Quercetin 2,3-dioxygenase is a copper-containing enzyme that catalyzes the insertion of molecular oxygen into polyphenolic flavonols. Dioxygenation catalyzed by iron-containing enzymes has been ...studied extensively, but dioxygenases employing other metal cofactors are poorly understood. We determined the crystal structure of quercetin 2,3-dioxygenase at 1.6 Å resolution. The enzyme forms homodimers, which are stabilized by an N-linked heptasaccharide at the dimer interface. The mononuclear type 2 copper center displays two distinct geometries: a distorted tetrahedral coordination, formed by His66, His68, His112, and a water molecule, and a distorted trigonal bipyramidal environment, which additionally comprises Glu73. Manual docking of the substrate quercetin into the active site showed that the different geometries of the copper site might be of catalytic importance.
Gene duplication and fusion are among the primary natural processes that generate new proteins from simpler ancestors. Here we adopted this strategy to evolve a promiscuous homohexameric ...4‐oxalocrotonate tautomerase (4‐OT) into an efficient biocatalyst for enantioselective Michael reactions. We first designed a tandem‐fused 4‐OT to allow independent sequence diversification of adjacent subunits by directed evolution. This fused 4‐OT was then subjected to eleven rounds of directed evolution to give variant 4‐OT(F11), which showed an up to 320‐fold enhanced activity for the Michael addition of nitromethane to cinnamaldehydes. Crystallographic analysis revealed that 4‐OT(F11) has an unusual asymmetric trimeric architecture in which one of the monomers is flipped 180° relative to the others. This gene duplication and fusion strategy to break structural symmetry is likely to become an indispensable asset of the enzyme engineering toolbox, finding wide use in engineering oligomeric proteins.
Less symmetry, more diversity: Here we show that gene duplication and fusion to break structural symmetry, allowing independent sequence diversification of neighboring subunits by directed evolution, is a powerful strategy to boost catalysis by a promiscuous homohexameric 4‐oxalocrotonate tautomerase (4‐OT), enabling efficient asymmetric Michael additions.
Nitroreductases (NRs) are NAD(P)H‐dependent flavoenzymes that reduce nitro aromatic compounds to their corresponding arylamines via the nitroso and hydroxylamine intermediates. Because of their broad ...substrate scope and versatility, NRs have found application in multiple fields such as biocatalysis, bioremediation, cell‐imaging and prodrug activation. However, only a limited number of members of the broad NR superfamily (> 24 000 sequences) have been experimentally characterized. Within this group of enzymes, only few are capable of amine synthesis, which is a fundamental chemical transformation for the pharmaceutical, agricultural, and textile industries. Herein, we provide a comprehensive description of a recently discovered NR from Bacillus tequilensis , named BtNR. This enzyme has previously been demonstrated to have the capability to fully convert nitro aromatic and heterocyclic compounds to their respective primary amines. In this study, we determined its biochemical, kinetic and structural properties, including its apparent melting temperature ( T m ) of 59 °C, broad pH activity range (from pH 3 to 10) and a notably low redox potential (−236 ± 1 mV) in comparison to other well‐known NRs. We also determined its steady‐state and pre‐steady‐state kinetic parameters, which are consistent with other NRs. Additionally, we elucidated the crystal structure of BtNR, which resembles the well‐characterized Escherichia coli oxygen‐insensitive NAD(P)H nitroreductase (NfsB), and investigated the substrate binding in its active site through docking and molecular dynamics studies with four nitro aromatic substrates. Guided by these structural analyses, we probed the functional roles of active site residues by site‐directed mutagenesis. Our findings provide valuable insights into the biochemical and structural properties of BtNR, as well as its potential applications in biotechnology.
The synthesis of thymosin-α
, an acetylated 28 amino acid long therapeutic peptide, via conventional chemical methods is exceptionally challenging. The enzymatic coupling of unprotected peptide ...segments in water offers great potential for a more efficient synthesis of peptides that are difficult to synthesize. Based on the design of a highly engineered peptide ligase, we developed a fully convergent chemo-enzymatic peptide synthesis (CEPS) process for the production of thymosin-α
via a 14-mer + 14-mer segment condensation strategy. Using structure-inspired enzyme engineering, the thiol-subtilisin variant peptiligase was tailored to recognize the respective 14-mer thymosin-α
segments in order to create a clearly improved biocatalyst, termed thymoligase. Thymoligase catalyzes peptide bond formation between both segments with a very high efficiency (>94% yield) and is expected to be well applicable to many other ligations in which residues with similar characteristics (e.g. Arg and Glu) are present in the respective positions P1 and P1'. The crystal structure of thymoligase was determined and shown to be in good agreement with the model used for the engineering studies. The combination of the solid phase peptide synthesis (SPPS) of the 14-mer segments and their thymoligase-catalyzed ligation on a gram scale resulted in a significantly increased, two-fold higher overall yield (55%) of thymosin-α
compared to those typical of existing industrial processes.
Soluble glucose dehydrogenase (s‐GDH; EC 1.1.99.17) is a classical quinoprotein which requires the cofactor pyrroloquinoline quinone (PQQ) to oxidize glucose to gluconolactone. The reaction mechanism ...of PQQ‐dependent enzymes has remained controversial due to the absence of comprehensive structural data. We have determined the X‐ray structure of s‐GDH with the cofactor at 2.2 Å resolution, and of a complex with reduced PQQ and glucose at 1.9 Å resolution. These structures reveal the active site of s‐GDH, and show for the first time how a functionally bound substrate interacts with the cofactor in a PQQ‐dependent enzyme. Twenty years after the discovery of PQQ, our results finally provide conclusive evidence for a reaction mechanism comprising general base‐catalyzed hydride transfer, rather than the generally accepted covalent addition‐elimination mechanism. Thus, PQQ‐dependent enzymes use a mechanism similar to that of nicotinamide‐ and flavin‐dependent oxidoreductases.
Most flavin-dependent enzymes contain a dissociable flavin cofactor. We present a new approach for installing in vivo a covalent bond between a flavin cofactor and its host protein. By using a flavin ...transferase and carving a flavinylation motif in target proteins, we demonstrate that “dissociable” flavoproteins can be turned into covalent flavoproteins. Specifically, four different flavin mononucleotide-containing proteins were engineered to undergo covalent flavinylation: a light-oxygen-voltage domain protein, a mini singlet oxygen generator, a nitroreductase, and an old yellow enzyme-type ene reductase. Optimizing the flavinylation motif and expression conditions led to the covalent flavinylation of all four flavoproteins. The engineered covalent flavoproteins retained function and often exhibited improved performance, such as higher thermostability or catalytic performance. The crystal structures of the designed covalent flavoproteins confirmed the designed threonyl-phosphate linkage. The targeted flavoproteins differ in fold and function, indicating that this method of introducing a covalent flavin-protein bond is a powerful new method to create flavoproteins that cannot lose their cofactor, boosting their performance.
Efficient bioethanol production from hemicellulose feedstocks by
requires xylose utilization. Whereas
does not metabolize xylose, engineered strains that express xylose isomerase can metabolize ...xylose by converting it to xylulose. For this, the type II xylose isomerase from
(PirXI) is used but the in vivo activity is rather low and very high levels of the enzyme are needed for xylose metabolism. In this study, we explore the use of protein engineering and in vivo selection to improve the performance of PirXI. Recently solved crystal structures were used to focus mutagenesis efforts.
We constructed focused mutant libraries of
xylose isomerase by substitution of second shell residues around the substrate- and metal-binding sites. Following library transfer to
and selection for enhanced xylose-supported growth under aerobic and anaerobic conditions, two novel xylose isomerase mutants were obtained, which were purified and subjected to biochemical and structural analysis. Apart from a small difference in response to metal availability, neither the new mutants nor mutants described earlier showed significant changes in catalytic performance under various in vitro assay conditions. Yet, in vivo performance was clearly improved. The enzymes appeared to function suboptimally in vivo due to enzyme loading with calcium, which gives poor xylose conversion kinetics. The results show that better in vivo enzyme performance is poorly reflected in kinetic parameters for xylose isomerization determined in vitro with a single type of added metal.
This study shows that in vivo selection can identify xylose isomerase mutants with only minor changes in catalytic properties measured under standard conditions. Metal loading of xylose isomerase expressed in yeast is suboptimal and strongly influences kinetic properties. Metal uptake, distribution and binding to xylose isomerase are highly relevant for rapid xylose conversion and may be an important target for optimizing yeast xylose metabolism.
Crystal structures of haloalkane dehalogenase were determined in the presence of the substrate 1,2-dichloroethane. At pH 5 and 4 degrees C, substrate is bound in the active site without being ...converted; warming to room temperature causes the substrate's carbon-chlorine bond to be broken, producing a chloride ion with concomitant alkylation of the active-site residue Asp124. At pH 6 and room temperature the alkylated enzyme is hydrolysed by a water molecule activated by the His289-Asp260 pair in the active site. These results show that catalysis by the dehalogenase proceeds by a two-step mechanism involving an ester intermediate covalently bound at Asp124.
Using a newly discovered encapsulin from Mycolicibacterium hassiacum, several biocatalysts were packaged in this robust protein cage. The encapsulin was found to be easy to produce as recombinant ...protein. Elucidation of its crystal structure revealed that it is a spherical protein cage of 60 protomers (diameter of 23 nm) with narrow pores. By developing an effective coexpression and isolation procedure, the effect of packaging a variety of biocatalysts could be evaluated. It was shown that encapsulation results in a significantly higher stability of the biocatalysts. Most of the targeted cofactor-containing biocatalysts remained active in the encapsulin. Due to the restricted diameters of the encapsulin pores (5–9 Å), the protein cage protects the encapsulated enzymes from bulky compounds. The work shows that encapsulins may be valuable tools to tune the properties of biocatalysts such as stability and substrate specificity.
•A novel, robust protein cage, encapsulin EncMh, was discovered and its structure was elucidated.•High expression of EncMh in Escherichia coli can be achieved.•Various cofactor-containing enzymes can be packaged in encapsulin EncMh.•Enzymes in encapsulin EncMh are more thermostable.•The pores of encapsulin, through a size-exclusion effect, can modulate the substrate profile of an encapsulated enzyme.
Chitin hydrolases have been identified in a variety of organisms ranging from bacteria to eukaryotes. They have been proposed to be possible targets for the design of novel chemotherapeutics against ...human pathogens such as fungi and protozoan parasites as mammals were not thought to possess chitin-processing enzymes. Recently, a human chitotriosidase was described as a marker for Gaucher disease with plasma levels of the enzyme elevated up to 2 orders of magnitude. The chitotriosidase was shown to be active against colloidal chitin and is inhibited by the family 18 chitinase inhibitor allosamidin. Here, the crystal structure of the human chitotriosidase and complexes with a chitooligosaccharide and allosamidin are described. The structures reveal an elongated active site cleft, compatible with the binding of long chitin polymers, and explain the inactivation of the enzyme through an inherited genetic deficiency. Comparison with YM1 and HCgp-39 shows how the chitinase has evolved into these mammalian lectins by the mutation of key residues in the active site, tuning the substrate binding specificity. The soaking experiments with allosamidin and chitooligosaccharides give insight into ligand binding properties and allow the evaluation of differential binding and design of species-selective chitinase inhibitors.
PDF
