Publications by authors named "Kai Tittmann"

65 Publications

A lysine-cysteine redox switch with an NOS bridge regulates enzyme function.

Nature 2021 May 5;593(7859):460-464. Epub 2021 May 5.

Department of Molecular Enzymology, Göttingen Center of Molecular Biosciences, Georg August University Göttingen, Göttingen, Germany.

Disulfide bonds between cysteine residues are important post-translational modifications in proteins that have critical roles for protein structure and stability, as redox-active catalytic groups in enzymes or allosteric redox switches that govern protein function. In addition to forming disulfide bridges, cysteine residues are susceptible to oxidation by reactive oxygen species, and are thus central not only to the scavenging of these but also to cellular signalling and communication in biological as well as pathological contexts. Oxidized cysteine species are highly reactive and may form covalent conjugates with, for example, tyrosines in the active sites of some redox enzymes. However, to our knowledge, regulatory switches with covalent crosslinks other than disulfides have not previously been demonstrated. Here we report the discovery of a covalent crosslink between a cysteine and a lysine residue with a NOS bridge that serves as an allosteric redox switch in the transaldolase enzyme of Neisseria gonorrhoeae, the pathogen that causes gonorrhoea. X-ray structure analysis of the protein in the oxidized and reduced state reveals a loaded-spring mechanism that involves a structural relaxation upon redox activation, which is propagated from the allosteric redox switch at the protein surface to the active site in the protein interior. This relaxation leads to a reconfiguration of key catalytic residues and elicits an increase in enzymatic activity of several orders of magnitude. The redox switch is highly conserved in related transaldolases from other members of the Neisseriaceae; for example, it is present in the transaldolase of Neisseria meningitides (a pathogen that is the primary cause of meningitis and septicaemia in children). We surveyed the Protein Data Bank and found that the NOS bridge exists in diverse protein families across all domains of life (including Homo sapiens) and that it is often located at catalytic or regulatory hotspots. Our findings will inform strategies for the design of proteins and peptides, as well as the development of new classes of drugs and antibodies that target the lysine-cysteine redox switch.
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http://dx.doi.org/10.1038/s41586-021-03513-3DOI Listing
May 2021

Structural basis for antibiotic action of the B antivitamin 2'-methoxy-thiamine.

Nat Chem Biol 2020 11 24;16(11):1237-1245. Epub 2020 Aug 24.

Department of Molecular Enzymology, Göttingen Center of Molecular Biosciences, University of Göttingen, Göttingen, Germany.

The natural antivitamin 2'-methoxy-thiamine (MTh) is implicated in the suppression of microbial growth. However, its mode of action and enzyme-selective inhibition mechanism have remained elusive. Intriguingly, MTh inhibits some thiamine diphosphate (ThDP) enzymes, while being coenzymatically active in others. Here we report the strong inhibition of Escherichia coli transketolase activity by MTh and unravel its mode of action and the structural basis thereof. The unique 2'-methoxy group of MTh diphosphate (MThDP) clashes with a canonical glutamate required for cofactor activation in ThDP-dependent enzymes. This glutamate is forced into a stable, anticatalytic low-barrier hydrogen bond with a neighboring glutamate, disrupting cofactor activation. Molecular dynamics simulations of transketolases and other ThDP enzymes identify active-site flexibility and the topology of the cofactor-binding locale as key determinants for enzyme-selective inhibition. Human enzymes either retain enzymatic activity with MThDP or preferentially bind authentic ThDP over MThDP, while core bacterial metabolic enzymes are inhibited, demonstrating therapeutic potential.
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http://dx.doi.org/10.1038/s41589-020-0628-4DOI Listing
November 2020

Hydrazides Are Potent Transition-State Analogues for Glutaminyl Cyclase Implicated in the Pathogenesis of Alzheimer's Disease.

Biochemistry 2020 07 29;59(28):2585-2591. Epub 2020 Jun 29.

Department of Molecular Enzymology, Georg-August University Göttingen, Julia-Lermontowa-Weg 3, 37077 Göttingen, Germany.

Amyloidogenic plaques are hallmarks of Alzheimer's disease (AD) and typically consist of high percentages of modified Aβ peptides bearing N-terminally cyclized glutamate residues. The human zinc(II) enzyme glutaminyl cyclase (QC) was shown in vivo to catalyze the cyclization of N-terminal glutamates of Aβ peptides in a pathophysiological side reaction establishing QC as a druggable target for therapeutic treatment of AD. Here, we report crystallographic snapshots of human QC catalysis acting on the neurohormone neurotensin that delineate the stereochemical course of catalysis and suggest that hydrazides could mimic the transition state of peptide cyclization and deamidation. This hypothesis is validated by a sparse-matrix inhibitor screening campaign that identifies hydrazides as the most potent metal-binding group compared to classic Zn binders. The structural basis of hydrazide inhibition is illuminated by X-ray structure analysis of human QC in complex with a hydrazide-bearing peptide inhibitor and reveals a pentacoordinated Zn complex. Our findings inform novel strategies in the design of potent and highly selective QC inhibitors by employing hydrazides as the metal-binding warhead.
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http://dx.doi.org/10.1021/acs.biochem.0c00337DOI Listing
July 2020

Crossing the Border: From Keto- to Imine Reduction in Short-Chain Dehydrogenases/Reductases.

Chembiochem 2020 09 2;21(18):2615-2619. Epub 2020 Jul 2.

Institute of Pharmaceutical Sciences, University of Freiburg, Albertstrasse 25, 79104, Freiburg, Germany.

The family of NAD(P)H-dependent short-chain dehydrogenases/reductases (SDRs) comprises numerous biocatalysts capable of C=O or C=C reduction. The highly homologous noroxomaritidine reductase (NR) from Narcissus sp. aff. pseudonarcissus and Zt_SDR from Zephyranthes treatiae, however, are SDRs with an extended imine substrate scope. Comparison with a similar SDR from Asparagus officinalis (Ao_SDR) exhibiting keto-reducing activity, yet negligible imine-reducing capability, and mining the Short-Chain Dehydrogenase/Reductase Engineering Database indicated that NR and Zt_SDR possess a unique active-site composition among SDRs. Adapting the active site of Ao_SDR accordingly improved its imine-reducing capability. By applying the same strategy, an unrelated SDR from Methylobacterium sp. 77 (M77_SDR) with distinct keto-reducing activity was engineered into a promiscuous enzyme with imine-reducing activity, thereby confirming that the ability to reduce imines can be rationally introduced into members of the "classical" SDR enzyme family. Thus, members of the SDR family could be a promising starting point for protein approaches to generate new imine-reducing enzymes.
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http://dx.doi.org/10.1002/cbic.202000233DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7540013PMC
September 2020

Discovery of a Regulatory Subunit of the Yeast Fatty Acid Synthase.

Cell 2020 03 10;180(6):1130-1143.e20. Epub 2020 Mar 10.

Department of Structural Dynamics, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany. Electronic address:

Fatty acid synthases (FASs) are central to metabolism but are also of biotechnological interest for the production of fine chemicals and biofuels from renewable resources. During fatty acid synthesis, the growing fatty acid chain is thought to be shuttled by the dynamic acyl carrier protein domain to several enzyme active sites. Here, we report the discovery of a γ subunit of the 2.6 megadalton α-βS. cerevisiae FAS, which is shown by high-resolution structures to stabilize a rotated FAS conformation and rearrange ACP domains from equatorial to axial positions. The γ subunit spans the length of the FAS inner cavity, impeding reductase activities of FAS, regulating NADPH turnover by kinetic hysteresis at the ketoreductase, and suppressing off-pathway reactions at the enoylreductase. The γ subunit delineates the functional compartment within FAS. As a scaffold, it may be exploited to incorporate natural and designed enzymatic activities that are not present in natural FAS.
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http://dx.doi.org/10.1016/j.cell.2020.02.034DOI Listing
March 2020

Low-barrier hydrogen bonds in enzyme cooperativity.

Nature 2019 09 18;573(7775):609-613. Epub 2019 Sep 18.

Department of Molecular Enzymology, Göttingen Centre for Molecular Biosciences and Albrecht-von-Haller Institute, Georg-August University Göttingen, Göttingen, Germany.

The underlying molecular mechanisms of cooperativity and allosteric regulation are well understood for many proteins, with haemoglobin and aspartate transcarbamoylase serving as prototypical examples. The binding of effectors typically causes a structural transition of the protein that is propagated through signalling pathways to remote sites and involves marked changes on the tertiary and sometimes even the quaternary level. However, the origin of these signals and the molecular mechanism of long-range signalling at an atomic level remain unclear. The different spatial scales and timescales in signalling pathways render experimental observation challenging; in particular, the positions and movement of mobile protons cannot be visualized by current methods of structural analysis. Here we report the experimental observation of fluctuating low-barrier hydrogen bonds as switching elements in cooperativity pathways of multimeric enzymes. We have observed these low-barrier hydrogen bonds in ultra-high-resolution X-ray crystallographic structures of two multimeric enzymes, and have validated their assignment using computational calculations. Catalytic events at the active sites switch between low-barrier hydrogen bonds and ordinary hydrogen bonds in a circuit that consists of acidic side chains and water molecules, transmitting a signal through the collective repositioning of protons by behaving as an atomistic Newton's cradle. The resulting communication synchronizes catalysis in the oligomer. Our studies provide several lines of evidence and a working model for not only the existence of low-barrier hydrogen bonds in proteins, but also a connection to enzyme cooperativity. This finding suggests new principles of drug and enzyme design, in which sequences of residues can be purposefully included to enable long-range communication and thus the regulation of engineered biomolecules.
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http://dx.doi.org/10.1038/s41586-019-1581-9DOI Listing
September 2019

Structural and Functional Analyses of the Human PDH Complex Suggest a "Division-of-Labor" Mechanism by Local E1 and E3 Clusters.

Structure 2019 07 23;27(7):1124-1136.e4. Epub 2019 May 23.

Department of Molecular Enzymology, Göttingen Centre for Molecular Biosciences and Albrecht-von-Haller Institute, Georg-August University Göttingen, Julia-Lermontowa-Weg 3, 37077 Göttingen, Germany; Department of Structural Dynamics, Max-Planck-Institute for Biophysical Chemistry Göttingen, Am Fassberg 11, 37077 Göttingen, Germany. Electronic address:

The pseudo-atomic structural model of human pyruvate dehydrogenase complex (PDHc) core composed of full-length E2 and E3BP components, calculated from our cryoelectron microscopy-derived density maps at 6-Å resolution, is similar to those of prokaryotic E2 structures. The spatial organization of human PDHc components as evidenced by negative-staining electron microscopy and native mass spectrometry is not homogeneous, and entails the unanticipated formation of local clusters of E1:E2 and E3BP:E3 complexes. Such uneven, clustered organization translates into specific duties for E1-E2 clusters (oxidative decarboxylation and acetyl transfer) and E3BP-E3 clusters (regeneration of reduced lipoamide) corresponding to half-reactions of the PDHc catalytic cycle. The addition of substrate coenzyme A modulates the conformational landscape of PDHc, in particular of the lipoyl domains, extending the postulated multiple random coupling mechanism. The conformational and associated chemical landscapes of PDHc are thus not determined entirely stochastically, but are restrained and channeled through an asymmetric architecture and further modulated by substrate binding.
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http://dx.doi.org/10.1016/j.str.2019.04.009DOI Listing
July 2019

Theoretical Studies of the Electronic Absorption Spectra of Thiamin Diphosphate in Pyruvate Decarboxylase.

Biochemistry 2017 04 22;56(13):1854-1864. Epub 2017 Mar 22.

Institute of Physical Chemistry, University of Goettingen , Tammannstraße 6, D-37077 Göttingen, Germany.

Electronic absorption spectra are oftentimes used to identify reaction intermediates or substrates/products in enzymatic systems, as long as absorption bands can be unequivocally assigned to the species being studied. The latter task is far from trivial given the transient nature of some states and the complexity of the surrounding environment around the active site. To identify unique spectral fingerprints, controlled experiments with model compounds have been used in the past, but even these can sometimes be unreliable. Circular dichroism (CD) and ultraviolet-visible spectra have been tools of choice in the study of the rich chemistry of thiamin diphosphate-dependent enzymes. In this study, we focus on the Zymomonas mobilis pyruvate decarboxylase, and mutant analogues thereof, as a prototypical representative of the thiamin diphosphate (ThDP) enzyme superfamily. Through the use of electronic structure methods, we analyze the nature of electronic excitations in the cofactor. We find that all the determining CD bands around the 280-340 nm spectral range correspond to charge-transfer excitations between the pyrimidine and thiazolium rings of ThDP, which, most likely, is a general property of related ThDP-dependent enzymes. While we can confirm the assignments of previously proposed bands to chemical states, our calculations further suggest that a hitherto unassigned band of enzyme-bound ThDP reports on the ionization state of the canonical glutamate that is required for cofactor activation. This finding expands the spectroscopic "library" of chemical states of ThDP enzymes, permitting a simultaneous assignment of both the cofactor ThDP and the activating glutamate. We anticipate this finding to be helpful for mechanistic analyses of related ThDP enzymes.
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http://dx.doi.org/10.1021/acs.biochem.6b00984DOI Listing
April 2017

PtdInsP and PtdSer cooperate to trap synaptotagmin-1 to the plasma membrane in the presence of calcium.

Elife 2016 10 28;5. Epub 2016 Oct 28.

Department of Neurobiology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.

The Ca-sensor synaptotagmin-1 that triggers neuronal exocytosis binds to negatively charged membrane lipids (mainly phosphatidylserine (PtdSer) and phosphoinositides (PtdIns)) but the molecular details of this process are not fully understood. Using quantitative thermodynamic, kinetic and structural methods, we show that synaptotagmin-1 (from and expressed in ) binds to PtdIns(4,5)P via a polybasic lysine patch in the C2B domain, which may promote the priming or docking of synaptic vesicles. Ca neutralizes the negative charges of the Ca-binding sites, resulting in the penetration of synaptotagmin-1 into the membrane, via binding of PtdSer, and an increase in the affinity of the polybasic lysine patch to phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P). These Ca-induced events decrease the dissociation rate of synaptotagmin-1 membrane binding while the association rate remains unchanged. We conclude that both membrane penetration and the increased residence time of synaptotagmin-1 at the plasma membrane are crucial for triggering exocytotic membrane fusion.
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http://dx.doi.org/10.7554/eLife.15886DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5123861PMC
October 2016

Structural Analysis of an Evolved Transketolase Reveals Divergent Binding Modes.

Sci Rep 2016 10 21;6:35716. Epub 2016 Oct 21.

Department of Biochemical Engineering, Gordon Street, University College London, WC1H 0AH, UK.

The S385Y/D469T/R520Q variant of E. coli transketolase was evolved previously with three successive smart libraries, each guided by different structural, bioinformatical or computational methods. Substrate-walking progressively shifted the target acceptor substrate from phosphorylated aldehydes, towards a non-phosphorylated polar aldehyde, a non-polar aliphatic aldehyde, and finally a non-polar aromatic aldehyde. Kinetic evaluations on three benzaldehyde derivatives, suggested that their active-site binding was differentially sensitive to the S385Y mutation. Docking into mutants generated in silico from the wild-type crystal structure was not wholly satisfactory, as errors accumulated with successive mutations, and hampered further smart-library designs. Here we report the crystal structure of the S385Y/D469T/R520Q variant, and molecular docking of three substrates. This now supports our original hypothesis that directed-evolution had generated an evolutionary intermediate with divergent binding modes for the three aromatic aldehydes tested. The new active site contained two binding pockets supporting π-π stacking interactions, sterically separated by the D469T mutation. While 3-formylbenzoic acid (3-FBA) preferred one pocket, and 4-FBA the other, the less well-accepted substrate 3-hydroxybenzaldehyde (3-HBA) was caught in limbo with equal preference for the two pockets. This work highlights the value of obtaining crystal structures of evolved enzyme variants, for continued and reliable use of smart library strategies.
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http://dx.doi.org/10.1038/srep35716DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5073344PMC
October 2016

The inhibition mechanism of human 20S proteasomes enables next-generation inhibitor design.

Science 2016 Aug;353(6299):594-8

Department of Structural Dynamics, Max Planck Institut für biophysikalische Chemie, Am Fassberg 11, D-37077 Göttingen, Germany.

The proteasome is a validated target for anticancer therapy, and proteasome inhibition is employed in the clinic for the treatment of tumors and hematological malignancies. Here, we describe crystal structures of the native human 20S proteasome and its complexes with inhibitors, which either are drugs approved for cancer treatment or are in clinical trials. The structure of the native human 20S proteasome was determined at an unprecedented resolution of 1.8 angstroms. Additionally, six inhibitor-proteasome complex structures were elucidated at resolutions between 1.9 and 2.1 angstroms. Collectively, the high-resolution structures provide new insights into the catalytic mechanisms of inhibition and necessitate a revised description of the proteasome active site. Knowledge about inhibition mechanisms provides insights into peptide hydrolysis and can guide strategies for the development of next-generation proteasome-based cancer therapeutics.
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http://dx.doi.org/10.1126/science.aaf8993DOI Listing
August 2016

Tuning and Switching Enantioselectivity of Asymmetric Carboligation in an Enzyme through Mutational Analysis of a Single Hot Spot.

Chembiochem 2015 Dec 18;16(18):2580-4. Epub 2015 Nov 18.

Abt. Molekulare Enzymologie, Georg-August-Universität Göttingen, Justus-von-Liebig-Weg 11, 37077, Göttingen, Germany.

Enantioselective bond making and breaking is a hallmark of enzyme action, yet switching the enantioselectivity of the reaction is a difficult undertaking, and typically requires extensive screening of mutant libraries and multiple mutations. Here, we demonstrate that mutational diversification of a single catalytic hot spot in the enzyme pyruvate decarboxylase gives access to both enantiomers of acyloins acetoin and phenylacetylcarbinol, important pharmaceutical precursors, in the case of acetoin even starting from the unselective wild-type protein. Protein crystallography was used to rationalize these findings and to propose a mechanistic model of how enantioselectivity is controlled. In a broader context, our studies highlight the efficiency of mechanism-inspired and structure-guided rational protein design for enhancing and switching enantioselectivity of enzymatic reactions, by systematically exploring the biocatalytic potential of a single hot spot.
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http://dx.doi.org/10.1002/cbic.201500529DOI Listing
December 2015

ProteoPlex: stability optimization of macromolecular complexes by sparse-matrix screening of chemical space.

Nat Methods 2015 Sep 3;12(9):859-65. Epub 2015 Aug 3.

Research Group of 3D Electron Cryomicroscopy, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.

Molecular machines or macromolecular complexes are supramolecular assemblies of biomolecules with a variety of functions. Structure determination of these complexes in a purified state is often tedious owing to their compositional complexity and the associated relative structural instability. To improve the stability of macromolecular complexes in vitro, we present a generic method that optimizes the stability, homogeneity and solubility of macromolecular complexes by sparse-matrix screening of their thermal unfolding behavior in the presence of various buffers and small molecules. The method includes the automated analysis of thermal unfolding curves based on a biophysical unfolding model for complexes. We found that under stabilizing conditions, even large multicomponent complexes reveal an almost ideal two-state unfolding behavior. We envisage an improved biochemical understanding of purified macromolecules as well as a substantial boost in successful macromolecular complex structure determination by both X-ray crystallography and cryo-electron microscopy.
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http://dx.doi.org/10.1038/nmeth.3493DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5136620PMC
September 2015

Converting Transaldolase into Aldolase through Swapping of the Multifunctional Acid-Base Catalyst: Common and Divergent Catalytic Principles in F6P Aldolase and Transaldolase.

Biochemistry 2015 Jul 20;54(29):4475-86. Epub 2015 Jul 20.

Göttingen Center for Molecular Biosciences, Department of Molecular Enzymology, Georg-August University Göttingen, Ernst-Caspari-Haus, Justus-von-Liebig-Weg 11, D-37077 Göttingen, Germany.

Transaldolase (TAL) and fructose-6-phosphate aldolase (FSA) both belong to the class I aldolase family and share a high degree of structural similarity and sequence identity. The molecular basis of the different reaction specificities (transferase vs aldolase) has remained enigmatic. A notable difference between the active sites is the presence of either a TAL-specific Glu (Gln in FSA) or a FSA-specific Tyr (Phe in TAL). Both residues seem to have analoguous multifunctional catalytic roles but are positioned at different faces of the substrate locale. We have engineered a TAL double variant (Glu to Gln and Phe to Tyr) with an active site resembling that of FSA. This variant indeed exhibits aldolase activity as its main activity with a catalytic efficiency even larger than that of authentic FSA, while TAL activity is greatly impaired. Structural analysis of this variant in complex with the dihydroxyacetone Schiff base formed upon substrate cleavage identifies the introduced Tyr (genuine in FSA) to catalyze protonation of the central carbanion-enamine intermediate as a key determinant of the aldolase reaction. Our studies pinpoint that the Glu in TAL and the Tyr in FSA, although located at different positions at the active site, similarly act as bona fide acid-base catalysts in numerous catalytic steps, including substrate binding, dehydration of the carbinolamine, and substrate cleavage. We propose that the different spatial positions of the multifunctional Glu in TAL and of the corresponding multifunctional Tyr in FSA relative to the substrate locale are critically controlling reaction specificity through either unfavorable (TAL) or favorable (FSA) geometry of proton transfer onto the common carbanion-enamine intermediate. The presence of both potential acid-base residues, Glu and Tyr, in the active site of TAL has deleterious effects on substrate binding and cleavage, most likely resulting from a differently organized H-bonding network. Large-scale motions of the protein associated with opening and closing of the active site that seem to bear relevance for catalysis are observed as covalent intermediates are exclusively observed in the "closed" conformation of the active site. Pre-steady-state kinetics are used to monitor catalytic processes and structural transitions and to refine the kinetic framework of TAL catalysis.
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http://dx.doi.org/10.1021/acs.biochem.5b00283DOI Listing
July 2015

Phosphate ions and glutaminyl cyclases catalyze the cyclization of glutaminyl residues by facilitating synchronized proton transfers.

Bioorg Chem 2015 Jun 24;60:98-101. Epub 2015 Apr 24.

Probiodrug AG, Weinbergweg 22, 06120 Halle, Germany. Electronic address:

Phosphate ions and glutaminyl cyclase (QC) both catalyze the formation of pyroglutamate (pE, pGlu) from N-terminal glutamine residues of peptides and proteins. Here, we studied the mechanism of glutamine cyclization using kinetic secondary deuterium and solvent isotope effects. The data suggest that proton transfer(s) are rate determining for the spontaneous reaction, and that phosphate and QC are accelerating the reaction by promoting synchronized proton transfers in a concerted mechanism. Thus, non-enzymatic and enzymatic catalysis of pyroglutamate formation exploit a similar mode of transition-state stabilization.
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http://dx.doi.org/10.1016/j.bioorg.2015.04.005DOI Listing
June 2015

Membrane enzymes: transformers at the interface.

Nat Chem Biol 2015 Feb 12;11(2):102-3. Epub 2015 Jan 12.

Department of Molecular Enzymology, Göttingen Center for Molecular Biosciences, Georg-August University Göttingen, Göttingen, Germany.

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http://dx.doi.org/10.1038/nchembio.1738DOI Listing
February 2015

Marvels of enzyme catalysis at true atomic resolution: distortions, bond elongations, hidden flips, protonation states and atom identities.

Curr Opin Struct Biol 2014 Dec 23;29:122-33. Epub 2014 Nov 23.

Abteilung Molekulare Enzymologie, Göttinger Zentrum für Molekulare Biowissenschaften (GZMB), Justus-von-Liebig-Weg 11, Georg-August-Universität Göttingen, Göttingen D-37077, Germany. Electronic address:

Although general principles of enzyme catalysis are fairly well understood nowadays, many important details of how exactly the substrate is bound and processed in an enzyme remain often invisible and as such elusive. In fortunate cases, structural analysis of enzymes can be accomplished at true atomic resolution thus making possible to shed light on otherwise concealed fine-structural traits of bound substrates, intermediates, cofactors and protein groups. We highlight recent structural studies of enzymes using ultrahigh-resolution X-ray protein crystallography showcasing its enormous potential as a tool in the elucidation of enzymatic mechanisms and in unveiling fundamental principles of enzyme catalysis. We discuss the observation of seemingly hyper-reactive, physically distorted cofactors and intermediates with elongated scissile substrate bonds, the detection of 'hidden' conformational and chemical equilibria and the analysis of protonation states with surprising findings. In delicate cases, atomic resolution is required to unambiguously disclose the identity of atoms as demonstrated for the metal cluster in nitrogenase. In addition to the pivotal structural findings and the implications for our understanding of enzyme catalysis, we further provide a practical framework for resolution enhancement through optimized data acquisition and processing.
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http://dx.doi.org/10.1016/j.sbi.2014.11.001DOI Listing
December 2014

Identification, characterization, and structure analysis of the cyclic di-AMP-binding PII-like signal transduction protein DarA.

J Biol Chem 2015 Jan 28;290(5):3069-80. Epub 2014 Nov 28.

Molecular Structural Biology, and

The cyclic dimeric AMP nucleotide c-di-AMP is an essential second messenger in Bacillus subtilis. We have identified the protein DarA as one of the prominent c-di-AMP receptors in B. subtilis. Crystal structure analysis shows that DarA is highly homologous to PII signal transducer proteins. In contrast to PII proteins, the functionally important B- and T-loops are swapped with respect to their size. DarA is a homotrimer that binds three molecules of c-di-AMP, each in a pocket located between two subunits. We demonstrate that DarA is capable to bind c-di-AMP and with lower affinity cyclic GMP-AMP (3'3'-cGAMP) but not c-di-GMP or 2'3'-cGAMP. Consistently the crystal structure shows that within the ligand-binding pocket only one adenine is highly specifically recognized, whereas the pocket for the other adenine appears to be promiscuous. Comparison with a homologous ligand-free DarA structure reveals that c-di-AMP binding is accompanied by conformational changes of both the fold and the position of the B-loop in DarA.
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http://dx.doi.org/10.1074/jbc.M114.619619DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4317042PMC
January 2015

Sweet siblings with different faces: the mechanisms of FBP and F6P aldolase, transaldolase, transketolase and phosphoketolase revisited in light of recent structural data.

Authors:
Kai Tittmann

Bioorg Chem 2014 Dec 16;57:263-280. Epub 2014 Sep 16.

Göttingen Center for Molecular Biosciences, Georg-August University Göttingen, Justus-von-Liebig-Weg 11, 37077 Göttingen, Germany. Electronic address:

Nature has evolved different strategies for the reversible cleavage of ketose phosphosugars as essential metabolic reactions in all domains of life. Prominent examples are the Schiff-base forming class I FBP and F6P aldolase as well as transaldolase, which all exploit an active center lysine to reversibly cleave the C3-C4 bond of fructose-1,6-bisphosphate or fructose-6-phosphate to give two 3-carbon products (aldolase), or to shuttle 3-carbon units between various phosphosugars (transaldolase). In contrast, transketolase and phosphoketolase make use of the bioorganic cofactor thiamin diphosphate to cleave the preceding C2-C3 bond of ketose phosphates. While transketolase catalyzes the reversible transfer of 2-carbon ketol fragments in a reaction analogous to that of transaldolase, phosphoketolase forms acetyl phosphate as final product in a reaction that comprises ketol cleavage, dehydration and phosphorolysis. In this review, common and divergent catalytic principles of these enzymes will be discussed, mostly, but not exclusively, on the basis of crystallographic snapshots of catalysis. These studies in combination with mutagenesis and kinetic analysis not only delineated the stereochemical course of substrate binding and processing, but also identified key catalytic players acting at the various stages of the reaction. The structural basis for the different chemical fates and lifetimes of the central enamine intermediates in all five enzymes will be particularly discussed, in addition to the mechanisms of substrate cleavage, dehydration and ring-opening reactions of cyclic substrates. The observation of covalent enzymatic intermediates in hyperreactive conformations such as Schiff-bases with twisted double-bond linkages in transaldolase and physically distorted substrate-thiamin conjugates with elongated substrate bonds to be cleaved in transketolase, which probably epitomize a canonical feature of enzyme catalysis, will be also highlighted.
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http://dx.doi.org/10.1016/j.bioorg.2014.09.001DOI Listing
December 2014

Control of the diadenylate cyclase CdaS in Bacillus subtilis: an autoinhibitory domain limits cyclic di-AMP production.

J Biol Chem 2014 Jul 16;289(30):21098-107. Epub 2014 Jun 16.

From the Department of General Microbiology, Institute of Microbiology and Genetics, Georg-August University, D-37077 Göttingen, Germany,

The Gram-positive bacterium Bacillus subtilis encodes three diadenylate cyclases that synthesize the essential signaling nucleotide cyclic di-AMP. The activities of the vegetative enzymes DisA and CdaA are controlled by protein-protein interactions with their conserved partner proteins. Here, we have analyzed the regulation of the unique sporulation-specific diadenylate cyclase CdaS. Very low expression of CdaS as the single diadenylate cyclase resulted in the appearance of spontaneous suppressor mutations. Several of these mutations in the cdaS gene affected the N-terminal domain of CdaS. The corresponding CdaS mutant proteins exhibited a significantly increased enzymatic activity. The N-terminal domain of CdaS consists of two α-helices and is attached to the C-terminal catalytically active diadenylate cyclase (DAC) domain. Deletion of the first or both helices resulted also in strongly increased activity indicating that the N-terminal domain serves to limit the enzyme activity of the DAC domain. The structure of YojJ, a protein highly similar to CdaS, indicates that the protein forms hexamers that are incompatible with enzymatic activity of the DAC domains. In contrast, the mutations and the deletions of the N-terminal domain result in conformational changes that lead to highly increased enzymatic activity. Although the full-length CdaS protein was found to form hexamers, a truncated version with a deletion of the first N-terminal helix formed dimers with high enzyme activity. To assess the role of CdaS in sporulation, we assayed the germination of wild type and cdaS mutant spores. The results indicate that cyclic di-AMP formed by CdaS is required for efficient germination.
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http://dx.doi.org/10.1074/jbc.M114.562066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4110313PMC
July 2014

Sub-ångström-resolution crystallography reveals physical distortions that enhance reactivity of a covalent enzymatic intermediate.

Nat Chem 2013 Sep 18;5(9):762-7. Epub 2013 Aug 18.

Albrecht-von-Haller Institute, Göttingen Center for Molecular Biosciences, Georg-August University Göttingen, 37077 Göttingen, Germany.

It is recognized widely that enzymes promote reactions by providing a pathway that proceeds through a transition state of lower energy. In principle, further rate enhancements could be achieved if intermediates are prevented from relaxing to their lowest energy state, and thereby reduce the barrier to the subsequent transition state. Here, we report sub-ångström-resolution crystal structures of genuine covalent reaction intermediates of transketolase. These structures reveal a pronounced out-of-plane distortion of over 20° for the covalent bond that links cofactor and substrate, and a specific elongation of the scissile substrate carbon-carbon bond (d > 1.6 Å). To achieve these distortions, the protein's conformation appears to prevent relaxation of a substrate-cofactor intermediate. The results implicate a reduced barrier to the subsequent step that is consistent with an intermediate of raised energy and leads to a more efficient overall process.
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http://dx.doi.org/10.1038/nchem.1728DOI Listing
September 2013

Observation of a stable carbene at the active site of a thiamin enzyme.

Nat Chem Biol 2013 Aug 9;9(8):488-90. Epub 2013 Jun 9.

Albrecht-von-Haller Institute, Göttingen Center for Molecular Biosciences, Ernst-Caspari-Haus, Georg-August University Göttingen, Göttingen, Germany.

Carbenes are highly reactive chemical compounds that are exploited as ligands in organometallic chemistry and are powerful organic catalysts. They were postulated to occur as transient intermediates in enzymes, yet their existence in a biological system could never be demonstrated directly. We present spectroscopic and structural data of a thiamin enzyme in a noncovalent complex with substrate, which implicate accumulation of a stable carbene as a major resonance contributor to deprotonated thiamin.
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http://dx.doi.org/10.1038/nchembio.1275DOI Listing
August 2013

Alternating sites reactivity is a common feature of thiamin diphosphate-dependent enzymes as evidenced by isothermal titration calorimetry studies of substrate binding.

Biochemistry 2013 Apr 1;52(15):2505-7. Epub 2013 Apr 1.

Göttingen Center for Molecular Biosciences, Georg-August-University Göttingen, Ernst-Caspari-Haus, Justus-von-Liebig-Weg 11,37077 Göttingen, Germany.

Thiamin diphosphate (ThDP)-dependent enzymes play vital roles in cellular metabolism in all kingdoms of life. In previous kinetic and structural studies, a communication between the active centers in terms of a negative cooperativity had been suggested for some but not all ThDP enzymes, which typically operate as functional dimers. To further underline this hypothesis and to test its universality, we investigated the binding of substrate analogue methyl acetylphosphonate (MAP) to three different ThDP-dependent enzymes acting on substrate pyruvate, namely, the Escherichia coli E1 component of the pyruvate dehydrogenase complex, E. coli acetohydroxyacid synthase isoenzyme I, and the Lactobacillus plantarum pyruvate oxidase using isothermal titration calorimetry. The results unambiguously show for all three enzymes studied that only one active center of the functional dimers accomplishes covalent binding of the substrate analogue, supporting the proposed alternating sites reactivity as a common feature of all ThDP enzymes and resolving the recent controversy in the field.
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http://dx.doi.org/10.1021/bi301591eDOI Listing
April 2013

A δ38 deletion variant of human transketolase as a model of transketolase-like protein 1 exhibits no enzymatic activity.

PLoS One 2012 31;7(10):e48321. Epub 2012 Oct 31.

Albrecht-von-Haller-Institute and Göttingen Center for Molecular Biosciences, Department of Bioanalytics, Georg-August-University Göttingen, Germany.

Besides transketolase (TKT), a thiamin-dependent enzyme of the pentose phosphate pathway, the human genome encodes for two closely related transketolase-like proteins, which share a high sequence identity with TKT. Transketolase-like protein 1 (TKTL1) has been implicated in cancerogenesis as its cellular expression levels were reported to directly correlate with invasion efficiency of cancer cells and patient mortality. It has been proposed that TKTL1 exerts its function by catalyzing an unusual enzymatic reaction, a hypothesis that has been the subject of recent controversy. The most striking difference between TKTL1 and TKT is a deletion of 38 consecutive amino acids in the N-terminal domain of the former, which constitute part of the active site in authentic TKT. Our structural and sequence analysis suggested that TKTL1 might not possess transketolase activity. In order to test this hypothesis in the absence of a recombinant expression system for TKTL1 and resilient data on its biochemical properties, we have engineered and biochemically characterized a "pseudo-TKTL1" Δ38 deletion variant of human TKT (TKTΔ38) as a viable model of TKTL1. Although the isolated protein is properly folded under in vitro conditions, both thermal stability as well as stability of the TKT-specific homodimeric assembly are markedly reduced. Circular dichroism and NMR spectroscopic analysis further indicates that TKTΔ38 is unable to bind the thiamin cofactor in a specific manner, even at superphysiological concentrations. No transketolase activity of TKTΔ38 can be detected for conversion of physiological sugar substrates thus arguing against an intrinsically encoded enzymatic function of TKTL1 in tumor cell metabolism.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0048321PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3485151PMC
April 2013

Unexpected tautomeric equilibria of the carbanion-enamine intermediate in pyruvate oxidase highlight unrecognized chemical versatility of thiamin.

Proc Natl Acad Sci U S A 2012 Jul 22;109(27):10867-72. Epub 2012 Jun 22.

Albrecht-von-Haller Institute and Göttingen Center for Molecular Biosciences, D-37077 Göttingen, Germany.

Thiamin diphosphate, the vitamin B1 coenzyme, plays critical roles in fundamental metabolic pathways that require acyl carbanion equivalents. Studies on chemical models and enzymes had suggested that these carbanions are resonance-stabilized as enamines. A crystal structure of this intermediate in pyruvate oxidase at 1.1 Å resolution now challenges this paradigm by revealing that the enamine does not accumulate. Instead, the intermediate samples between the ketone and the carbanion both interlocked in a tautomeric equilibrium. Formation of the keto tautomer is associated with a loss of aromaticity of the cofactor. The alternate confinement of electrons to neighboring atoms rather than π-conjugation seems to be of importance for the enzyme-catalyzed, redox-coupled acyl transfer to phosphate, which requires a dramatic inversion of polarity of the reacting substrate carbon in two subsequent catalytic steps. The ability to oscillate between a nucleophilic (carbanion) and an electrophilic (ketone) substrate center highlights a hitherto unrecognized versatility of the thiamin cofactor. It remains to be studied whether formation of the keto tautomer is a general feature of all thiamin enzymes, as it could provide for stable storage of the carbanion state, or whether this feature represents a specific trait of thiamin oxidases. In addition, the protonation state of the two-electron reduced flavin cofactor can be fully assigned, demonstrating the power of high-resolution cryocrystallography for elucidation of enzymatic mechanisms.
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http://dx.doi.org/10.1073/pnas.1201280109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3390845PMC
July 2012

Many of the functional differences between acetohydroxyacid synthase (AHAS) isozyme I and other AHASs are a result of the rapid formation and breakdown of the covalent acetolactate-thiamin diphosphate adduct in AHAS I.

FEBS J 2012 Jun 20;279(11):1967-79. Epub 2012 Apr 20.

Department of Life Sciences, Ben-Gurion University of the Negev, Beer-Sheva, Israel.

Acetohydroxy acid synthase (AHAS; EC 2.2.1.6) is a thiamin diphosphate (ThDP)-dependent decarboxylase-ligase that catalyzes the first common step in the biosynthesis of branched-chain amino acids. In the first stage of the reaction, pyruvate is decarboxylated and the reactive intermediate hydroxyethyl-ThDP carbanion/enamine is formed. In the second stage, the intermediate is ligated to another 2-ketoacid to form either acetolactate or acetohydroxybutyrate. AHAS isozyme I from Escherichia coli is unique among the AHAS isozymes in that it is not specific for 2-ketobutyrate (2-KB) over pyruvate as an acceptor substrate. It also appears to have a different mechanism for inhibition by valine than does AHAS III from E. coli. An investigation of this enzyme by directed mutagenesis and knowledge of detailed kinetics using the rapid mixing-quench NMR method or stopped-flow spectroscopy, as well as the use of alternative substrates, suggests that two residues determine most of the unique properties of AHAS I. Gln480 and Met476 in AHAS I replace the Trp and Leu residues conserved in other AHASs and lead to accelerated ligation and product release steps. This difference in kinetics accounts for the unique specificity, reversibility and allosteric response of AHAS I. The rate of decarboxylation of the initially formed 2-lactyl-ThDP intermediate is, in some AHAS I mutants, different for the alternative acceptors pyruvate and 2-KB, putting into question whether AHAS operates via a pure ping-pong mechanism. This finding might be compatible with a concerted mechanism (i.e. the formation of a ternary donor-acceptor:enzyme complex followed by covalent, ThDP-promoted catalysis with concerted decarboxylation-carboligation). It might alternatively be explained by an allosteric interaction between the multiple catalytic sites in AHAS.
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http://dx.doi.org/10.1111/j.1742-4658.2012.08577.xDOI Listing
June 2012

Bifunctionality of the thiamin diphosphate cofactor: assignment of tautomeric/ionization states of the 4'-aminopyrimidine ring when various intermediates occupy the active sites during the catalysis of yeast pyruvate decarboxylase.

J Am Chem Soc 2012 Feb 17;134(8):3873-85. Epub 2012 Feb 17.

Department of Chemistry, Rutgers University, Newark, New Jersey 07102, USA.

Thiamin diphosphate (ThDP) dependent enzymes perform crucial C-C bond forming and breaking reactions in sugar and amino acid metabolism and in biosynthetic pathways via a sequence of ThDP-bound covalent intermediates. A member of this superfamily, yeast pyruvate decarboxylase (YPDC) carries out the nonoxidative decarboxylation of pyruvate and is mechanistically a simpler ThDP enzyme. YPDC variants created by substitution at the active center (D28A, E51X, and E477Q) and on the substrate activation pathway (E91D and C221E) display varying activity, suggesting that they stabilize different covalent intermediates. To test the role of both rings of ThDP in YPDC catalysis (the 4'-aminopyrimidine as acid-base, and thiazolium as electrophilic covalent catalyst), we applied a combination of steady state and time-resolved circular dichroism experiments (assessing the state of ionization and tautomerization of enzyme-bound ThDP-related intermediates), and chemical quench of enzymatic reaction mixtures followed by NMR characterization of the ThDP-bound intermediates released from YPDC (assessing occupancy of active centers by these intermediates and rate-limiting steps). Results suggest the following: (1) Pyruvate and analogs induce active site asymmetry in YPDC and variants. (2) The rare 1',4'-iminopyrimidine ThDP tautomer participates in formation of ThDP-bound intermediates. (3) Propionylphosphinate also binds at the regulatory site and its binding is reflected by catalytic events at the active site 20 Å away. (4) YPDC stabilizes an electrostatic model for the 4'-aminopyrimidinium ionization state, an important contribution of the protein to catalysis. The combination of tools used provides time-resolved details about individual events during ThDP catalysis; the methods are transferable to other ThDP superfamily members.
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http://dx.doi.org/10.1021/ja211139cDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3295232PMC
February 2012

Twisted Schiff base intermediates and substrate locale revise transaldolase mechanism.

Nat Chem Biol 2011 Aug 21;7(10):678-84. Epub 2011 Aug 21.

Albrecht-von-Haller-Institut and Göttingen Center for Molecular Biosciences, Georg-August-Universität Göttingen, Göttingen, Germany.

We examined the catalytic cycle of transaldolase (TAL) from Thermoplasma acidophilum by cryocrystallography and were able to structurally characterize--for the first time, to our knowledge--different genuine TAL reaction intermediates. These include the Schiff base adducts formed between the catalytic lysine and the donor ketose substrates fructose-6-phosphate and sedoheptulose-7-phosphate as well as the Michaelis complex with acceptor aldose erythrose-4-phosphate. These structural snapshots necessitate a revision of the accepted reaction mechanism with respect to functional roles of active site residues, and they further reveal fundamental insights into the general structural features of enzymatic Schiff base intermediates and the role of conformational dynamics in enzyme catalysis, substrate binding and discrimination. A nonplanar arrangement of the substituents around the Schiff base double bond was observed, suggesting that a structurally encoded reactant-state destabilization is a driving force of catalysis. Protein dynamics and the intrinsic hydrogen-bonding pattern appear to be crucial for selective recognition and binding of ketose as first substrate.
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http://dx.doi.org/10.1038/nchembio.633DOI Listing
August 2011

Crystallization and preliminary X-ray diffraction analysis of transaldolase from Thermoplasma acidophilum.

Acta Crystallogr Sect F Struct Biol Cryst Commun 2011 May 27;67(Pt 5):584-6. Epub 2011 Apr 27.

Göttinger Zentrum für Molekulare Biowissenschaften, Georg-August-Universität Göttingen, Ernst-Caspari-Haus, Justus-von-Liebig-Weg 11, D-37077 Göttingen, Germany.

The metabolic enzyme transaldolase from Thermoplasma acidophilum was recombinantly expressed in Escherichia coli and could be crystallized in two polymorphic forms. Crystals were grown by the hanging-drop vapour-diffusion method using PEG 6000 as precipitant. Native data sets for crystal forms 1 and 2 were collected in-house to resolutions of 3.0 and 2.7 Å, respectively. Crystal form 1 belonged to the orthorhombic space group C222(1) with five monomers per asymmetric unit and crystal form 2 belonged to the monoclinic space group P2(1) with ten monomers per asymmetric unit.
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http://dx.doi.org/10.1107/S1744309111009274DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3087646PMC
May 2011

Significant catalytic roles for Glu47 and Gln 110 in all four of the C-C bond-making and -breaking steps of the reactions of acetohydroxyacid synthase II.

Biochemistry 2011 Apr 23;50(15):3250-60. Epub 2011 Mar 23.

Department of Life Sciences, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel.

Acetohydroxy acid synthase (AHAS) is a thiamin diphosphate (ThDP)-dependent enzyme that catalyzes the first common step in the biosynthesis of branched-chain amino acids, condensation of pyruvate with a second 2-ketoacid to form either acetolactate or acetohydroxybutyrate. AHAS isozyme II from Escherichia coli is specific for pyruvate as the first donor substrate but exhibits a 60-fold higher specificity for 2-ketobutyrate (2-KB) over pyruvate as an acceptor substrate. In previous studies relying on steady state and transient kinetics, substrate competition and detailed analysis of the distribution of intermediates in the steady-state, we have identified several residues which confer specificity for the donor and acceptor substrates, respectively. Here, we examine the roles of active site polar residues Glu47, Gln110, Lys159, and His251 for elementary steps of catalysis using similar approaches. While Glu47, the conserved essential glutamate conserved in all ThDP-dependent enzymes whose carboxylate is in H-bonding distance of the ThDP iminopyrimidine N1', is involved as expected in cofactor activation, substrate binding, and product elimination, our studies further suggest a crucial catalytic role for it in the carboligation of the acceptor and the hydroxyethyl-ThDP enamine intermediate. The Glu47-cofactor proton shuttle acts in concert with Gln110 in the carboligation. We suggest that either the transient oxyanion on the acceptor carbonyl is stabilized by H-bonding to the glutamine side chain, or carboligation involves glutamine tautomerization and the elementary reactions of addition and protonation occur in a concerted manner. This is in contrast to the situation in other ThDP enzymes that catalyze a carboligation, such as, e.g., transketolase or benzaldehyde lyase, where histidines act as general acid/base catalysts. Our studies further suggest global catalytic roles for Gln110 and Glu47, which are engaged in all major bond-breaking and bond-making steps. In contrast to earlier suggestions, Lys159 has a minor effect on the kinetics and specificity of AHAS II, far less than does Arg276, previously shown to influence the specificity for a 2-ketoacid as a second substrate. His251 has a large effect on donor substrate binding, but this effect masks any other effects of replacement of His251.
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http://dx.doi.org/10.1021/bi102051hDOI Listing
April 2011
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