scholarly journals The ABCs (and Ds) of Class I Terpene Cyclase (αC), Trans- Head-to-Tail (αHT) and Head-to-Head (αHH) Prenyltransferase Structures and Mechanisms of Action

2019 ◽  
Author(s):  
Chun-Chi Chen ◽  
Satish Malwal ◽  
Xu Han ◽  
Weidong Liu ◽  
Lixin Ma ◽  
...  
Keyword(s):  
Substrate Binding ◽  
Mechanisms Of Action ◽  
Farnesyl Diphosphate ◽  
Class I ◽  
General Interest ◽  
Santalum Album ◽  
Conserved Residues ◽  
Terpene Cyclase ◽  
Sequential Mechanism ◽  
Bond Network

We report the structures of the terpene cyclases <i>Santalum album</i> L. sesquisabinene synthases 1 and 2 and of santalene synthase, in apo forms, and with the sesquisabinene synthases, bound to either farnesyl diphosphate (FPP), farnesyl S-thiolo-diphosphate, FPP containing a POP bridging O-to-CCl<sub>2</sub> substitution, or to sabinene, leading to a sequential mechanism for substrate binding and catalysis. We trapped early pre-catalytic inactive open forms that show how ligands initially bind to the apo-proteins, then when the pocket closes, catalysis can proceed. We also show that there are strong structural similarities between the most highly conserved residues in class I cyclases and those in head-to-tail (aHT) trans-prenyl transferases—outside the well-known DDXXD-like and NSE/DTE-like domains. In the aHT prenyltransferases there is a highly conserved Thr>Gln>Asp>Tyr motif and in the cyclases, a similar Thr>Arg>Asp>Tyr domain, these residues forming very similar, extended H-bond networks (rmsd ~1.4 Å) that are involved in catalysis, leading to the proposal that there are 3 key domains in both the cyclases and the aHT prenyltransferases: The AC-domain that binds MgA and MgC; the B domain that binds MgB and leads to pocket closure, ionization, and condensation or cyclization; and the D-domain H-bond network, involved in H<sup>+</sup> elimination. In aHH prenyltransferases the overall folds and MgABC motifs are similar to those found in the cyclase and aHT proteins, but the full Thr>Arg/Gln>Asp>Tyr domain is absent and instead there are Tyr/Asp or Tyr/Glu residues that bind to MgC and are highly conserved. Overall, the results are of general interest since they show unexpected similarities between the enzymes that produce the most diverse molecules on Earth: aHT and aHH prenyltransferases, and terpenoid cyclases.

scholarly journals The ABCs (and Ds) of Class I Terpene Cyclase (αC), Trans- Head-to-Tail (αHT) and Head-to-Head (αHH) Prenyltransferase Structures and Mechanisms of Action

2019 ◽  
Author(s):  
Chun-Chi Chen ◽  
Satish Malwal ◽  
Xu Han ◽  
Weidong Liu ◽  
Lixin Ma ◽  
...  
Keyword(s):  
Substrate Binding ◽  
Mechanisms Of Action ◽  
Farnesyl Diphosphate ◽  
Class I ◽  
General Interest ◽  
Santalum Album ◽  
Conserved Residues ◽  
Terpene Cyclase ◽  
Sequential Mechanism ◽  
Bond Network

We report the structures of the terpene cyclases <i>Santalum album</i> L. sesquisabinene synthases 1 and 2 and of santalene synthase, in apo forms, and with the sesquisabinene synthases, bound to either farnesyl diphosphate (FPP), farnesyl S-thiolo-diphosphate, FPP containing a POP bridging O-to-CCl<sub>2</sub> substitution, or to sabinene, leading to a sequential mechanism for substrate binding and catalysis. We trapped early pre-catalytic inactive open forms that show how ligands initially bind to the apo-proteins, then when the pocket closes, catalysis can proceed. We also show that there are strong structural similarities between the most highly conserved residues in class I cyclases and those in head-to-tail (aHT) trans-prenyl transferases—outside the well-known DDXXD-like and NSE/DTE-like domains. In the aHT prenyltransferases there is a highly conserved Thr>Gln>Asp>Tyr motif and in the cyclases, a similar Thr>Arg>Asp>Tyr domain, these residues forming very similar, extended H-bond networks (rmsd ~1.4 Å) that are involved in catalysis, leading to the proposal that there are 3 key domains in both the cyclases and the aHT prenyltransferases: The AC-domain that binds MgA and MgC; the B domain that binds MgB and leads to pocket closure, ionization, and condensation or cyclization; and the D-domain H-bond network, involved in H<sup>+</sup> elimination. In aHH prenyltransferases the overall folds and MgABC motifs are similar to those found in the cyclase and aHT proteins, but the full Thr>Arg/Gln>Asp>Tyr domain is absent and instead there are Tyr/Asp or Tyr/Glu residues that bind to MgC and are highly conserved. Overall, the results are of general interest since they show unexpected similarities between the enzymes that produce the most diverse molecules on Earth: aHT and aHH prenyltransferases, and terpenoid cyclases.

Structure and mechanism of the γ-glutamyl-γ-aminobutyrate hydrolase SpuA from Pseudomonas aeruginosa

2021 ◽  
Vol 77 (10) ◽  
pp. 1305-1316
Author(s):  
Yujing Chen ◽  
Haizhu Jia ◽  
Jianyu Zhang ◽  
Yakun Liang ◽  
Ruihua Liu ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Catalytic Mechanism ◽  
Substrate Binding ◽  
Binding Pocket ◽  
Class I ◽  
Polyamine Metabolism ◽  
Binding Assays ◽  
Starting Point ◽  
Family Activity ◽  
Living Organisms

Polyamines are important regulators in all living organisms and are implicated in essential biological processes including cell growth, differentiation and apoptosis. Pseudomonas aeruginosa possesses an spuABCDEFGHI gene cluster that is involved in the metabolism and uptake of two polyamines: spermidine and putrescine. In the proposed γ-glutamylation–putrescine metabolism pathway, SpuA hydrolyzes γ-glutamyl-γ-aminobutyrate (γ-Glu-GABA) to glutamate and γ-aminobutyric acid (GABA). In this study, crystal structures of P. aeruginosa SpuA are reported, confirming it to be a member of the class I glutamine amidotransferase (GAT) family. Activity and substrate-binding assays confirm that SpuA exhibits a preference for γ-Glu-GABA as a substrate. Structures of an inactive H221N mutant were determined with bound glutamate thioester intermediate or glutamate product, thus delineating the active site and substrate-binding pocket and elucidating the catalytic mechanism. The crystal structure of another bacterial member of the class I GAT family from Mycolicibacterium smegmatis (MsGATase) in complex with glutamine was determined for comparison and reveals a binding site for glutamine. Activity assays confirm that MsGATase has activity for glutamine as a substrate but not for γ-Glu-GABA. The work reported here provides a starting point for further investigation of polyamine metabolism in P. aeruginosa.

scholarly journals Functional role of residues involved in substrate binding of human 4-hydroxyphenylpyruvate dioxygenase

2021 ◽  
Author(s):  
Chih-Wei Huang ◽  
Chi-Ching Hwang ◽  
Yung-Lung Chang ◽  
Jen-Tzu Liu ◽  
Sheng-Peng Wu ◽  
...  
Keyword(s):  
Binding Affinity ◽  
Functional Role ◽  
Substrate Binding ◽  
Critical Role ◽  
Intermediate Formation ◽  
Hydroxyl Group ◽  
Double Mutation ◽  
Closed Conformation ◽  
Bond Network

4-Hydroxylphenylpyruvate dioxygenase (HPPD) catalyzes the conversion of 4-hydroxylphenylpyruvate (HPP) to homogentisate, the important step for tyrosine catabolism. Comparison of the structure of human HPPD with the substrate-bound structure of A. thaliana HPPD revealed notably different orientations of the C-terminal helix. This helix performed as a closed conformation in human enzyme. Simulation revealed a different substrate-binding mode in which the carboxyl group of HPP interacted by a H-bond network formed by Gln334, Glu349 (the metal-binding ligand), and Asn363 (in the C-terminal helix). The 4-hydroxyl group of HPP interacted with Gln251 and Gln265. The relative activity and substrate-binding affinity were preserved for the Q334A mutant, implying the alternative role of Asn363 for HPP binding and catalysis. The reduction in kcat/Km of the Asn363 mutants confirmed the critical role in catalysis. Compared to the N363A mutant, the dramatic reduction in the Kd and thermal stability of the N363D mutant implies the side-chain effect in the hinge region rotation of the C-terminal helix. The activity and binding affinity were not recovered by double mutation; however, the 4-hydroxyphenylacetate intermediate formation by the uncoupled reaction of Q334N/N363Q and Q334A/N363D mutants indicated the importance of the H-bond network in the electrophilic reaction. These results highlight the functional role of the H-bond network in a closed conformation of the C-terminal helix to stabilize the bound substrate. The extremely low activity and reduction in Q251E’s Kd suggest that interaction coupled with the H-bond network is crucial to locate the substrate for nucleophilic reaction.

scholarly journals Insights into substrate binding and catalysis in bacterial type I dehydroquinase

2014 ◽  
Vol 462 (3) ◽  
pp. 415-424 ◽  
Author(s):  
María Maneiro ◽  
Antonio Peón ◽  
Emilio Lence ◽  
José M. Otero ◽  
Mark J. Van Raaij ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Substrate Binding ◽  
Detailed Knowledge ◽  
Type I ◽  
Conserved Residues ◽  
Binding Process ◽  
Bacterial Type

The crystal structure of S. typhi type I dehydroquinase in complex with (2R)-3-methyl-3-dehydroquinic acid is described. A previously unknown key role of several conserved residues and a detailed knowledge of the substrate binding process is detailed.

scholarly journals Elucidation of the roles of conserved residues in the biosynthesis of the lasso peptide paeninodin

2018 ◽  
Vol 54 (65) ◽  
pp. 9007-9010 ◽  
Author(s):  
Julian D. Hegemann ◽  
Christopher J. Schwalen ◽  
Douglas A. Mitchell ◽  
Wilfred A. van der Donk
Keyword(s):  
Substrate Binding ◽  
Proteolytic Processing ◽  
Binding Assays ◽  
Conserved Residues ◽  
Lasso Peptide ◽  
Precursor Peptide

Substrate binding assays, in vitro proteolytic processing assays, and heterologous lasso peptide production were used to investigate the roles of conserved precursor peptide residues during paeninodin maturation.

Biochemical and structural investigation of sulfoacetaldehyde reductase from Klebsiella oxytoca

2019 ◽  
Vol 476 (4) ◽  
pp. 733-746 ◽  
Author(s):  
Yan Zhou ◽  
Yifeng Wei ◽  
Lianyun Lin ◽  
Tong Xu ◽  
Ee Lui Ang ◽  
...  
Keyword(s):  
Nitrogen Assimilation ◽  
Structural Investigation ◽  
Bioinformatics Analysis ◽  
Substrate Binding ◽  
Klebsiella Oxytoca ◽  
Waste Product ◽  
Sulfonate Group ◽  
Metabolic Functions ◽  
Bond Network ◽  
Β Sheet

Abstract Sulfoacetaldehyde reductase (IsfD) is a member of the short-chain dehydrogenase/reductase (SDR) family, involved in nitrogen assimilation from aminoethylsulfonate (taurine) in certain environmental and human commensal bacteria. IsfD catalyzes the reversible NADPH-dependent reduction of sulfoacetaldehyde, which is generated by transamination of taurine, forming hydroxyethylsulfonate (isethionate) as a waste product. In the present study, the crystal structure of Klebsiella oxytoca IsfD in a ternary complex with NADPH and isethionate was solved at 2.8 Å, revealing residues important for substrate binding. IsfD forms a homotetramer in both crystal and solution states, with the C-terminal tail of each subunit interacting with the C-terminal tail of the diagonally opposite subunit, forming an antiparallel β sheet that constitutes part of the substrate-binding site. The sulfonate group of isethionate is stabilized by a hydrogen bond network formed by the residues Y148, R195, Q244 and a water molecule. In addition, F249 from the diagonal subunit restrains the conformation of Y148 to further stabilize the orientation of the sulfonate group. Mutation of any of these four residues into alanine resulted in a complete loss of catalytic activity for isethionate oxidation. Biochemical investigations of the substrate scope of IsfD, and bioinformatics analysis of IsfD homologs, suggest that IsfD is related to the promiscuous 3-hydroxyacid dehydrogenases with diverse metabolic functions.

Crystal structure of the catalytic core of Saccharomyces cerevesiae histone demethylase Rph1: insights into the substrate specificity and catalytic mechanism

2010 ◽  
Vol 433 (2) ◽  
pp. 295-302 ◽  
Author(s):  
Yuanyuan Chang ◽  
Jian Wu ◽  
Xia-Jing Tong ◽  
Jin-Qiu Zhou ◽  
Jianping Ding
Keyword(s):  
Substrate Specificity ◽  
Substrate Binding ◽  
Histone Demethylase ◽  
Structural Motif ◽  
Catalytic Core ◽  
Conserved Residues ◽  
Demethylase Activity ◽  
Jmjc Domain ◽  
Saccharomyces Cerevesiae ◽  
Binding Cleft

Saccharomyces cerevesiae Rph1 is a histone demethylase orthologous to human JMJD2A (Jumonji-domain-containing protein 2A) that can specifically demethylate tri- and di-methylated Lys36 of histone H3. c-Rph1, the catalytic core of Rph1, is responsible for the demethylase activity, which is essential for the transcription elongation of some actively transcribed genes. In the present work, we report the crystal structures of c-Rph1 in apo form and in complex with Ni2+ and α-KG [2-oxoglutarate (α-ketoglutarate)]. The structure of c-Rph1 is composed of a JmjN (Jumonji N) domain, a long β-hairpin, a mixed structural motif and a JmjC domain. The α-KG cofactor forms hydrogen-bonding interactions with the side chains of conserved residues, and the Ni2+ ion at the active site is chelated by conserved residues and the cofactor. Structural comparison of Rph1 with JMJD2A indicates that the substrate-binding cleft of Rph1 is formed with several structural elements of the JmjC domain, the long β-hairpin and the mixed structural motif; and the methylated Lys36 of H3 is recognized by several conserved residues of the JmjC domain. In vitro biochemical results show that mutations of the key residues at the catalytic centre and in the substrate-binding cleft abolish the demethylase activity. In vivo growth phenotype analyses also demonstrate that these residues are essential for its functional roles in transcription elongation. Taken together, our structural and biological data provide insights into the molecular basis of the histone demethylase activity and the substrate specificity of Rph1.

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