reconstruction to resurrect ancestors of two colocalizing proteins, Aurora A kinase and its allosteric activator TPX2 (targeting protein for Xklp2), to experimentally characterize the evolutionary path of allosteric activation. Autophosphorylation of the activation loop is the most ancient activation mechanism; it is fully developed in the oldest kinase ancestor and has remained stable over 1 billion years of evolution. As the microtubule-associated protein TPX2 appeared, efficient kinase binding to TPX2 evolved, likely owing to increased fitness by virtue of colocalization. Subsequently, TPX2-mediated allosteric kinase regulation gradually evolved. Surprisingly, evolution of this regulation is encoded in the kinase and did not arise by a dominating mechanism of coevolution.


Subject(s)
Aurora Kinase A/classification , Aurora Kinase A/metabolism , Evolution, Molecular , Allosteric Regulation , Animals , Aurora Kinase A/chemistry , Cell Cycle Proteins/metabolism , Enzyme Activation , Humans , Microtubule-Associated Proteins/metabolism , Phylogeny
7.
J Biol Chem ; 295(12): 3965-3981, 2020 03 20.
Article in English | MEDLINE | ID: mdl-32014994

ABSTRACT

Hydroxylation of substituted phenols by flavin-dependent monooxygenases is the first step of their biotransformation in various microorganisms. The reaction is thought to proceed via electrophilic aromatic substitution, catalyzed by enzymatic deprotonation of substrate, in single-component hydroxylases that use flavin as a cofactor (group A). However, two-component hydroxylases (group D), which use reduced flavin as a co-substrate, are less amenable to spectroscopic investigation. Herein, we employed 19F NMR in conjunction with fluorinated substrate analogs to directly measure pKa values and to monitor protein events in hydroxylase active sites. We found that the single-component monooxygenase 3-hydroxybenzoate 6-hydroxylase (3HB6H) depresses the pKa of the bound substrate analog 4-fluoro-3-hydroxybenzoate (4F3HB) by 1.6 pH units, consistent with previously proposed mechanisms. 19F NMR was applied anaerobically to the two-component monooxygenase 4-hydroxyphenylacetate 3-hydroxylase (HPAH), revealing depression of the pKa of 3-fluoro-4-hydroxyphenylacetate by 2.5 pH units upon binding to the C2 component of HPAH. 19F NMR also revealed a pKa of 8.7 ± 0.05 that we attributed to an active-site residue involved in deprotonating bound substrate, and assigned to His-120 based on studies of protein variants. Thus, in both types of hydroxylases, we confirmed that binding favors the phenolate form of substrate. The 9 and 14 kJ/mol magnitudes of the effects for 3HB6H and HPAH-C2, respectively, are consistent with pKa tuning by one or more H-bonding interactions. Our implementation of 19F NMR in anaerobic samples is applicable to other two-component flavin-dependent hydroxylases and promises to expand our understanding of their catalytic mechanisms.


Subject(s)
Bacterial Proteins/metabolism , Flavins/metabolism , Mixed Function Oxygenases/metabolism , 4-Hydroxybenzoate-3-Monooxygenase/genetics , 4-Hydroxybenzoate-3-Monooxygenase/metabolism , Bacterial Proteins/genetics , Binding Sites , Biocatalysis , Catalytic Domain , Hydrogen Bonding , Hydrogen-Ion Concentration , Kinetics , Mixed Function Oxygenases/genetics , Mutagenesis, Site-Directed , Nuclear Magnetic Resonance, Biomolecular , Phenylacetates/chemistry , Phenylacetates/metabolism , Rhodococcus/enzymology , Substrate Specificity
8.
Nat Commun ; 9(1): 4507, 2018 10 30.
Article in English | MEDLINE | ID: mdl-30375376

ABSTRACT

Protein tyrosine phosphatase SHP2 functions as a key regulator of cell cycle control, and activating mutations cause several cancers. Here, we dissect the energy landscape of wild-type SHP2 and the oncogenic mutation E76K. NMR spectroscopy and X-ray crystallography reveal that wild-type SHP2 exchanges between closed, inactive and open, active conformations. E76K mutation shifts this equilibrium toward the open state. The previously unknown open conformation is characterized, including the active-site WPD loop in the inward and outward conformations. Binding of the allosteric inhibitor SHP099 to E76K mutant, despite much weaker, results in an identical structure as the wild-type complex. A conformational selection to the closed state reduces drug affinity which, combined with E76K's much higher activity, demands significantly greater SHP099 concentrations to restore wild-type  activity levels. The differences in structural ensembles and drug-binding kinetics of cancer-associated SHP2 forms may stimulate innovative ideas for developing more potent inhibitors for activated SHP2 mutants.


Subject(s)
Allosteric Regulation/genetics , Mutation , Piperidines/metabolism , Protein Tyrosine Phosphatase, Non-Receptor Type 11/genetics , Pyrimidines/metabolism , Crystallography, X-Ray , Humans , Magnetic Resonance Spectroscopy , Piperidines/pharmacology , Protein Conformation , Protein Tyrosine Phosphatase, Non-Receptor Type 11/antagonists & inhibitors , Protein Tyrosine Phosphatase, Non-Receptor Type 11/metabolism , Protein Tyrosine Phosphatase, Non-Receptor Type 11/ultrastructure , Pyrimidines/pharmacology
9.
Elife ; 72018 06 14.
Article in English | MEDLINE | ID: mdl-29901437

ABSTRACT

Protein kinases are major drug targets, but the development of highly-selective inhibitors has been challenging due to the similarity of their active sites. The observation of distinct structural states of the fully-conserved Asp-Phe-Gly (DFG) loop has put the concept of conformational selection for the DFG-state at the center of kinase drug discovery. Recently, it was shown that Gleevec selectivity for the Tyr-kinase Abl was instead rooted in conformational changes after drug binding. Here, we investigate whether protein dynamics after binding is a more general paradigm for drug selectivity by characterizing the binding of several approved drugs to the Ser/Thr-kinase Aurora A. Using a combination of biophysical techniques, we propose a universal drug-binding mechanism, that rationalizes selectivity, affinity and long on-target residence time for kinase inhibitors. These new concepts, where protein dynamics in the drug-bound state plays the crucial role, can be applied to inhibitor design of targets outside the kinome.


Subject(s)
Aurora Kinase A/antagonists & inhibitors , Imatinib Mesylate/pharmacology , Molecular Dynamics Simulation , Protein Kinase Inhibitors/pharmacology , Aurora Kinase A/chemistry , Aurora Kinase A/metabolism , Crystallography, X-Ray , Drug Discovery/methods , Humans , Imatinib Mesylate/chemistry , Imatinib Mesylate/metabolism , Kinetics , Protein Binding , Protein Conformation , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/metabolism
10.
Molecules ; 23(2)2018 Jan 24.
Article in English | MEDLINE | ID: mdl-29364838

ABSTRACT

Nitroreductases (NRs) hold promise for converting nitroaromatics to aromatic amines. Nitroaromatic reduction rate increases with Hammett substituent constant for NRs from two different subgroups, confirming substrate identity as a key determinant of reactivity. Amine yields were low, but compounds yielding amines tend to have a large π system and electron withdrawing substituents. Therefore, we also assessed the prospects of varying the enzyme. Several different subgroups of NRs include members able to produce aromatic amines. Comparison of four NR subgroups shows that they provide contrasting substrate binding cavities with distinct constraints on substrate position relative to the flavin. The unique architecture of the NR dimer produces an enormous contact area which we propose provides the stabilization needed to offset the costs of insertion of the active sites between the monomers. Thus, we propose that the functional diversity included in the NR superfamily stems from the chemical versatility of the flavin cofactor in conjunction with a structure that permits tremendous active site variability. These complementary properties make NRs exceptionally promising enzymes for development for biocatalysis in prodrug activation and conversion of nitroaromatics to valuable aromatic amines. We provide a framework for identifying NRs and substrates with the greatest potential to advance.


Subject(s)
Amines/metabolism , Fermentation , Nitroreductases/metabolism , Amines/chemistry , Binding Sites , Biosynthetic Pathways , Models, Molecular , Molecular Conformation , Molecular Structure , NAD/chemistry , NAD/metabolism , Nitroreductases/chemistry , Oxidation-Reduction , Protein Binding , Structure-Activity Relationship , Substrate Specificity
11.
Structure ; 25(7): 978-987.e4, 2017 07 05.
Article in English | MEDLINE | ID: mdl-28578873

ABSTRACT

Nitroreductase (NR) from Enterobacter cloacae reduces diverse nitroaromatics including herbicides, explosives, and prodrugs, and holds promise for bioremediation, prodrug activation, and enzyme-assisted synthesis. We solved crystal structures of NR complexes with bound substrate or analog for each of its two half-reactions. We complemented these with kinetic isotope effect (KIE) measurements elucidating H-transfer steps essential to each half-reaction. KIEs indicate hydride transfer from NADH to the flavin consistent with our structure of NR with the NADH analog nicotinic acid adenine dinucleotide (NAAD). The KIE on reduction of p-nitrobenzoic acid (p-NBA) also indicates hydride transfer, and requires revision of prior computational mechanisms. Our mechanistic information provided a structural restraint for the orientation of bound substrate, placing the nitro group closer to the flavin N5 in the pocket that binds the amide of NADH. KIEs show that solvent provides a proton, enabling accommodation of different nitro group placements, consistent with the broad repertoire of NR.


Subject(s)
Bacterial Proteins/chemistry , Nitroreductases/chemistry , Bacterial Proteins/metabolism , Binding Sites , Enterobacter cloacae/enzymology , Flavins/metabolism , NAD/metabolism , Nitrobenzoates/metabolism , Nitroreductases/metabolism , Protein Binding , Substrate Specificity
12.
J Biol Chem ; 289(22): 15203-14, 2014 May 30.
Article in English | MEDLINE | ID: mdl-24706760

ABSTRACT

The oxygen-insensitive nitroreductase from Enterobacter cloacae (NR) catalyzes two-electron reduction of nitroaromatics to the corresponding nitroso compounds and, subsequently, to hydroxylamine products. NR has an unusually broad substrate repertoire, which may be related to protein dynamics (flexibility) and/or a simple non-selective kinetic mechanism. To investigate the possible role of mechanism in the broad substrate repertoire of NR, the kinetics of oxidation of NR by para-nitrobenzoic acid (p-NBA) were investigated using stopped-flow techniques at 4 °C. The results revealed a hyperbolic dependence on the p-NBA concentration with a limiting rate of 1.90 ± 0.09 s(-1), indicating one-step binding before the flavin oxidation step. There is no evidence for a distinct binding step in which specificity might be enforced. The reduction of p-NBA is rate-limiting in steady-state turnover (1.7 ± 0.3 s(-1)). The pre-steady-state reduction kinetics of NR by NADH indicate that NADH reduces the enzyme with a rate constant of 700 ± 20 s(-1) and a dissociation constant of 0.51 ± 0.04 mM. Thus, we demonstrate simple transient kinetics in both the reductive and oxidative half-reactions that help to explain the broad substrate repertoire of NR. Finally, we tested the ability of NR to reduce para-hydroxylaminobenzoic acid, demonstrating that the corresponding amine does not accumulate to significant levels even under anaerobic conditions. Thus E. cloacae NR is not a good candidate for enzymatic production of aromatic amines.


Subject(s)
Enterobacter cloacae/enzymology , Escherichia coli/enzymology , Models, Chemical , Nitroreductases/chemistry , Nitroreductases/metabolism , Aerobiosis , Amines/metabolism , Anaerobiosis , Biodegradation, Environmental , Dinitrocresols/metabolism , Enzyme Activation/physiology , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Kinetics , NAD/metabolism , Nitrobenzoates/metabolism , Oxidation-Reduction , Protein Structure, Tertiary , Substrate Specificity
13.
Biochemistry ; 52(39): 6834-43, 2013 Oct 01.
Article in English | MEDLINE | ID: mdl-24004065

ABSTRACT

Bacterial luciferase (LuxAB) is a two-component flavin mononucleotide (FMN)-dependent monooxygenase that catalyzes the oxidation of reduced FMN (FMNH(-)) and a long-chain aliphatic aldehyde by molecular oxygen to generate oxidized FMN, the corresponding aliphatic carboxylic acid, and concomitant emission of light. The LuxAB reaction requires a flavin reductase to generate FMNH(-) to serve as a luciferin in its reaction. However, FMNH(-) is unstable and can react with oxygen to generate H2O2, so that it is important to transfer it efficiently to LuxAB. Recently, LuxG has been identified as a NADH:FMN oxidoreductase that supplies FMNH(-) to luciferase in vivo. In this report, the mode of transfer of FMNH(-) between LuxG from Photobacterium leiognathi TH1 and LuxABs from both P. leiognathi TH1 and Vibrio campbellii (PlLuxAB and VcLuxAB, respectively) was investigated using single-mixing and double-mixing stopped-flow spectrophotometry. The oxygenase component of p-hydroxyphenylacetate hydroxylase (C2) from Acinetobacter baumannii, which has no structural similarity to LuxAB, was used to measure the kinetics of release of FMNH(-) from LuxG. With all FMNH(-) acceptors used (C2, PlLuxAB, and VcLuxAB), the kinetics of FMN reduction on LuxG were the same, showing that LuxG releases FMNH(-) with a rate constant of 4.5-6 s(-1). Our data showed that the kinetics of binding of FMNH(-)to PlLuxAB and VcLuxAB and the subsequent reactions with oxygen were the same with either free FMNH(-) or FMNH(-) generated in situ by LuxG. These results strongly suggest that no complexes between LuxG and the various species are necessary to transfer FMNH(-) to the acceptors. The kinetics of the overall reactions and the individual rate constants correlate well with a free diffusion model for the transfer of FMNH(-) from LuxG to either LuxAB.


Subject(s)
Bacterial Proteins/metabolism , Diffusion , Flavin Mononucleotide/metabolism , Luciferases/metabolism , Oxidoreductases/metabolism , Biocatalysis , Molecular Structure , Photobacterium/enzymology , Vibrio/enzymology
14.
J Mol Biol ; 402(3): 578-94, 2010 Sep 24.
Article in English | MEDLINE | ID: mdl-20708626

ABSTRACT

Flavoenzymes perform a wide range of redox reactions in nature, and a subclass of flavoenzymes carry covalently bound cofactor. The enzyme-flavin bond helps to increase the flavin's redox potential to facilitate substrate oxidation in several oxidases. The formation of the enzyme-flavin covalent bond--the flavinylation reaction--has been studied for the past 40 years. For the most advocated mechanism of autocatalytic flavinylation, the quinone methide mechanism, appropriate stabilization of developing negative charges at the flavin N(1) and N(5) loci is crucial. Whereas the structural basis for stabilization at N(1) is relatively well studied, the structural requisites for charge stabilization at N(5) remain less clear. Here, we show that flavinylation of histidine 167 of pyranose 2-oxidase from Trametes multicolor requires hydrogen bonding at the flavin N(5)/O(4) locus, which is offered by the side chain of Thr169 when the enzyme is in its closed, but not open, state. Moreover, our data show that additional stabilization at N(5) by histidine 548 is required to ensure high occupancy of the histidyl-flavin bond. The combination of structural and spectral data on pyranose 2-oxidase mutants supports the quinone methide mechanism. Our results demonstrate an elaborate structural fine-tuning of the active site to complete its own formation that couples efficient holoenzyme synthesis to conformational substates of the substrate-recognition loop and concerted movements of side chains near the flavinylation ligand.


Subject(s)
Carbohydrate Dehydrogenases , Flavin-Adenine Dinucleotide , Fungal Proteins , Trametes/enzymology , Binding Sites , Carbohydrate Dehydrogenases/chemistry , Carbohydrate Dehydrogenases/metabolism , Catalysis , Catalytic Domain , Flavin-Adenine Dinucleotide/chemistry , Flavin-Adenine Dinucleotide/metabolism , Fungal Proteins/chemistry , Fungal Proteins/metabolism , Histidine , Hydrogen Bonding , Molecular Conformation , Protein Binding , Trametes/chemistry
15.
J Biol Chem ; 285(13): 9697-9705, 2010 Mar 26.
Article in English | MEDLINE | ID: mdl-20089849

ABSTRACT

Pyranose 2-oxidase (P2O) catalyzes the oxidation by O(2) of d-glucose and several aldopyranoses to yield the 2-ketoaldoses and H(2)O(2). Based on crystal structures, in one rotamer conformation, the threonine hydroxyl of Thr(169) forms H-bonds to the flavin-N5/O4 locus, whereas, in a different rotamer, it may interact with either sugar or other parts of the P2O.sugar complex. Transient kinetics of wild-type (WT) and Thr(169) --> S/N/G/A replacement variants show that D-Glc binds to T169S, T169N, and WT with the same K(d) (45-47 mm), and the hydride transfer rate constants (k(red)) are similar (15.3-9.7 s(-1) at 4 degrees C). k(red) of T169G with D-glucose (0.7 s(-1), 4 degrees C) is significantly less than that of WT but not as severely affected as in T169A (k(red) of 0.03 s(-1) at 25 degrees C). Transient kinetics of WT and mutants using d-galactose show that P2O binds d-galactose with a one-step binding process, different from binding of d-glucose. In T169S, T169N, and T169G, the overall turnover with d-Gal is faster than that of WT due to an increase of k(red). In the crystal structure of T169S, Ser(169) O gamma assumes a position identical to that of O gamma 1 in Thr(169); in T169G, solvent molecules may be able to rescue H-bonding. Our data suggest that a competent reductive half-reaction requires a side chain at position 169 that is able to form an H-bond within the ES complex. During the oxidative half-reaction, all mutants failed to stabilize a C4a-hydroperoxyflavin intermediate, thus suggesting that the precise position and geometry of the Thr(169) side chain are required for intermediate stabilization.


Subject(s)
Carbohydrate Dehydrogenases/chemistry , Carbohydrates/chemistry , Flavins/chemistry , Oxygen/chemistry , Threonine/chemistry , Catalytic Domain , Crystallography, X-Ray/methods , Galactose/chemistry , Glucose/chemistry , Hydrogen Bonding , Hydrogen Peroxide/chemistry , Kinetics , Mutagenesis, Site-Directed , Temperature , Trametes/enzymology
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