Probing the location and function of the conserved histidine residue of phosphoglucose isomerase by using an active site directed inhibitor N-bromoacetylethanolamine phosphate

Abstract Objective Non-haem iron(II)- and 2-oxoglutarate-dependent dioxygenases (i2OGdd), are a taxonomically and functionally diverse group of enzymes. The active site comprises ferrous iron in a hexa-coordinated distorted octahedron with the apoenzyme, 2-oxoglutarate and a displaceable water molecule. Current information on novel i2OGdd members is sparse and relies on computationally-derived annotation schema. The dissimilar amino acid composition and variable active site geometry thereof, results in differing reaction chemistries amongst i2OGdd members. An additional need of researchers is a curated list of sequences with putative i2OGdd function which can be probed further for empirical data. Results This work reports the implementation of $$Fe\left(2\right)OG$$ F e 2 O G , a web server with dual functionality and an extension of previous work on i2OGdd enzymes $$\left(Fe\left(2\right)OG\equiv \{H2OGpred,DB2OG\}\right)$$ F e 2 O G ≡ { H 2 O G p r e d , D B 2 O G } . $$Fe\left(2\right)OG$$ F e 2 O G , in this form is completely revised, updated (URL, scripts, repository) and will strengthen the knowledge base of investigators on i2OGdd biochemistry and function. $$Fe\left(2\right)OG$$ F e 2 O G , utilizes the superior predictive propensity of HMM-profiles of laboratory validated i2OGdd members to predict probable active site geometries in user-defined protein sequences. $$Fe\left(2\right)OG$$ F e 2 O G , also provides researchers with a pre-compiled list of analyzed and searchable i2OGdd-like sequences, many of which may be clinically relevant. $$Fe(2)OG$$ F e ( 2 ) O G , is freely available (http://204.152.217.16/Fe2OG.html) and supersedes all previous versions, i.e., H2OGpred, DB2OG.

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A Histidine Residue at the Active Site of Avian Liver Phosphoenolpyruvate Carboxykinase

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)47165-2 ◽

1989 ◽

Vol 264 (33) ◽

pp. 19666-19676

Author(s):

K C Cheng ◽

T Nowak

Keyword(s):

Active Site ◽

Phosphoenolpyruvate Carboxykinase ◽

Histidine Residue

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Three Conserved Regions in Baculovirus Sulfhydryl Oxidase P33 Are Critical for Enzymatic Activity and Function

Journal of Virology ◽

10.1128/jvi.01158-17 ◽

2017 ◽

Vol 91 (23) ◽

Cited By ~ 6

Author(s):

Wenhua Kuang ◽

Huanyu Zhang ◽

Manli Wang ◽

Ning-Yi Zhou ◽

Fei Deng ◽

...

Keyword(s):

Enzymatic Activity ◽

Active Site ◽

Salt Bridge ◽

Sulfhydryl Oxidase ◽

Dimer Interface ◽

Conserved Regions ◽

Occlusion Bodies ◽

Budded Virus ◽

And Function ◽

Substrate Proteins

ABSTRACT Baculoviruses encode a conserved sulfhydryl oxidase, P33, which is necessary for budded virus (BV) production and multinucleocapsid occlusion-derived virus (ODV) formation. Here, the structural and functional relationship of P33 was revealed by X-ray crystallography, site-directed mutagenesis, and functional analysis. Based on crystallographic characterization and structural analysis, a series of P33 mutants within three conserved regions, i.e., the active site, the dimer interface, and the R127-E183 salt bridge, were constructed. In vitro experiments showed that mutations within the active site and dimer interface severely impaired the sulfhydryl oxidase activity of P33, while the mutations in the salt bridge had a relatively minor influence. Recombinant viruses containing mutated P33 were constructed and assayed in vivo. Except for the active-site mutant AXXA, all other mutants produced infectious BVs, although certain mutants had a decreased BV production. The active-site mutant H114A, the dimer interface mutant H227D, and the salt bridge mutant R127A-E183A were further analyzed by electron microscopy and bioassays. The occlusion bodies (OBs) of mutants H114A and R127A-E183A had a ragged surface and contained mostly ODVs with a single nucleocapsid. The OBs of all three mutants contained lower numbers of ODVs and had a significantly reduced oral infectivity in comparison to control virus. Crystallographic analyses further revealed that all three regions may coordinate with one another to achieve optimal function of P33. Taken together, our data revealed that all the three conserved regions are involved in P33 activity and are crucial for virus morphogenesis and peroral infectivity. IMPORTANCE Sulfhydryl oxidase catalyzes disulfide bond formation of substrate proteins. P33, a baculovirus-encoded sulfhydryl oxidase, is different from other cellular and viral sulfhydryl oxidases, bearing unique features in tertiary and quaternary structure organizations. In this study, we found that three conserved regions, i.e., the active site, dimer interface, and the R127-E183 salt bridge, play important roles in the enzymatic activity and function of P33. Previous observations showed that deletion of p33 results in a total loss of budded virus (BV) production and in morphological changes in occlusion-derived virus (ODV). Our study revealed that certain P33 mutants lead to occlusion bodies (OBs) with a ragged surface, decreased embedded ODVs, and reduced oral infectivity. Interestingly, some P33 mutants with impaired ODV/OB still retained BV productivity, indicating that the impacts on BV and on ODV/OB are two distinctly different functions of P33, which are likely to be performed via different substrate proteins.

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Loss of a histidine residue at the active site of S-locus ribonuclease is associated with self-compatibility in Lycopersicon peruvianum.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.91.14.6511 ◽

1994 ◽

Vol 91 (14) ◽

pp. 6511-6514 ◽

Cited By ~ 89

Author(s):

J. Royo ◽

C. Kunz ◽

Y. Kowyama ◽

M. Anderson ◽

A. E. Clarke ◽

...

Keyword(s):

Active Site ◽

Histidine Residue ◽

Lycopersicon Peruvianum ◽

S Locus ◽

Self Compatibility

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Isocitrate Lyase from Cephalosporium acremonium. Role of Mg2+ Ions, Kinetics, and Evidence for a Histidine Residue in the Active Site of the Enzyme

Biochemistry ◽

10.1021/bi00018a008 ◽

1995 ◽

Vol 34 (18) ◽

pp. 6059-6068 ◽

Cited By ~ 7

Author(s):

Eusebio Perdiguero ◽

Dolores de Arriaga ◽

Felix Busto ◽

Joaquin Soler

Keyword(s):

Active Site ◽

Isocitrate Lyase ◽

Histidine Residue ◽

Cephalosporium Acremonium

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Characterization of anEscherichia coliSulfite Oxidase Homologue Reveals the Role of a Conserved Active Site Cysteine in Assembly and Function†

Biochemistry ◽

10.1021/bi050621a ◽

2005 ◽

Vol 44 (30) ◽

pp. 10339-10348 ◽

Cited By ~ 51

Author(s):

Stephen J. Brokx ◽

Richard A. Rothery ◽

Guijin Zhang ◽

Derek P. Ng ◽

Joel H. Weiner

Keyword(s):

Active Site ◽

Active Site Cysteine ◽

And Function

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Reverse evolution leads to genotypic incompatibility despite functional and active site convergence

eLife ◽

10.7554/elife.06492 ◽

2015 ◽

Vol 4 ◽

Cited By ~ 43

Author(s):

Miriam Kaltenbach ◽

Colin J Jackson ◽

Eleanor C Campbell ◽

Florian Hollfelder ◽

Nobuhiko Tokuriki

Keyword(s):

Active Site ◽

Adaptive Landscape ◽

Sequence Structure ◽

Activity Increase ◽

Enzyme Active Site ◽

Fundamental Relationship ◽

And Function ◽

Alternative Set ◽

Shed Light ◽

New Mutations

Understanding the extent to which enzyme evolution is reversible can shed light on the fundamental relationship between protein sequence, structure, and function. Here, we perform an experimental test of evolutionary reversibility using directed evolution from a phosphotriesterase to an arylesterase, and back, and examine the underlying molecular basis. We find that wild-type phosphotriesterase function could be restored (>104-fold activity increase), but via an alternative set of mutations. The enzyme active site converged towards its original state, indicating evolutionary constraints imposed by catalytic requirements. We reveal that extensive epistasis prevents reversions and necessitates fixation of new mutations, leading to a functionally identical sequence. Many amino acid exchanges between the new and original enzyme are not tolerated, implying sequence incompatibility. Therefore, the evolution was phenotypically reversible but genotypically irreversible. Our study illustrates that the enzyme's adaptive landscape is highly rugged, and different functional sequences may constitute separate fitness peaks.

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Mechanistic and active-site studies on d(–)-mandelate dehydrogenase from Rhodotorula graminis

Biochemical Journal ◽

10.1042/bj2810211 ◽

1992 ◽

Vol 281 (1) ◽

pp. 211-218 ◽

Cited By ~ 6

Author(s):

D P Baker ◽

C Kleanthous ◽

J N Keen ◽

E Weinhold ◽

C A Fewson

Keyword(s):

Active Site ◽

Complete Sequence ◽

Histidine Residue ◽

Extended Version ◽

Order Process ◽

First Order ◽

Tryptic Digest ◽

Complete Inactivation ◽

Hydrolysis Of ◽

High Voltage Electrophoresis

D(–)-Mandelate dehydrogenase, the first enzyme of the mandelate pathway in the yeast Rhodotorula graminis, catalyses the NAD(+)-dependent oxidation of D(–)-mandelate to phenylglyoxylate. D(–)-2-(Bromoethanoyloxy)-2-phenylethanoic acid [‘D(–)-bromoacetylmandelic acid’], an analogue of the natural substrate, was synthesized as a probe for reactive and accessible nucleophilic groups within the active site of the enzyme. D(–)-Mandelate dehydrogenase was inactivated by D(–)-bromoacetylmandelate in a psuedo-first-order process. D(–)-Mandelate protected against inactivation, suggesting that the residue that reacts with the inhibitor is located at or near the active site. Complete inactivation of the enzyme resulted in the incorporation of approx. 1 mol of label/mol of enzyme subunit. D(–)-Mandelate dehydrogenase that had been inactivated with 14C-labelled D(–)-bromoacetylmandelate was digested with trypsin; there was substantial incorporation of 14C into two tryptic-digest peptides, and this was lowered in the presence of substrate. One of the tryptic peptides had the sequence Val-Xaa-Leu-Glu-Ile-Gly-Lys, with the residue at the second position being the site of radiolabel incorporation. The complete sequence of the second peptide was not determined, but it was probably an N-terminally extended version of the first peptide. High-voltage electrophoresis of the products of hydrolysis of modified protein showed that the major peak of radioactivity co-migrated with N tau-carboxymethylhistidine, indicating that a histidine residue at the active site of the enzyme is the most likely nucleophile with which D(–)-bromoacetylmandelate reacts. D(–)-Mandelate dehydrogenase was incubated with phenylglyoxylate and either (4S)-[4-3H]NADH or (4R)-[4-3H]NADH and then the resulting D(–)-mandelate and NAD+ were isolated. The enzyme transferred the pro-R-hydrogen atom from NADH during the reduction of phenylglyoxylate. The results are discussed with particular reference to the possibility that this enzyme evolved by the recruitment of a 2-hydroxy acid dehydrogenase from another metabolic pathway.

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Protein Kinase G Phosphorylates Mosquito-Borne Flavivirus NS5

Journal of Virology ◽

10.1128/jvi.00271-09 ◽

2009 ◽

Vol 83 (18) ◽

pp. 9195-9205 ◽

Cited By ~ 23

Author(s):

Dipankar Bhattacharya ◽

Mayuri ◽

S. M. Best ◽

R. Perera ◽

R. J. Kuhn ◽

...

Keyword(s):

Protein Kinase ◽

Yellow Fever Virus ◽

Nonstructural Protein ◽

Protein Kinase G ◽

Phosphorylation Sites ◽

Histidine Residue ◽

Phosphorylation Event ◽

Langat Virus ◽

And Function

ABSTRACT Serine/threonine phosphorylation of the nonstructural protein 5 (NS5) is a conserved feature of flaviviruses, but the kinase(s) responsible and function(s) remain unknown. Mass spectrometry was used to compare the phosphorylation sites of the NS5 proteins of yellow fever virus (YFV) and dengue virus (DENV), two flaviviruses transmitted by mosquitoes. Seven DENV phosphopeptides were identified, but only one conserved phosphoacceptor site (threonine 449 in DENV) was identified in both viruses. This site is predicted to be a protein kinase G (PKG) recognition site and is a strictly conserved serine/threonine phosphoacceptor site in mosquito-borne flaviviruses. In contrast, in tick-borne flaviviruses, this residue is typically a histidine. A DENV replicon engineered to have the tick-specific histidine residue at this position is replication defective. We show that DENV NS5 purified from Escherichia coli is a substrate for PKG in vitro and facilitates the autophosphorylation of PKG as seen with cellular substrates. Phosphorylation in vitro by PKG also occurs at threonine 449. Activators and inhibitors of PKG modulate DENV replication in cell culture but not replication of the tick-borne langat virus. Collectively, these data argue that PKG mediates a conserved serine/threonine phosphorylation event specifically for flaviviruses spread by mosquitoes.

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