I04-1-Macromolecular Crystallography (fixed wavelength)
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Abstract: Xylonolactonase Cc XylC from Caulobacter crescentus catalyzes the hydrolysis of the intramolecular ester bond of d-xylonolactone. We have determined crystal structures of Cc XylC in complex with d-xylonolactone isomer analogues d-xylopyranose and (r)-(+)-4-hydroxy-2-pyrrolidinone at high resolution. Cc XylC has a 6-bladed β-propeller architecture, which contains a central open channel having the active site at one end. According to our previous native mass spectrometry studies, Cc XylC is able to specifically bind Fe2+. The crystal structures, presented here, revealed an active site bound metal ion with an octahedral binding geometry. The side-chains of three amino acid residues, Glu18, Asn146, and Asp196 which participate in binding of metal ion are located in the same plane. The solved complex structures allowed suggesting a reaction mechanism for intramolecular ester bond hydrolysis in which the major contribution for catalysis arises from the carbonyl oxygen coordination of the xylonolactone substrate to the Fe2+. The structure of Cc XylC was compared with eight other ester hydrolases of the β-propeller hydrolase family. The previously published crystal structures of other β-propeller hydrolases contain either Ca2+, Mg2+ or Zn2+ and show clear similarities in ligand and metal ion binding geometries to that of Cc XylC. It would be interesting to reinvestigate the metal binding specificity of these enzymes and clarify whether they are also able to use Fe2+ as a catalytic metal. This could further expand our understanding of utilization of Fe2+ not only in oxidative enzymes but also in hydrolases.
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Nov 2021
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[19951]
Open Access
Abstract: Background: Nowadays there is a strong trend towards a circular economy using lignocellulosic biowaste for the production of biofuels and other bio-based products. The use of enzymes at several stages of the production process (e.g., saccharification) can offer a sustainable route due to avoidance of harsh chemicals and high temperatures. For novel enzyme discovery, physically linked gene clusters targeting carbohydrate degradation in bacteria, polysaccharide utilization loci (PULs), are recognized ‘treasure troves’ in the era of exponentially growing numbers of sequenced genomes. Results: We determined the biochemical properties and structure of a protein of unknown function (PUF) encoded within PULs of metagenomes from beaver droppings and moose rumen enriched on poplar hydrolysate. The corresponding novel bifunctional carbohydrate esterase (CE), now named BD-FAE, displayed feruloyl esterase (FAE) and acetyl esterase activity on simple, synthetic substrates. Whereas acetyl xylan esterase (AcXE) activity was detected on acetylated glucuronoxylan from birchwood, only FAE activity was observed on acetylated and feruloylated xylooligosaccharides from corn fiber. The genomic contexts of 200 homologs of BD-FAE revealed that the 33 closest homologs appear in PULs likely involved in xylan breakdown, while the more distant homologs were found either in alginate-targeting PULs or else outside PUL contexts. Although the BD-FAE structure adopts a typical α/β-hydrolase fold with a catalytic triad (Ser-Asp-His), it is distinct from other biochemically characterized CEs. Conclusions: The bifunctional CE, BD-FAE, represents a new candidate for biomass processing given its capacity to remove ferulic acid and acetic acid from natural corn and birchwood xylan substrates, respectively. Its detailed biochemical characterization and solved crystal structure add to the toolbox of enzymes for biomass valorization as well as structural information to inform the classification of new CEs.
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May 2021
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I24-Microfocus Macromolecular Crystallography
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Sanni
Voutilainen
,
Markus
Heinonen
,
Martina
Andberg
,
Emmi
Jokinen
,
Hannu
Maaheimo
,
Johan
Pääkkönen
,
Nina
Hakulinen
,
Juha
Rouvinen
,
Harri
Lähdesmäki
,
Samuel
Kaski
,
Juho
Rousu
,
Merja
Penttilä
,
Anu
Koivula
Open Access
Abstract: In this work, deoxyribose-5-phosphate aldolase (Ec DERA, EC 4.1.2.4) from Escherichia coli was chosen as the protein engineering target for improving the substrate preference towards smaller, non-phosphorylated aldehyde donor substrates, in particular towards acetaldehyde. The initial broad set of mutations was directed to 24 amino acid positions in the active site or in the close vicinity, based on the 3D complex structure of the E. coli DERA wild-type aldolase. The specific activity of the DERA variants containing one to three amino acid mutations was characterised using three different substrates. A novel machine learning (ML) model utilising Gaussian processes and feature learning was applied for the 3rd mutagenesis round to predict new beneficial mutant combinations. This led to the most clear-cut (two- to threefold) improvement in acetaldehyde (C2) addition capability with the concomitant abolishment of the activity towards the natural donor molecule glyceraldehyde-3-phosphate (C3P) as well as the non-phosphorylated equivalent (C3). The Ec DERA variants were also tested on aldol reaction utilising formaldehyde (C1) as the donor. Ec DERA wild-type was shown to be able to carry out this reaction, and furthermore, some of the improved variants on acetaldehyde addition reaction turned out to have also improved activity on formaldehyde.
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Nov 2020
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[10291]
Abstract: Catechol oxidases and tyrosinases are coupled binuclear copper enzymes that oxidize various o‐diphenolic compounds to corresponding o‐quinones. Tyrosinases have an additional monooxygenation ability to hydroxylate monophenol to o‐diphenol. It is still not clear what causes the difference in the catalytic activities. We solved a complex structure of Aspergillus oryzae catechol oxidase with resorcinol bound into the active site. Catalytic activity of A. oryzae catechol oxidase was studied, for the first time, by high‐resolution FT‐ICR mass spectrometry to shed light on the reaction mechanism. The enzyme was also found to catalyze monooxygenation of small phenolics, which provides a novel perspective for the discussion of differences in the catalytic activity between tyrosinases and catechol oxidases. According to the results, two binding modes for resorcinol are suggested and a reaction mechanism for coupled binuclear copper enzymes is discussed.
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Sep 2018
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I02-Macromolecular Crystallography
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Diamond Proposal Number(s):
[10291]
Open Access
Abstract: Coupled binuclear copper (CBC) enzymes have a conserved type 3 copper site that binds molecular oxygen to oxidize various mono- and diphenolic compounds. In this study, we found a new crystal form of catechol oxidase from Aspergillus oryzae (AoCO4) and solved two new structures from two different crystals at 1.8-Å and at 2.5-Å resolutions. These structures showed different copper site forms (met/deoxy and deoxy) and also differed from the copper site observed in the previously solved structure of AoCO4. We also analysed the electron density maps of all of the 56 CBC enzyme structures available in the protein data bank (PDB) and found that many of the published structures have vague copper sites. Some of the copper sites were then re-refined to find a better fit to the observed electron density. General problems in the refinement of metalloproteins and metal centres are discussed.
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May 2018
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[10291]
Open Access
Abstract: The Ilv/ED dehydratase protein family includes dihydroxy acid-, gluconate-, 6-phosphogluconate- and pentonate dehydratases. The members of this family are involved in various biosynthetic and carbohydrate metabolic pathways. Here, we describe the first crystal structure of D-xylonate dehydratase from Caulobacter crescentus (CcXyDHT) at 2.7 Å resolution and compare it with other available enzyme structures from the IlvD/EDD protein family. The quaternary structure of CcXyDHT is a tetramer, and each monomer is composed of two domains in which the N-terminal domain forms a binding site for a [2Fe-2S] cluster and a Mg2+ ion. The active site is located at the monomer-monomer interface and contains residues from both the N-terminal recognition helix and the C-terminus of the dimeric counterpart. The active site also contains a conserved Ser490, which probably acts as a base in catalysis. Importantly, the cysteines that participate in the binding and formation of the [2Fe-2S] cluster are not all conserved within the Ilv/ED dehydratase family, which suggests that some members of the IlvD/EDD family may bind different types of [Fe-S] clusters.
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Jan 2018
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I04-Macromolecular Crystallography
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Yindi
Chu
,
Tao
Tu
,
Leena
Penttinen
,
Xianli
Xue
,
Xiaoyu
Wang
,
Zhuolin
Yi
,
Li
Gong
,
Juha
Rouvinen
,
Huiying
Luo
,
Nina
Hakulinen
,
Bin
Yao
,
Xiaoyun
Su
Abstract: Bifunctional glycoside hydrolases have potential for cost saving in enzymatic decomposition of plant cell wall polysaccharides for biofuels and bio-based chemicals. The N-terminal GH10 domain of a bifunctional multimodular enzyme CbXyn10C/Cel48B from Caldicellulosiruptor bescii, is an enzyme able to degrade xylan and cellulose simultaneously. However, the molecular mechanism underlying its substrate promiscuity has not been elucidated. Herein, we discovered that the binding cleft of CbXyn10C would have at least six sugar binding subsites by using isothermal titration calorimetry analysis of the inactive E140Q/E248Q mutant with xylo- and cellooligosaccharides. This was confirmed by determining the catalytic efficiency of the wild-type enzyme on these oligosaccharides. The free form and complex structures of CbXyn10C with xylose- or glucose-configured oligosaccharide ligands were further obtained by crystallographic analysis and molecular modeling and docking. CbXyn10C was found to have a typical (β/α)8-TIM barrel fold and "salad-bowl" shape of GH10 enzymes. In complex structure with xylo-oligosaccharides, seven sugar-binding subsites were found and many residues responsible for substrate interactions were identified. Site-directed mutagenesis indicated that six and ten amino acid residues were key residues for xylan and cellulose hydrolysis, respectively. The most important residues are centered on the subsites -2 and -1 near the cleavage site, while residues playing moderate roles could be located at more distal regions of the binding cleft. Manipulating the residues directly or indirectly interacting with substrates in the distal regions improved the activity of CbXyn10C on xylan and cellulose. Most of the key residues for cellulase activity are conserved across GH10 xylanases. Revisiting randomly selected GH10 enzymes revealed unreported cellulase activity, indicating that the dual function may be a more common phenomenon than has been expected.
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Oct 2017
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[10291]
Abstract: We present a novel crystal structure of the IlvD/EDD family enzyme, L-arabinonate dehydratase from Rhizobium leguminosarum bv. trifolii (RlArDHT, EC 4.2.1.25), which catalyzes the conversion of L-arabinonate to 2-dehydro-3-deoxy-L-arabinonate. The enzyme is a tetramer consisting of a dimer of dimers, where each monomer is composed of two domains. The active site contains a catalytically important [2Fe-2S] cluster and Mg2+ ion, and is buried between two domains, and also at the dimer interface. The active site Lys129 was found to be carbamylated. Ser480 and Thr482 were shown to be essential residues for catalysis, and the S480A mutant structure showed an unexpected open conformation in which the active site was more accessible for the substrate. This structure showed the partial binding of L-arabinonate, which allowed us to suggest that the alkoxide ion form of the Ser480 side chain functions as a base and the [2Fe-2S] cluster functions as a Lewis acid in the elimination reaction.
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Jun 2017
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[8030, 10291]
Open Access
Abstract: L-Arabinonate dehydratase (EC 4.2.1.25) and D-xylonate dehydratase (EC 4.2.1.82) are two enzymes that are involved in a nonphosphorylative oxidation pathway of pentose sugars. L-Arabinonate dehydratase converts L-arabinonate into 2-dehydro-3-deoxy-L-arabinonate, and D-xylonate dehydratase catalyzes the dehydration of D-xylonate to 2-dehydro-3-deoxy-D-xylonate. L-Arabinonate and D-xylonate dehydratases belong to the IlvD/EDD family, together with 6-phosphogluconate dehydratases and dihydroxyacid dehydratases. No crystal structure of any L-arabinonate or D-xylonate dehydratase is available in the PDB. In this study, recombinant L-arabinonate dehydratase from Rhizobium leguminosarum bv. trifolii (RlArDHT) and D-xylonate dehydratase from Caulobacter crescentus (CcXyDHT) were heterologously expressed in Escherichia coli and purified by the use of affinity chromatography followed by gel-filtration chromatography. The purified proteins were crystallized using the hanging-drop vapour-diffusion method at 293 K. Crystals of RlArDHT that diffracted to 2.40 Å resolution were obtained using sodium formate as a precipitating agent. They belonged to space group P21, with unit-cell parameters a = 106.07, b = 208.61, c = 147.09 Å, [beta] = 90.43°. Eight RlArDHT molecules (two tetramers) in the asymmetric unit give a VM value of 3.2 Å3 Da-1 and a solvent content of 62%. Crystals of CcXyDHT that diffracted to 2.66 Å resolution were obtained using sodium formate and polyethylene glycol 3350. They belonged to space group C2, with unit-cell parameters a = 270.42, b = 236.13, c = 65.17 Å, [beta] = 97.38°. Four CcXyDHT molecules (a tetramer) in the asymmetric unit give a VM value of 4.0 Å3 Da-1 and a solvent content of 69%.
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Aug 2016
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I03-Macromolecular Crystallography
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Abstract: Aldose-aldose oxidoreductase ( Cc AAOR) is a recently characterized enzyme from the bacterial strain of Caulobacter crescentus CB15 belonging to the Gfo/Idh/MocA protein family. Cc AAOR catalyzes the oxidation and reduction of a panel of aldose monosaccharides using a tightly bound NADP(H) cofactor that is regenerated in the catalytic cycle. Furthermore, Cc AAOR can also oxidize 1,4-linked oligosaccharides. Here, we present novel crystal structures of the dimeric Cc AAOR in complex with the cofactor and glycerol, D-xylose, D-glucose, maltotriose, and D-sorbitol determined to 2.0, 1.8, 1.7, 1.9, and 1.8 Å resolutions, respectively. These complex structures allowed for a detailed analysis of the ligand-binding interactions. The structures showed that the C1 carbon of a substrate, which is either reduced or oxidized, is close to the reactive C4 carbon of the nicotinamide ring of NADP(H). In addition, the O1 hydroxyl group of the substrate, which is either protonated or deprotonated, is unexpectedly close to both Lys-104 and Tyr-189, which may both act as a proton donor or acceptor. This led us to hypothesize that this intriguing feature could be beneficial for Cc AAOR to catalyze the reduction of a linear form of a monosaccharide substrate and the oxidation of a pyranose form of the same substrate in a reaction cycle, during which the bound cofactor is regenerated.
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Oct 2015
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