scholarly journals Campylobacter jejuni PglH Is a Single Active Site Processive Polymerase that Utilizes Product Inhibition to Limit Sequential Glycosyl Transfer Reactions†

Biochemistry ◽  
2009 ◽  
Vol 48 (12) ◽  
pp. 2807-2816 ◽  
Author(s):  
Jerry M. Troutman ◽  
Barbara Imperiali
Keyword(s):  
Campylobacter Jejuni ◽  
Active Site ◽  
Product Inhibition ◽  
Transfer Reactions

Sirtuin-dependent reversible lysine acetylation controls the activity of acetyl-Coenzyme A synthetase in Campylobacter jejuni

2021 ◽  
Author(s):  
Victoria L. Jeter ◽  
Jorge C. Escalante-Semerena
Keyword(s):  
Campylobacter Jejuni ◽  
Posttranslational Modifications ◽  
Active Site ◽  
Protein Function ◽  
Metabolic Enzyme ◽  
Lysine Acetylation ◽  
Class Iii ◽  
Acetyl Coa

Posttranslational modifications are mechanisms for rapid control of protein function used by cells from all domains of life. Acetylation of the epsilon amino group ( N ε ) of an active-site lysine of the AMP-forming acetyl-CoA synthetase (Acs) enzyme is the paradigm for the posttranslational control of the activity of metabolic enzymes. In bacteria, the alluded active-site lysine of Acs enzymes can be modified by a number of different GCN5-type N -acetyltransferases (GNATs). Acs activity is lost as a result of acetylation, and restored by deacetylation. Using a heterologous host, we show that Campylobacter jejuni NCTC11168 synthesizes enzymes that control Acs function by reversible lysine acetylation (RLA). This work validates the function of gene products encoded by the cj1537c , cj1715, and cj1050c loci, namely the AMP-forming acetate:CoA ligase ( Cj Acs), a type IV GCN5-type lysine acetyltransferase (GNAT, hereafter Cj LatA), and a NAD + -dependent (class III) sirtuin deacylase ( Cj CobB), respectively. To our knowledge, these are the first in vivo and in vitro data on C. jejuni enzymes that control the activity of Cj Acs. IMPORTANCE This work is important because it provides the experimental evidence needed to support the assignment of function to three key enzymes, two of which control the reversible posttranslational modification of an active-site lysyl residue of the central metabolic enzyme acetyl-CoA synthetase ( Cj Acs). We can now generate Campylobacter jejuni mutant strains defective in these functions, so we can establish the conditions in which this mode of regulation of Cj Acs is triggered in this bacterium. Such knowledge may provide new therapeutic strategies for the control of this pathogen.

Functional analysis of box I mutations in yeast site-specific recombinases Flp and R: pairwise complementation with recombinase variants lacking the active-site tyrosine

1992 ◽  
Vol 12 (9) ◽  
pp. 3757-3765
Author(s):  
J W Chen ◽  
B R Evans ◽  
S H Yang ◽  
H Araki ◽  
Y Oshima ◽  
...  
Keyword(s):  
Active Site ◽  
Dna Cleavage ◽  
Substrate Binding ◽  
Point Mutations ◽  
Transfer Reactions ◽  
Strand Transfer ◽  
Site Specific ◽  
Arginine Residues ◽  
Active Site Tyrosine ◽  
Half Site

The site-specific recombinases Flp and R from Saccharomyces cerevisiae and Zygosaccharomyces rouxii, respectively, are related proteins that belong to the yeast family of site-specific recombinases. They share approximately 30% amino acid matches and exhibit a common reaction mechanism that appears to be conserved within the larger integrase family of site-specific recombinases. Two regions of the proteins, designated box I and box II, also harbor a significantly high degree of homology at the nucleotide sequence level. We have analyzed the properties of Flp and R variants carrying point mutations within the box I segment in substrate-binding, DNA cleavage, and full-site and half-site strand transfer reactions. All mutations abolish or seriously diminish recombinase function either at the substrate-binding step or at the catalytic steps of strand cleavage or strand transfer. Of particular interest are mutations of Arg-191 of Flp and R, residues which correspond to one of the two invariant arginine residues of the integrase family. These variant proteins bind substrate with affinities comparable to those of the corresponding wild-type recombinases. Among the binding-competent variants, only Flp(R191K) is capable of efficient substrate cleavage in a full recombination target. However, this protein does not cleave a half recombination site and fails to complete strand exchange in a full site. Strikingly, the Arg-191 mutants of Flp and R can be rescued in half-site strand transfer reactions by a second point mutant of the corresponding recombinase that lacks its active-site tyrosine (Tyr-343). Similarly, Flp and R variants of Cys-189 and Flp variants at Asp-194 and Asp-199 can also be complemented by the corresponding Tyr-343-to-phenylalanine recombinase mutant.

scholarly journals Streptomyces K15 active-site serine dd-transpeptidase: specificity profile for peptide, thiol ester and ester carbonyl donors and pathways of the transfer reactions

1995 ◽  
Vol 307 (2) ◽  
pp. 335-339 ◽  
Author(s):  
J Grandchamps ◽  
M Nguyen-Distèche ◽  
C Damblon ◽  
J M Frère ◽  
J M Ghuysen
Keyword(s):  
Active Site ◽  
Kinetic Data ◽  
Transfer Reactions ◽  
Ester Bond ◽  
Penicillin Binding Protein ◽  
Thiol Ester ◽  
Ester Carbonyl ◽  
Simple Alternative ◽  
Scissile Bond ◽  
Enzyme Intermediate

The Streptomyces K15 transferase is a penicillin-binding protein presumed to be involved in bacterial wall peptidoglycan crosslinking. It catalyses cleavage of the peptide, thiol ester or ester bond of carbonyl donors Z-R1-CONH-CHR2-COX-CHR3-COO- (where X is NH, S or O) and transfers the electrophilic group Z-R1-CONH-CHR2-CO to amino acceptors via an acyl-enzyme intermediate. Kinetic data suggest that the amino acceptor behaves as a simple alternative nucleophile at the level of the acyl-enzyme in the case of thiol ester and ester donors, and that it binds to the enzyme.carbonyl donor Michaelis complex and influences the rate of enzyme acylation by the carbonyl donor in the case of amide donors. Depending on the nature of the scissile bond, the enzyme has different requirements for substituents at positions R1, R2 and R3.

scholarly journals The Molybdenum Active Site of Formate Dehydrogenase Is Capable of Catalyzing C–H Bond Cleavage and Oxygen Atom Transfer Reactions

Biochemistry ◽  
2016 ◽  
Vol 55 (16) ◽  
pp. 2381-2389 ◽  
Author(s):  
Tobias Hartmann ◽  
Peer Schrapers ◽  
Tillmann Utesch ◽  
Manfred Nimtz ◽  
Yvonne Rippers ◽  
...  
Keyword(s):  
Oxygen Atom ◽  
Active Site ◽  
Formate Dehydrogenase ◽  
Bond Cleavage ◽  
Transfer Reactions ◽  
Atom Transfer ◽  
Oxygen Atom Transfer ◽  
Oxygen Atom Transfer Reactions ◽  
Atom Transfer Reactions

Crystal structures of pyrrolidone-carboxylate peptidase I from Deinococcus radiodurans reveal the mechanism of L-pyroglutamate recognition

2019 ◽  
Vol 75 (3) ◽  
pp. 308-316
Author(s):  
Richa Agrawal ◽  
Rahul Singh ◽  
Ashwani Kumar ◽  
Amit Kumar ◽  
Ravindra D. Makde
Keyword(s):  
Crystal Structures ◽  
Active Site ◽  
Deinococcus Radiodurans ◽  
Product Inhibition ◽  
Recognition Mechanism ◽  
Flexible Loop ◽  
Tetrameric Structure ◽  
Functional Regulation ◽  
Polar Interactions ◽  
Unusual Amino Acid

Pyrrolidone-carboxylate peptidase (PCP) catalyzes the removal of an unusual amino acid, L-pyroglutamate (pG), from the N-termini of peptides and proteins. It has implications in the functional regulation of different peptides in both prokaryotes and eukaryotes. However, the pG-recognition mechanism of the PCP enzyme remains largely unknown. Here, crystal structures of PCP I from Deinococcus radiodurans (PCPdr) are reported in pG-free and pG-bound forms at resolutions of 1.73 and 1.55 Å, respectively. Four protomers in PCPdr form a tetrameric structure. The residues responsible for recognizing the pG residue are mostly contributed by a flexible loop (loop A) that is present near the active site. These residues are conserved in all known PCPs I, including those from mammals. Phe9 and Phe12 of loop A form stacking interactions with the pyrrolidone ring of pG, while Asn18 forms a hydrogen bond to OE of pG. The main chain of a nonconserved residue, Leu71, forms two hydrogen bonds to NH and OE of pG. Thus, pG is recognized in the S1 substrate subsite of the enzyme by both van der Waals and polar interactions, which provide specificity for the pG residue of the peptide. In contrast to previously reported PCP I structures, the PCPdr tetramer is in a closed conformation with an inaccessible active site. The structures show that the active site can be accessed by the substrates via disordering of loop A. This disordering could also prevent product inhibition by releasing the bound pG product from the S1 subsite, thus allowing the enzyme to engage a fresh substrate.

scholarly journals The nature of chemical innovation: new enzymes by evolution

2015 ◽  
Vol 48 (4) ◽  
pp. 404-410 ◽  
Author(s):  
Frances H. Arnold
Keyword(s):  
Cytochrome P450 ◽  
Active Site ◽  
Reactive Intermediates ◽  
Transfer Reactions ◽  
Amino Acid Substitutions ◽  
Chemical Catalysis ◽  
Nitrene Transfer ◽  
Enzyme Systems ◽  
Natural Enzyme ◽  
And Function

AbstractI describe how we direct the evolution of non-natural enzyme activities, using chemical intuition and information on structure and mechanism to guide us to the most promising reaction/enzyme systems. With synthetic reagents to generate new reactive intermediates and just a few amino acid substitutions to tune the active site, a cytochrome P450 can catalyze a variety of carbene and nitrene transfer reactions. The cyclopropanation, N–H insertion, C–H amination, sulfimidation, and aziridination reactions now demonstrated are all well known in chemical catalysis but have no counterparts in nature. The new enzymes are fully genetically encoded, assemble and function inside of cells, and can be optimized for different substrates, activities, and selectivities. We are learning how to use nature's innovation mechanisms to marry some of the synthetic chemists’ favorite transformations with the exquisite selectivity and tunability of enzymes.

scholarly journals Structural studies reveal flexible roof of active site responsible for ω-transaminase CrmG overcoming by-product inhibition

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Jinxin Xu ◽  
Xiaowen Tang ◽  
Yiguang Zhu ◽  
Zhijun Yu ◽  
Kai Su ◽  
...  
Keyword(s):  
Pharmaceutical Industry ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Product Inhibition ◽  
Strong Interactions ◽  
Structural Studies ◽  
Biochemical Studies ◽  
Fundamental Challenge ◽  
Amine Compounds

AbstractAmine compounds biosynthesis using ω-transaminases has received considerable attention in the pharmaceutical industry. However, the application of ω-transaminases was hampered by the fundamental challenge of severe by-product inhibition. Here, we report that ω-transaminase CrmG from Actinoalloteichus cyanogriseus WH1-2216-6 is insensitive to inhibition from by-product α-ketoglutarate or pyruvate. Combined with structural and QM/MM studies, we establish the detailed catalytic mechanism for CrmG. Our structural and biochemical studies reveal that the roof of the active site in PMP-bound CrmG is flexible, which will facilitate the PMP or by-product to dissociate from PMP-bound CrmG. Our results also show that amino acceptor caerulomycin M (CRM M), but not α-ketoglutarate or pyruvate, can form strong interactions with the roof of the active site in PMP-bound CrmG. Based on our results, we propose that the flexible roof of the active site in PMP-bound CrmG may facilitate CrmG to overcome inhibition from the by-product.

scholarly journals Structural basis for glucose tolerance in GH1 β-glucosidases

2014 ◽  
Vol 70 (6) ◽  
pp. 1631-1639 ◽  
Author(s):  
Priscila Oliveira de Giuseppe ◽  
Tatiana de Arruda Campos Brasil Souza ◽  
Flavio Henrique Moreira Souza ◽  
Leticia Maria Zanphorlin ◽  
Carla Botelho Machado ◽  
...  
Keyword(s):  
Glucose Tolerance ◽  
Active Site ◽  
Plant Cell Wall ◽  
Plant Biomass ◽  
Product Inhibition ◽  
Structural Basis ◽  
Clear Correlation ◽  
Biomass Saccharification ◽  
Glucose Tolerant ◽  
Shed Light

Product inhibition of β-glucosidases (BGs) by glucose is considered to be a limiting step in enzymatic technologies for plant-biomass saccharification. Remarkably, some β-glucosidases belonging to the GH1 family exhibit unusual properties, being tolerant to, or even stimulated by, high glucose concentrations. However, the structural basis for the glucose tolerance and stimulation of BGs is still elusive. To address this issue, the first crystal structure of a fungal β-glucosidase stimulated by glucose was solved in native and glucose-complexed forms, revealing that the shape and electrostatic properties of the entrance to the active site, including the +2 subsite, determine glucose tolerance. The aromatic Trp168 and the aliphatic Leu173 are conserved in glucose-tolerant GH1 enzymes and contribute to relieving enzyme inhibition by imposing constraints at the +2 subsite that limit the access of glucose to the −1 subsite. The GH1 family β-glucosidases are tenfold to 1000-fold more glucose tolerant than GH3 BGs, and comparative structural analysis shows a clear correlation between active-site accessibility and glucose tolerance. The active site of GH1 BGs is located in a deep and narrow cavity, which is in contrast to the shallow pocket in the GH3 family BGs. These findings shed light on the molecular basis for glucose tolerance and indicate that GH1 BGs are more suitable than GH3 BGs for biotechnological applications involving plant cell-wall saccharification.

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