Transthyretin is a metallopeptidase with an inducible active site

TTR (transthyretin) was found recently to possess proteolytic competency besides its well-known transport capabilities. It was described as a cryptic serine peptidase cleaving multiple natural substrates (including β-amyloid and apolipoprotein A-I) involved in diseases such as Alzheimer's disease and atherosclerosis. In the present study, we aimed to elucidate the catalytic machinery of TTR. All attempts to identify a catalytic serine residue were unsuccessful. However, metal chelators abolished TTR activity. Proteolytic inhibition by EDTA or 1,10-phenanthroline could be reversed with Zn2+ and Mn2+. These observations, supported by analysis of three-dimensional structures of TTR complexed with Zn2+, led to the hypothesis that TTR is a metallopeptidase. Site-directed mutagenesis of selected amino acids unambiguously confirmed this hypothesis. The TTR active site is inducible and constituted via a protein rearrangement resulting in ~7% of proteolytically active TTR at pH 7.4. The side chain of His88 is shifted near His90 and Glu92 establishing a Zn2+-chelating pattern HXHXE not found previously in any metallopeptidase and only conserved in TTR of humans and some other primates. Point mutations of these three residues yielded proteins devoid of proteolytic activity. Glu72 was identified as the general base involved in activation of the catalytic water. Our results unveil TTR as a metallopeptidase and define its catalytic machinery.

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Point mutations of two arginine residues in the Streptomyces R61 dd-peptidase

Biochemical Journal ◽

10.1042/bj2830123 ◽

1992 ◽

Vol 283 (1) ◽

pp. 123-128 ◽

Cited By ~ 5

Author(s):

C Bourguignon-Bellefroid ◽

B Joris ◽

J Van Beeumen ◽

J M Ghuysen ◽

J M Frère

Keyword(s):

Enzyme Activity ◽

Enzyme Stability ◽

Point Mutations ◽

Site Directed Mutagenesis ◽

Side Chain ◽

Directed Mutagenesis ◽

Serine Residue ◽

Wild Type Enzyme ◽

Arginine Residues

Incubation of the exocellular DD-carboxypeptidase/transpeptidase of Streptomyces R61 with phenylglyoxal resulted in a time-dependent decrease in the enzyme activity. This inactivation was demonstrated to be due to modification of the Arg-99 side chain. In consequence, the role of that residue was investigated by site-directed mutagenesis. Mutation of Arg-99 into leucine appeared to be highly detrimental to enzyme stability, reflecting a determining structural role for this residue. The conserved Arg-103 residue was also substituted by using site-directed mutagenesis. The modification to a serine residue yielded a stable enzyme, the catalytic properties of which were similar to those of the wild-type enzyme. Thus Arg-103, although strictly conserved or replaced by a lysine residue in most of the active-site penicillin-recognizing proteins, did not appear to fulfil any essential role in either the enzyme activity or structure.

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Active-site serine mutants of the Streptomyces albus G β-lactamase

Biochemical Journal ◽

10.1042/bj2770647 ◽

1991 ◽

Vol 277 (3) ◽

pp. 647-652 ◽

Cited By ~ 10

Author(s):

F Jacob ◽

B Joris ◽

J M Frère

Keyword(s):

Active Site ◽

Specific Activity ◽

Site Directed Mutagenesis ◽

Beta Lactamase ◽

Wild Type ◽

Serine Residue ◽

Wild Type Enzyme ◽

Ph Values ◽

Streptomyces Albus ◽

Small Production

By using site-directed mutagenesis, the active-site serine residue of the Streptomyces albus G beta-lactamase was substituted by alanine and cysteine. Both mutant enzymes were produced in Streptomyces lividans and purified to homogeneity. The cysteine beta-lactamase exhibited a substrate-specificity profile distinct from that of the wild-type enzyme, and its kcat./Km values at pH 7 were never higher than 0.1% of that of the serine enzyme. Unlike the wild-type enzyme, the activity of the mutant increased at acidic pH values. Surprisingly, the alanine mutant exhibited a weak but specific activity for benzylpenicillin and ampicillin. In addition, a very small production of wild-type enzyme, probably due to mistranslation, was detected, but that activity could be selectively eliminated. Both mutant enzymes were nearly as thermostable as the wild-type.

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Chemical modification of serine at the active site of penicillin acylase from Kluyvera citrophila

Biochemical Journal ◽

10.1042/bj2800659 ◽

1991 ◽

Vol 280 (3) ◽

pp. 659-662 ◽

Cited By ~ 15

Author(s):

J Martín ◽

A Slade ◽

A Aitken ◽

R Arche ◽

R Virden

Keyword(s):

Active Site ◽

Tertiary Structure ◽

Thiol Group ◽

Iodoacetic Acid ◽

Penicillin Acylase ◽

Protein Product ◽

Side Chain ◽

Serine Residue ◽

Conserved Sequence ◽

Ester Substrate

The site of reaction of penicillin acylase from Kluyvera citrophila with the potent inhibitor phenylmethanesulphonyl fluoride was investigated by incubating the inactivated enzyme with thioacetic acid to convert the side chain of the putative active-site serine residue to that of cysteine. The protein product contained one thiol group, which was reactive towards 2,2′-dipyridyl disulphide and iodoacetic acid. Carboxymethylcysteine was identified as the N-terminal residue of the beta-subunit of the carboxy[3H]methylthiol-protein. No significant changes in tertiary structure were detected in the modified penicillin acylase using near-u.v. c.d. spectroscopy. However, the catalytic activity (kcat) with either an anilide or an ester substrate was decreased in the thiol-protein by a factor of more than 10(4). A comparison of sequences of apparently related acylases shows no other extensive regions of conserved sequence containing an invariant serine residue. The side chain of this residue is proposed as a candidate nucleophile in the formation of an acyl-enzyme during catalysis.

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Threonine 57 is required for the post-translational activation ofEscherichia coliaspartate α-decarboxylase

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s1399004713034275 ◽

2014 ◽

Vol 70 (4) ◽

pp. 1166-1172 ◽

Cited By ~ 5

Author(s):

Michael E. Webb ◽

Briony A. Yorke ◽

Tom Kershaw ◽

Sarah Lovelock ◽

Carina M. C. Lobley ◽

...

Keyword(s):

Crystal Structure ◽

Active Site ◽

Site Directed Mutagenesis ◽

Intramolecular Rearrangement ◽

Directed Mutagenesis ◽

Vitamin B ◽

Biosynthesis Pathway ◽

Serine Residue ◽

Translational Activation

Aspartate α-decarboxylase is a pyruvoyl-dependent decarboxylase required for the production of β-alanine in the bacterial pantothenate (vitamin B5) biosynthesis pathway. The pyruvoyl group is formedviathe intramolecular rearrangement of a serine residue to generate a backbone ester intermediate which is cleaved to generate an N-terminal pyruvoyl group. Site-directed mutagenesis of residues adjacent to the active site, including Tyr22, Thr57 and Tyr58, reveals that only mutation of Thr57 leads to changes in the degree of post-translational activation. The crystal structure of the site-directed mutant T57V is consistent with a non-rearranged backbone, supporting the hypothesis that Thr57 is required for the formation of the ester intermediate in activation.

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An Adaptive Geometric Search Algorithm for Macromolecular Scaffold Selection

10.1101/099762 ◽

2017 ◽

Author(s):

Tian Jiang ◽

P. Douglas Renfrew ◽

Kevin Drew ◽

Noah Youngs ◽

Glenn Butterfoss ◽

...

Keyword(s):

Protein Design ◽

Active Site ◽

Metal Binding ◽

Search Algorithm ◽

Three Dimensional ◽

Side Chain ◽

Enzyme Design ◽

Hydrogen Binding ◽

Performance Improvements ◽

Geometric Search

AbstractA wide variety of protein and peptidomimetic design tasks require matching functional three-dimensional motifs to potential oligomeric scaffolds. Enzyme design, for example, aims to graft active-site patterns typically consisting of 3 to 15 residues onto new protein surfaces. Identifying suitable proteins capable of scaffolding such active-site engraftment requires costly searches to identify protein folds that can provide the correct positioning of side chains to host the desired active site. Other examples of biodesign tasks that require simpler fast exact geometric searches of potential side chain positioning include mimicking binding hotspots, design of metal binding clusters and the design of modular hydrogen binding networks for specificity. In these applications the speed and scaling of geometric search limits downstream design to small patterns. Here we present an adaptive algorithm to searching for side chain take-off angles compatible with an arbitrarily specified functional pattern that enjoys substantive performance improvements over previous methods. We demonstrate this method in both genetically encoded (protein) and synthetic (peptidomimetic) design scenarios. Examples of using this method with the Rosetta framework for protein design are provided but our implementation is compatible with multiple protein design frameworks and is freely available as a set of python scripts (https://github.com/JiangTian/adaptive-geometric-search-for-protein-design).

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Structure-Function Studies of Ser-289 in the Class C β-Lactamase from Enterobacter cloacae P99

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.43.3.543 ◽

1999 ◽

Vol 43 (3) ◽

pp. 543-548 ◽

Cited By ~ 13

Author(s):

Sonia Trépanier ◽

James R. Knox ◽

Natalie Clairoux ◽

François Sanschagrin ◽

Roger C. Levesque ◽

...

Keyword(s):

Three Dimensional ◽

Catalytic Efficiency ◽

Enterobacter Cloacae ◽

Acid Group ◽

Site Directed Mutagenesis ◽

Side Chain ◽

Class C ◽

Three Dimensional Models ◽

Hydrolytic Mechanism

ABSTRACT Site-directed mutagenesis of Ser-289 of the class C β-lactamase from Enterobacter cloacae P99 was performed to investigate the role of this residue in β-lactam hydrolysis. This amino acid lies near the active site of the enzyme, where it can interact with the C-3 substituent of cephalosporins. Kinetic analysis of six mutant β-lactamases with five cephalosporins showed that Ser-289 can be substituted by amino acids with nonpolar or polar uncharged side chains without altering the catalytic efficiency of the enzyme. These data suggest that Ser-289 is not essential in the binding or hydrolytic mechanism of AmpC β-lactamase. However, replacement by Lys or Arg decreased by two- to threefold the k cat of four of the five β-lactams tested, particularly cefoperazone, cephaloridine, and cephalothin. Three-dimensional models of the mutant β-lactamases revealed that the length and positive charge of the side chain of Lys and Arg could create an electrostatic linkage to the C-4 carboxylic acid group of the dihydrothiazine ring of the acyl intermediate which could slow the deacylation step or hinder release of the product.

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Trinuclear copper biocatalytic center forms an active site of thiocyanate dehydrogenase

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1922133117 ◽

2020 ◽

Vol 117 (10) ◽

pp. 5280-5290 ◽

Cited By ~ 2

Author(s):

Tamara V. Tikhonova ◽

Dimitry Y. Sorokin ◽

Wilfred R. Hagen ◽

Maria G. Khrenova ◽

Gerard Muyzer ◽

...

Keyword(s):

Active Site ◽

Copper Ions ◽

Soda Lakes ◽

Three Dimensional ◽

Kinetic Studies ◽

Isosceles Triangle ◽

Site Directed Mutagenesis ◽

Paramagnetic Resonance ◽

Mechanism Of Catalysis ◽

Copper Center

Biocatalytic copper centers are generally involved in the activation and reduction of dioxygen, with only few exceptions known. Here we report the discovery and characterization of a previously undescribed copper center that forms the active site of a copper-containing enzyme thiocyanate dehydrogenase (suggested EC 1.8.2.7) that was purified from the haloalkaliphilic sulfur-oxidizing bacterium of the genus Thioalkalivibrio ubiquitous in saline alkaline soda lakes. The copper cluster is formed by three copper ions located at the corners of a near-isosceles triangle and facilitates a direct thiocyanate conversion into cyanate, elemental sulfur, and two reducing equivalents without involvement of molecular oxygen. A molecular mechanism of catalysis is suggested based on high-resolution three-dimensional structures, electron paramagnetic resonance (EPR) spectroscopy, quantum mechanics/molecular mechanics (QM/MM) simulations, kinetic studies, and the results of site-directed mutagenesis.

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Crystal structures of two monomeric triosephosphate isomerase variants identifiedviaa directed-evolution protocol selecting forL-arabinose isomerase activity

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x16007548 ◽

2016 ◽

Vol 72 (6) ◽

pp. 490-499

Author(s):

Mirja Krause ◽

Tiila-Riikka Kiema ◽

Peter Neubauer ◽

Rik K. Wierenga

Keyword(s):

Crystal Structure ◽

Escherichia Coli ◽

Directed Evolution ◽

Crystal Structures ◽

Active Site ◽

Triosephosphate Isomerase ◽

Point Mutations ◽

Van Der Waals Interactions ◽

Side Chain ◽

Arabinose Isomerase

The crystal structures are described of two variants of A-TIM: Ma18 (2.7 Å resolution) and Ma21 (1.55 Å resolution). A-TIM is a monomeric loop-deletion variant of triosephosphate isomerase (TIM) which has lost the TIM catalytic properties. Ma18 and Ma21 were identified after extensive directed-evolution selection experiments using anEscherichia coliL-arabinose isomerase knockout strain expressing a randomly mutated A-TIM gene. These variants facilitate better growth of theEscherichia coliselection strain in medium supplemented with 40 mML-arabinose. Ma18 and Ma21 differ from A-TIM by four and one point mutations, respectively. Ma18 and Ma21 are more stable proteins than A-TIM, as judged from CD melting experiments. Like A-TIM, both proteins are monomeric in solution. In the Ma18 crystal structure loop 6 is open and in the Ma21 crystal structure loop 6 is closed, being stabilized by a bound glycolate molecule. The crystal structures show only small differences in the active site compared with A-TIM. In the case of Ma21 it is observed that the point mutation (Q65L) contributes to small structural rearrangements near Asn11 of loop 1, which correlate with different ligand-binding properties such as a loss of citrate binding in the active site. The Ma21 structure also shows that its Leu65 side chain is involved in van der Waals interactions with neighbouring hydrophobic side-chain moieties, correlating with its increased stability. The experimental data suggest that the increased stability and solubility properties of Ma21 and Ma18 compared with A-TIM cause better growth of the selection strain when coexpressing Ma21 and Ma18 instead of A-TIM.

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Structure-function studies of the myosin motor domain: importance of the 50-kDa cleft.

Molecular Biology of the Cell ◽

10.1091/mbc.7.7.1123 ◽

1996 ◽

Vol 7 (7) ◽

pp. 1123-1136 ◽

Cited By ~ 58

Author(s):

K M Ruppel ◽

J A Spudich

Keyword(s):

Active Site ◽

Three Dimensional ◽

Point Mutations ◽

Random Mutagenesis ◽

Motor Domain ◽

Actin Binding ◽

Dimensional Structure ◽

Null Cell ◽

Myosin Motor Domain ◽

Cell Phenotypes

We used random mutagenesis to create 21 point mutations in a highly conserved region of the motor domain of Dictyostelium myosin and classified them into three distinct groups based on the ability to complement myosin null cell phenotypes: wild type, intermediate, and null. Biochemical analysis of the mutated myosins also revealed three classes of mutants that correlated well with the phenotypic classification. The mutated myosins that were not fully functional showed defects ranging from ATP nonhydrolyzers to myosins whose enzymatic and mechanical properties are uncoupled. Placement of the mutations onto the three-dimensional structure of myosin showed that the mutated region lay along the cleft that separates the active site from the actin-binding domain and that has been shown to move in response to changes at the active site. These results demonstrate that this region of myosin plays a key role in transduction of chemical energy to mechanical displacement.

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