The integrity of the bacterial cell wall depends on the balanced action of several peptidoglycan (murein) synthesizing and degrading enzymes. Penicillin inhibits the enzymes responsible for peptide ...crosslinks in the peptidoglycan polymer. Enzymes that act solely on the glycosidic bonds are insensitive to this antibiotic, thus offering a target for the design of antibiotics distinct from the beta-lactams. Here we report the X-ray structure of the periplasmic soluble lytic transglycosylase (SLT; M(r) 70,000) from Escherichia coli. This unique bacterial exomuramidase cleaves the beta-1,4-glycosidic bonds of peptidoglycan to produce small 1,6-anhydromuropeptides. The structure of SLT reveals a 'superhelical' ring of alpha-helices with a separate domain on top which resembles the fold of lysozyme. Site-directed mutagenesis and a crystallographic inhibitor-binding study confirmed that the lysozyme-like domain contains the active site of SLT.
The l-2-haloacid dehalogenase from the 1,2-dichloroethane-degrading bacterium Xanthobacter autotrophicus GJ10 catalyzes the hydrolytic dehalogenation of small l-2-haloalkanoates to their ...correspondingd-2-hydroxyalkanoates, with inversion of the configuration at the C2 atom. The structure of the apoenzyme at pH 8 was refined at 1.5-Å resolution. By lowering the pH, the catalytic activity of the enzyme was considerably reduced, allowing the crystal structure determination of the complexes withl-2-monochloropropionate and monochloroacetate at 1.7 and 2.1 Å resolution, respectively. Both complexes showed unambiguous electron density extending from the nucleophile Asp8 to the C2 atom of the dechlorinated substrates corresponding to a covalent enzyme-ester reaction intermediate. The halide ion that is cleaved off is found in line with the Asp8Oδ1–C2 bond in a halide-stabilizing cradle made up of Arg39, Asn115, and Phe175. In both complexes, the Asp8 Oδ2 carbonyl oxygen atom interacts with Thr12, Ser171, and Asn173, which possibly constitute the oxyanion hole in the hydrolysis of the ester bond. The carboxyl moiety of the substrate is held in position by interactions with Ser114, Lys147, and main chain NH groups. The l-2-monochloropropionate CH3 group is located in a small pocket formed by side chain atoms of Lys147, Asn173, Phe175, and Asp176. The size and position of the pocket explain the stereospecificity and the limited substrate specificity of the enzyme. These crystallographic results demonstrate that the reaction of the enzyme proceeds via the formation of a covalent enzyme-ester intermediate at the nucleophile Asp8.
The cyclodextrin glycosyltransferase (CGTase, EC 2.4.1.19) gene from Bacillus circulans strain 251 was cloned and sequenced. It was found to code for a mature protein of 686 amino acid residues, ...showing 75% identity to the CGTase from B. circulans strain 8. The X-ray structure of the CGTase was elucidated in a maltodextrin-dependent crystal form and refined against X-ray diffraction data to 2.0 angstroms resolution. The structure of the enzyme is nearly identical to the CGTase from B. circulans strain 8. Three maltose binding sites are observed at the protein surface, two in domain E and one in domain C. The maltose-dependence of CGTase crystallization can be ascribed to the proximity of two of the maltose binding sites to intermolecular crystal contacts. The maltose molecules bound in the E domain interact with several residues implicated in a raw starch binding motif conserved among a diverse group of starch converting enzymes.
The α-amylase family is a large group of starch processing enzymes Svensson, B. (1994) Plant Mol. Biol. 25, 141–157. It is characterized by four short sequence motifs that contain the seven fully ...conserved amino acid residues in this family: two catalytic carboxylic acid residues and four substrate binding residues. The seventh conserved residue (Asp135) has no direct interactions with either substrates or products, but it is hydrogen-bonded to Arg227, which does bind the substrate in the catalytic site. Using cyclodextrin glycosyltransferase as an example, this paper provides for the first time definite biochemical and structural evidence that Asp135 is required for the proper conformation of several catalytic site residues and therefore for activity.
Abstract
The quinone‐dependent alcohol dehydrogenase (PQQ‐ADH, E.C. 1.1.5.2) from the Gram‐negative bacterium
Pseudogluconobacter saccharoketogenes
IFO 14464 oxidizes primary alcohols (e.g. ethanol, ...butanol), secondary alcohols (monosaccharides), as well as aldehydes, polysaccharides, and cyclodextrins. The recombinant protein, expressed in
Pichia pastoris
, was crystallized, and three‐dimensional (3D) structures of the native form, with PQQ and a Ca
2+
ion, and of the enzyme in complex with a Zn
2+
ion and a bound substrate mimic were determined at 1.72 Å and 1.84 Å resolution, respectively. PQQ‐ADH displays an eight‐bladed β‐propeller fold, characteristic of Type I quinone‐dependent methanol dehydrogenases. However, three of the four ligands of the Ca
2+
ion differ from those of related dehydrogenases and they come from different parts of the polypeptide chain. These differences result in a more open, easily accessible active site, which explains why PQQ‐ADH can oxidize a broad range of substrates. The bound substrate mimic suggests Asp333 as the catalytic base. Remarkably, no vicinal disulfide bridge is present near the PQQ, which in other PQQ‐dependent alcohol dehydrogenases has been proposed to be necessary for electron transfer. Instead an associated cytochrome
c
can approach the PQQ for direct electron transfer.
Interactive Figure 1
;
Interactive Figure 5
| PDB Code(s):
4CVB
;
4CVC
The synthesis of thymosin-α
1
, 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
1
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
1
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
1
compared to those typical of existing industrial processes.
A substrate-tailored peptide ligase for the chemo-enzymatic peptide synthesis (CEPS) of thymosin-α
1
.
Die Genfusion ist ein wichtiger natürlicher Prozess zur Erzeugung neuer Enzyme aus einfachen Vorläufern. Gerrit J. Poelarends und Mitarbeiter haben diese Strategie in ihrer Zuschrift (e202113970) ...angewendet, um eine promiskuitive homohexamere 4‐Oxalocrotonat‐Tautomerase (4‐OT) zu einem effizienten Enzym für enantioselektive Michael‐Reaktionen zu entwickeln. Sie entwarfen eine tandemfusionierte 4‐OT mit reduzierter Symmetrie, die eine unabhängige Sequenzdiversifizierung benachbarter Untereinheiten ermöglicht und so den Proteinsequenzraum vergrößert, der durch gerichtete Evolution erforscht werden kann.
The vast majority of characterized oxygenases use bound cofactors to activate molecular oxygen to carry out oxidation chemistry. Here, we show that an enzyme of unknown activity, RhCC from ...Rhodococcus jostii RHA1, functions as an oxygenase, using 4-hydroxyphenylenolpyruvate as a substrate. This unique and complex reaction yields 3-hydroxy-3-(4-hydroxyphenyl)-pyruvate, 4-hydroxybenzaldehyde, and oxalic acid as major products. Incubations with H2 18O, 18O2, and a substrate analogue suggest that this enzymatic oxygenation reaction likely involves a peroxide anion intermediate. Analysis of sequence similarity and the crystal structure of RhCC (solved at 1.78 Å resolution) reveal that this enzyme belongs to the tautomerase superfamily. Members of this superfamily typically catalyze tautomerization, dehalogenation, or decarboxylation reactions rather than oxygenation reactions. The structure shows the absence of cofactors, establishing RhCC as a rare example of a redox-metal- and coenzyme-free oxygenase. This sets the stage to study the mechanistic details of cofactor-independent oxygen activation in the unusual context of the tautomerase superfamily.
Die Genfusion ist ein wichtiger natürlicher Prozess zur Erzeugung neuer Enzyme aus einfachen Vorläufern. Gerrit J. Poelarends und Mitarbeiter haben diese Strategie in ihrer Zuschrift (e202113970) ...angewendet, um eine promiskuitive homohexamere 4‐Oxalocrotonat‐Tautomerase (4‐OT) zu einem effizienten Enzym für enantioselektive Michael‐Reaktionen zu entwickeln. Sie entwarfen eine tandemfusionierte 4‐OT mit reduzierter Symmetrie, die eine unabhängige Sequenzdiversifizierung benachbarter Untereinheiten ermöglicht und so den Proteinsequenzraum vergrößert, der durch gerichtete Evolution erforscht werden kann.
Cyclodextrin-glycosyltransferase (CGTase) catalyzes the formation of α-, β-, and γ-cyclodextrins (cyclic α-(1,4)-linked oligosaccharides
of 6, 7, or 8 glucose residues, respectively) from starch. ...Nine substrate binding subsites were observed in an x-ray structure
of the CGTase from Bacillus circulans strain 251 complexed with a maltononaose substrate. Subsite â6 is conserved in CGTases, suggesting its importance for the
reactions catalyzed by the enzyme. To investigate this in detail, we made six mutant CGTases (Y167F, G179L, G180L, N193G,
N193L, and G179L/G180L). All subsite â6 mutants had decreased k cat values for β-cyclodextrin formation, as well as for the disproportionation and coupling reactions, but not for hydrolysis.
Especially G179L, G180L, and G179L/G180L affected the transglycosylation activities, most prominently for the coupling reactions.
The results demonstrate that (i) subsite â6 is important for all three CGTase-catalyzed transglycosylation reactions, (ii)
Gly-180 is conserved because of its importance for the circularization of the linear substrates, (iii) it is possible to independently
change cyclization and coupling activities, and (iv) substrate interactions at subsite â6 activate the enzyme in catalysis
via an induced-fit mechanism. This article provides for the first time definite biochemical evidence for such an induced-fit
mechanism in the α-amylase family.
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