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.

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 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.

Recognition of the amyloid precursor protein by human γ-secretase

Science ◽  
2019 ◽  
Vol 363 (6428) ◽  
pp. eaaw0930 ◽  
Author(s):  
Rui Zhou ◽  
Guanghui Yang ◽  
Xuefei Guo ◽  
Qiang Zhou ◽  
Jianlin Lei ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Amyloid Precursor Protein ◽  
Atomic Structure ◽  
Substrate Binding ◽  
Peptide Bond ◽  
Catalytic Subunit ◽  
Precursor Protein ◽  
Β Sheet ◽  
Specific Inhibitors

Cleavage of amyloid precursor protein (APP) by the intramembrane protease γ-secretase is linked to Alzheimer’s disease (AD). We report an atomic structure of human γ-secretase in complex with a transmembrane (TM) APP fragment at 2.6-angstrom resolution. The TM helix of APP closely interacts with five surrounding TMs of PS1 (the catalytic subunit of γ-secretase). A hybrid β sheet, which is formed by a β strand from APP and two β strands from PS1, guides γ-secretase to the scissile peptide bond of APP between its TM and β strand. Residues at the interface between PS1 and APP are heavily targeted by recurring mutations from AD patients. This structure, together with that of γ-secretase bound to Notch, reveal contrasting features of substrate binding, which may be applied toward the design of substrate-specific inhibitors.

New structural insights into bacterial sulfoacetaldehyde and taurine metabolism

2020 ◽  
Vol 477 (8) ◽  
pp. 1367-1371
Author(s):  
Thore Rohwerder
Keyword(s):  
Nitrogen Assimilation ◽  
Klebsiella Oxytoca ◽  
Structural Elucidation ◽  
Degradation Pathways ◽  
Biochemical Journal ◽  
Structural Insights ◽  
Reversible Reduction

In last year's issue 4 of Biochemical Journal, Zhou et al. (Biochem J. 476, 733–746) kinetically and structurally characterized the reductase IsfD from Klebsiella oxytoca that catalyzes the reversible reduction in sulfoacetaldehyde to the corresponding alcohol isethionate. This is a key step in detoxification of the carbonyl intermediate formed in bacterial nitrogen assimilation from the α-aminoalkanesulfonic acid taurine. In 2019, the work on sulfoacetaldehyde reductase IsfD was the exciting start to a quite remarkable series of articles dealing with structural elucidation of proteins involved in taurine metabolism as well as the discovery of novel degradation pathways in bacteria.

A cluster of diagnostic Hsp68 amino acid sites that are identified in Drosophila from the melanogaster species group are concentrated around β-sheet residues involved with substrate binding

Genome ◽  
2005 ◽  
Vol 48 (2) ◽  
pp. 226-233 ◽  
Author(s):  
Mark Kellett ◽  
Stephen W McKechnie
Keyword(s):  
Amino Acid ◽  
Substrate Binding ◽  
Acid Sites ◽  
Amino Acid Sequences ◽  
Length Variation ◽  
Functional Sites ◽  
Structure Conservation ◽  
Α Helix ◽  
Montium Subgroup ◽  
Β Sheet

The coding region of the hsp68 gene has been amplified, cloned, and sequenced from 10 Drosophila species, 5 from the melanogaster subgroup and 5 from the montium subgroup. When the predicted amino acid sequences are compared with available Hsp70 sequences, patterns of conservation suggest that the C-terminal region should be subdivided according to predominant secondary structure. Conservation levels between Hsp68 and Hsp70 proteins were high in the N-terminal ATPase and adjacent β-sheet domains, medium in the α-helix domain, and low in the C-terminal mobile domain (78%, 72%, 41%, and 21% identity, respectively). A number of amino acid sites were found to be "diagnostic" for Hsp68 (28 of ~635 residues). A few of these occur in the ATPase domain (385 residues) but most (75%) are concentrated in the β-sheet and α-helix domains (34% of the protein) with none in the short mobile domain. Five of the diagnostic sites in the β-sheet domain are clustered around, but not coincident with, functional sites known to be involved in substrate binding. Nearly all of the Hsp70 family length variation occurs in the mobile domain. Within montium subgroup species, 2 nearly identical hsp68 PCR products that differed in length are either different alleles or products of an ancestral hsp68 duplication.Key words: Hsp70, Hsp68, diagnostic sites, Drosophila melanogaster, montium subgroup.

Chemical, Spectroscopic and Structural Investigation of the Substrate-Binding Site in Ascorbate Peroxidase

1997 ◽  
Vol 248 (2) ◽  
pp. 347-354 ◽  
Author(s):  
Adrian P. Hill ◽  
Sandeep Modi ◽  
Michael J. Sutcliffe ◽  
Daniel D. Turner ◽  
David J. Gilfoyle ◽  
...  
Keyword(s):  
Binding Site ◽  
Ascorbate Peroxidase ◽  
Structural Investigation ◽  
Substrate Binding ◽  
Substrate Binding Site

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.

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