Nicotinamide mononucleotide adenylyltransferase displays alternate binding modes for nicotinamide nucleotides

Nicotinamide mononucleotide adenylyltransferase (NMNAT) catalyzes the biosynthesis of NAD+and NaAD+. The crystal structure of NMNAT fromMethanobacterium thermoautotrophicumcomplexed with NAD+and SO42−revealed the active-site residues involved in binding and catalysis. Site-directed mutagenesis was used to further characterize the roles played by several of these residues. Arg11 and Arg136 were implicated in binding the phosphate groups of the ATP substrate. Both of these residues were mutated to lysine individually. Arg47 does not interact with either NMN or ATP substrates directly, but was deemed to play a role in binding as it is proximal to Arg11 and Arg136. Arg47 was mutated to lysine and glutamic acid. Surprisingly, when expressed inEscherichia coliall of these NMNAT mutants trapped a molecule of NADP+in their active sites. This NADP+was bound in a conformation that was quite different from that displayed by NAD+in the native enzyme complex. When NADP+was co-crystallized with wild-type NMNAT, the same structural arrangement was observed. These studies revealed a different conformation of NADP+in the active site of NMNAT, indicating plasticity of the active site.

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Site-directed mutagenesis of proposed active-site residues of penicillin-binding protein 5 from Escherichia coli

Biochemical Journal ◽

10.1042/bj3030357 ◽

1994 ◽

Vol 303 (2) ◽

pp. 357-362 ◽

Cited By ~ 29

Author(s):

M P G van der Linden ◽

L de Haan ◽

O Dideberg ◽

W Keck

Keyword(s):

Active Site ◽

Catalytic Mechanism ◽

Binding Capacity ◽

Binding Protein ◽

Complete Loss ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Penicillin Binding Protein ◽

Wild Type ◽

Active Site Residues

Alignment of the amino acid sequence of penicillin-binding protein 5 (PBP5) with the sequences of other members of the family of active-site-serine penicillin-interacting enzymes predicted the residues playing a role in the catalytic mechanism of PBP5. Apart from the active-site (Ser44), Lys47, Ser110-Gly-Asn, Asp175 and Lys213-Thr-Gly were identified as the residues making up the conserved boxes of this protein family. To determine the role of these residues, they were replaced using site-directed mutagenesis. The mutant proteins were assayed for their penicillin-binding capacity and DD-carboxypeptidase activity. The Ser44Cys and the Ser44Gly mutants showed a complete loss of both penicillin-binding capacity and DD-carboxypeptidase activity. The Lys47Arg mutant also lost its DD-carboxypeptidase activity but was able to bind and hydrolyse penicillin, albeit at a considerably reduced rate. Mutants in the Ser110-Gly-Asn fingerprint were affected in both acylation and deacylation upon reaction with penicillin and lost their DD-carboxypeptidase activity with the exception of Asn112Ser and Asn112Thr. The Asp175Asn mutant showed wild-type penicillin-binding but a complete loss of DD-carboxypeptidase activity. Mutants of Lys213 lost both penicillin-binding and DD-carboxypeptidase activity except for Lys213His, which still bound penicillin with a k+2/K' of 0.2% of the wild-type value. Mutation of His216 and Thr217 also had a strong effect on DD-carboxypeptidase activity. Thr217Ser and Thr217Ala showed augmented hydrolysis rates for the penicillin acyl-enzyme. This study reveals the residues in the conserved fingerprints to be very important for both DD-carboxypeptidase activity and penicillin-binding, and confirms them to play crucial roles in catalysis.

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Development of Novel Sugar Isomerases by Optimization of Active Sites in Phosphosugar Isomerases for Monosaccharides

Applied and Environmental Microbiology ◽

10.1128/aem.02539-12 ◽

2012 ◽

Vol 79 (3) ◽

pp. 982-988 ◽

Cited By ~ 11

Author(s):

Soo-Jin Yeom ◽

Yeong-Su Kim ◽

Deok-Kun Oh

Keyword(s):

Active Site ◽

Binding Sites ◽

Active Sites ◽

Pyrococcus Furiosus ◽

Wild Type ◽

Active Site Residues ◽

Phosphate Binding ◽

Wild Type Enzyme ◽

Content Type

ABSTRACTPhosphosugar isomerases can catalyze the isomerization of not only phosphosugar but also of monosaccharides, suggesting that the phosphosugar isomerases can be used as sugar isomerases that do not exist in nature. Determination of active-site residues of phosphosugar isomerases, including ribose-5-phosphate isomerase fromClostridium difficile(CDRPI), mannose-6-phosphate isomerase fromBacillus subtilis(BSMPI), and glucose-6-phosphate isomerase fromPyrococcus furiosus(PFGPI), was accomplished by docking of monosaccharides onto the structure models of the isomerases. The determinant residues, including Arg133 of CDRPI, Arg192 of BSMPI, and Thr85 of PFGPI, were subjected to alanine substitutions and found to act as phosphate-binding sites. R133D of CDRPI, R192 of BSMPI, and T85Q of PFGPI displayed the highest catalytic efficiencies for monosaccharides at each position. These residues exhibited 1.8-, 3.5-, and 4.9-fold higher catalytic efficiencies, respectively, for the monosaccharides than the wild-type enzyme. However, the activities of these 3 variant enzymes for phosphosugars as the original substrates disappeared. Thus, R133D of CDRPI, R192 of BSMPI, and T85Q of PFGPI are no longer phosphosugar isomerases; instead, they are changed to ad-ribose isomerase, anl-ribose isomerase, and anl-talose isomerase, respectively. In this study, we used substrate-tailored optimization to develop novel sugar isomerases which are not found in nature based on phosphosugar isomerases.

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Probing the role of the residues in the active site of the transaminase from Thermobaculum terrenum

PLoS ONE ◽

10.1371/journal.pone.0255098 ◽

2021 ◽

Vol 16 (7) ◽

pp. e0255098

Author(s):

Ekaterina Yu. Bezsudnova ◽

Alena Yu. Nikolaeva ◽

Alina K. Bakunova ◽

Tatiana V. Rakitina ◽

Dmitry A. Suplatov ◽

...

Keyword(s):

Amino Acids ◽

Substrate Specificity ◽

Active Site ◽

Active Sites ◽

Site Directed Mutagenesis ◽

Primary Amines ◽

Active Site Residues ◽

Type Iv ◽

Fold Type

Creating biocatalysts for (R)-selective amination effectively is highly desirable in organic synthesis. Despite noticeable progress in the engineering of (R)-amine activity in pyridoxal-5’-phosphate-dependent transaminases of fold type IV, the specialization of the activity is still an intuitive task, as there is poor understanding of sequence-structure-function relationships. In this study, we analyzed this relationship in transaminase from Thermobaculum terrenum, distinguished by expanded substrate specificity and activity in reactions with L-amino acids and (R)-(+)-1-phenylethylamine using α-ketoglutarate and pyruvate as amino acceptors. We performed site-directed mutagenesis to create a panel of the enzyme variants, which differ in the active site residues from the parent enzyme to a putative transaminase specific to (R)-primary amines. The variants were examined in the overall transamination reactions and half-reaction with (R)-(+)-1-phenylethylamine. A structural analysis of the most prominent variants revealed a spatial reorganization in the active sites, which caused changes in activity. Although the specialization to (R)-amine transaminase was not implemented, we succeeded in understanding the role of the particular active site residues in expanding substrate specificity of the enzyme. We showed that the specificity for (R)-(+)-1-phenylethylamine in transaminase from T. terrenum arises without sacrificing the specificity for L-amino acids and α-ketoglutarate and in consensus with it.

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Endogenous Synthesis of Erythritol, a Novel Biomarker of Weight Gain (P15-016-19)

Current Developments in Nutrition ◽

10.1093/cdn/nzz037.p15-016-19 ◽

2019 ◽

Vol 3 (Supplement_1) ◽

Author(s):

Semira Ortiz ◽

Doletha Szebenyi ◽

Martha Field

Keyword(s):

Molecular Modeling ◽

Active Site ◽

Active Sites ◽

A549 Cells ◽

Site Directed Mutagenesis ◽

Central Adiposity ◽

Wild Type ◽

Reduction Activity

Abstract Objectives Serum erythritol is associated with central adiposity gain in young adults. Erythritol, a 4-carbon polyol, is synthesized endogenously from erythrose through the pentose phosphate pathway. We have identified two enzymes which catalyze this reaction: alcohol dehydrogenase 1 (ADH1) and sorbitol dehydrogenase (SORD). Interestingly, ADH1 isoforms ADH1B1 and ADH1C2 catalyze NADPH-dependent synthesis of erythritol in vitro, but ADH1B1 does not. In A549 cells, siRNA knockdown of SORD levels to less than 15% of control levels reduced erythritol by 50%. A549 cells also have low levels of ADH1 expression. This indicates that other enzymes may be capable of endogenous erythritol production. Based on its high degree of homology to ADH1, we hypothesize that ADH4 also catalyzes erythritol synthesis. The objective of this study was to further elucidate the mechanism of erythritol synthesis by: determining key differences between the active site of ADH1B2 compared to ADH1B1 and SORD, and screening a new candidate enzyme, ADH4. Methods This study used molecular modeling to evaluate the enzyme, erythrose, NADPH complex. Site-directed mutagenesis was performed to replace Arg208 (R208) of recombinant human SORD with histidine. Recombinant SORD R208H mutant and ADH4 were then expressed, purified, and their erythrose reduction activity was kinetically characterized. Results Molecular modeling of the active sites of ADH1B1 and ADH1B2 revealed that the Arg48 residue of ADH1B1 makes three contacts with NADPH, whereas His48 of ADH1B2 only makes one contact. Mutation of the SORD active-site Arg208 to histidine reduced the kcat by more than 80% compared to wild-type (95 ± 21 min−1 wild-type, 16 ± 2 min−1 R208H, P < 0.05). ADH4 erythrose reduction activity reduced by more than 95% compared to ADH1 variant proteins in vitro (kcat 2 ± 1 min−1). Conclusions Mutation of arginine 208 to histidine in the active site of SORD significantly reduced the kcat, indicating that the presence of an arginine residue is essential for binding NADPH, the cofactor required in vivo for erythritol synthesis. The low erythrose reduction activity of ADH4 suggests that ADH4 does not contribute to erythritol synthesis in humans. Funding Sources None.

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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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ADP-Ribose-1"-Monophosphatase: a Conserved Coronavirus Enzyme That Is Dispensable for Viral Replication in Tissue Culture

Journal of Virology ◽

10.1128/jvi.79.20.12721-12731.2005 ◽

2005 ◽

Vol 79 (20) ◽

pp. 12721-12731 ◽

Cited By ~ 99

Author(s):

Ákos Putics ◽

Witold Filipowicz ◽

Jonathan Hall ◽

Alexander E. Gorbalenya ◽

John Ziebuhr

Keyword(s):

Active Site ◽

Biological Significance ◽

Virus Titer ◽

Site Directed Mutagenesis ◽

Replicase Gene ◽

Active Site Residues ◽

Nonstructural Proteins ◽

Trna Splicing

ABSTRACT Replication of the ∼30-kb plus-strand RNA genome of coronaviruses and synthesis of an extensive set of subgenome-length RNAs are mediated by the replicase-transcriptase, a membrane-bound protein complex containing several cellular proteins and up to 16 viral nonstructural proteins (nsps) with multiple enzymatic activities, including protease, polymerase, helicase, methyltransferase, and RNase activities. To get further insight into the replicase gene-encoded functions, we characterized the coronavirus X domain, which is part of nsp3 and has been predicted to be an ADP-ribose-1"-monophosphate (Appr-1"-p) processing enzyme. Bacterially expressed forms of human coronavirus 229E (HCoV-229E) and severe acute respiratory syndrome-coronavirus X domains were shown to dephosphorylate Appr-1"-p, a side product of cellular tRNA splicing, to ADP-ribose in a highly specific manner. The enzyme had no detectable activity on several other nucleoside phosphates. Guided by the crystal structure of AF1521, an X domain homolog from Archaeoglobus fulgidus, potential active-site residues of the HCoV-229E X domain were targeted by site-directed mutagenesis. The data suggest that the HCoV-229E replicase polyprotein residues, Asn 1302, Asn 1305, His 1310, Gly 1312, and Gly 1313, are part of the enzyme's active site. Characterization of an Appr-1"-pase-deficient HCoV-229E mutant revealed no significant effects on viral RNA synthesis and virus titer, and no reversion to the wild-type sequence was observed when the mutant virus was passaged in cell culture. The apparent dispensability of the conserved X domain activity in vitro indicates that coronavirus replicase polyproteins have evolved to include nonessential functions. The biological significance of the novel enzymatic activity in vivo remains to be investigated.

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Enzyme activity enhancement of chondroitinase ABC I from Proteus vulgaris by site-directed mutagenesis

RSC Advances ◽

10.1039/c5ra15220h ◽

2015 ◽

Vol 5 (93) ◽

pp. 76040-76047 ◽

Cited By ~ 11

Author(s):

Zhenya Chen ◽

Ye Li ◽

Yue Feng ◽

Liang Chen ◽

Qipeng Yuan

Keyword(s):

Active Site ◽

Simulation Analysis ◽

Docking Simulation ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Wild Type ◽

Proteus Vulgaris ◽

Molecular Docking Simulation ◽

Activity Enhancement ◽

First Time

Arg660 was found as a new active site and Asn795Ala and Trp818Ala mutants showed higher activities than the wild type based on molecular docking simulation analysis for the first time.

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Catalytic and structural contributions for glutathione-binding residues in a Delta class glutathione S-transferase

Biochemical Journal ◽

10.1042/bj20040697 ◽

2004 ◽

Vol 382 (2) ◽

pp. 751-757 ◽

Cited By ~ 23

Author(s):

Pakorn WINAYANUWATTIKUN ◽

Albert J. KETTERMAN

Keyword(s):

Binding Site ◽

Active Site ◽

Structural Integrity ◽

Catalytic Mechanism ◽

Tertiary Structure ◽

Site Directed Mutagenesis ◽

Active Site Residues ◽

Anopheles Dirus ◽

Steady State Kinetics ◽

Cellular Detoxification

Glutathione S-transferases (GSTs) are dimeric proteins that play a major role in cellular detoxification. The GSTs in mosquito Anopheles dirus species B, an important malaria vector in South East Asia, are of interest because they can play an important role in insecticide resistance. In the present study, we characterized the Anopheles dirus (Ad)GST D3-3 which is an alternatively spliced product of the adgst1AS1 gene. The data from the crystal structure of GST D3-3 shows that Ile-52, Glu-64, Ser-65, Arg-66 and Met-101 interact directly with glutathione. To study the active-site function of these residues, alanine substitution site-directed mutagenesis was performed resulting in five mutants: I52A (Ile-52→Ala), E64A, S65A, R66A and M101A. Interestingly, the E64A mutant was expressed in Escherichia coli in inclusion bodies, suggesting that this residue is involved with the tertiary structure or folding property of this enzyme. However, the I52A, S65A, R66A and M101A mutants were purified by glutathione affinity chromatography and the enzyme activity characterized. On the basis of steady-state kinetics, difference spectroscopy, unfolding and refolding studies, it was concluded that these residues: (1) contribute to the affinity of the GSH-binding site (‘G-site’) for GSH, (2) influence GSH thiol ionization, (3) participate in kcat regulation by affecting the rate-limiting step of the reaction, and in the case of Ile-52 and Arg-66, influenced structural integrity and/or folding of the enzyme. The structural perturbations from these mutants are probably transmitted to the hydrophobic-substrate-binding site (‘H-site’) through changes in active site topology or through effects on GSH orientation. Therefore these active site residues appear to contribute to various steps in the catalytic mechanism, as well as having an influence on the packing of the protein.

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Identification of the active site and characterization of a novel sporulation-specific cysteine protease YabG from Bacillus subtilis

The Journal of Biochemistry ◽

10.1093/jb/mvab135 ◽

2021 ◽

Author(s):

Ryuji Yamazawa ◽

Ritsuko Kuwana ◽

Kenji Takeuchi ◽

Hiromu Takamatsu ◽

Yoshitaka Nakajima ◽

...

Keyword(s):

Bacillus Subtilis ◽

Chemical Modification ◽

Cysteine Protease ◽

Active Site ◽

Amino Acid Sequences ◽

Site Directed Mutagenesis ◽

Protease Gene ◽

Active Site Residues ◽

Protein Determination ◽

35 Kda Protein

Abstract In order to characterize the probable protease gene yabG found in the genomes of spore-forming bacteria, Bacillus subtilis yabG was expressed as a 35 kDa His-tagged protein (BsYabG) in Escherichia coli cells. During purification using Ni-affinity chromatography, the 35 kDa protein was degraded via several intermediates to form a 24 kDa protein. Furthermore, it was degraded after an extended incubation period. The effect of protease inhibitors, including certain chemical modification reagents, on the conversion of the 35 kDa protein to the 24 kDa protein was investigated. Reagents reacting with sulfhydryl groups exerted significant effects, strongly suggesting that the yabG gene product is a cysteine protease with autolytic activity. Site-directed mutagenesis of the conserved Cys and His residues indicated that Cys218 and His172 are active site residues. No degradation was observed in the C218A/S and H172A mutants. In addition to the chemical modification reagents, benzamidine inhibited the degradation of the 24 kDa protein. Determination of the N-terminal amino acid sequences of the intermediates revealed trypsin-like specificity for YabG protease. Based on the relative positions of His172 and Cys218 and their surrounding sequences, we propose the classification of YabG as a new family of clan CD in the Merops peptidase database.

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