Structure, substrate specificity, and catalytic mechanism of human D-2-HGDH and insights into pathogenicity of disease-associated mutations

AbstractD-2-hydroxyglutarate dehydrogenase (D-2-HGDH) catalyzes the oxidation of D-2-hydroxyglutarate (D-2-HG) into 2-oxoglutarate, and genetic D-2-HGDH deficiency leads to abnormal accumulation of D-2-HG which causes type I D-2-hydroxyglutaric aciduria and is associated with diffuse large B-cell lymphoma. This work reports the crystal structures of human D-2-HGDH in apo form and in complexes with D-2-HG, D-malate, D-lactate, L-2-HG, and 2-oxoglutarate, respectively. D-2-HGDH comprises a FAD-binding domain, a substrate-binding domain, and a small C-terminal domain. The active site is located at the interface of the FAD-binding domain and the substrate-binding domain. The functional roles of the key residues involved in the substrate binding and catalytic reaction and the mutations identified in D-2-HGDH-deficient diseases are analyzed by biochemical studies. The structural and biochemical data together reveal the molecular mechanism of the substrate specificity and catalytic reaction of D-2-HGDH and provide insights into the pathogenicity of the disease-associated mutations.

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Structures of trans-2-enoyl-CoA reductases from Clostridium acetobutylicum and Treponema denticola: insights into the substrate specificity and the catalytic mechanism

Biochemical Journal ◽

10.1042/bj20120871 ◽

2012 ◽

Vol 449 (1) ◽

pp. 79-89 ◽

Cited By ~ 6

Author(s):

Kuan Hu ◽

Meng Zhao ◽

Tianlong Zhang ◽

Manwu Zha ◽

Chen Zhong ◽

...

Keyword(s):

Substrate Specificity ◽

Clostridium Acetobutylicum ◽

Catalytic Mechanism ◽

Acyl Carrier Protein ◽

Substrate Binding ◽

Binding Domain ◽

Treponema Denticola ◽

Sequence Comparisons ◽

Cofactor Binding ◽

Catalytic Active Site

TERs (trans-2-enoyl-CoA reductases; EC 1.3.1.44), which specifically catalyse the reduction of crotonyl-CoA to butyryl-CoA using NADH as cofactor, have recently been applied in the design of robust synthetic pathways to produce butan-1-ol as a biofuel. We report in the present paper the characterization of a CaTER (a TER homologue in Clostridium acetobutylicum), the structures of CaTER in apo form and in complexes with NADH and NAD+, and the structure of TdTER (Treponema denticola TER) in complex with NAD+. Structural and sequence comparisons show that CaTER and TdTER share approximately 45% overall sequence identity and high structural similarities with the FabV class enoyl-acyl carrier protein reductases in the bacterial fatty acid synthesis pathway, suggesting that both types of enzymes belong to the same family. CaTER and TdTER function as monomers and consist of a cofactor-binding domain and a substrate-binding domain with the catalytic active site located at the interface of the two domains. Structural analyses of CaTER together with mutagenesis and biochemical data indicate that the conserved Glu75 determines the cofactor specificity, and the conserved Tyr225, Tyr235 and Lys244 play critical roles in catalysis. Upon cofactor binding, the substrate-binding loop changes from an open conformation to a closed conformation, narrowing a hydrophobic channel to the catalytic site. A modelling study shows that the hydrophobic channel is optimal in both width and length for the binding of crotonyl-CoA. These results provide molecular bases for the high substrate specificity and the catalytic mechanism of TERs.

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Structural and mechanistic basis of the high catalytic activity of monooxygenase Tet(X4) on tigecycline

BMC Biology ◽

10.1186/s12915-021-01199-7 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Qipeng Cheng ◽

Yanchu Cheung ◽

Chenyu Liu ◽

Qingjie Xiao ◽

Bo Sun ◽

...

Keyword(s):

Rational Design ◽

Substrate Binding ◽

Catalytic Efficiency ◽

Binding Domain ◽

Multiple Drug ◽

High Catalytic Activity ◽

Multiple Drug Resistant ◽

Mechanistic Basis ◽

Substrate Binding Domain ◽

Fad Binding

Abstract Background Tigecycline is a tetracycline derivative that constitutes one of the last-resort antibiotics used clinically to treat infections caused by both multiple drug-resistant (MDR) Gram-negative and Gram-positive bacteria. Resistance to this drug is often caused by chromosome-encoding mechanisms including over-expression of efflux pumps and ribosome protection. However, a number of variants of the flavin adenine dinucleotide (FAD)-dependent monooxygenase TetX, such as Tet(X4), emerged in recent years as conferring resistance to tigecycline in strains of Enterobacteriaceae, Acinetobacter sp., Pseudomonas sp., and Empedobacter sp. To date, mechanistic details underlying the improvement of catalytic activities of new TetX enzymes are not available. Results In this study, we found that Tet(X4) exhibited higher affinity and catalytic efficiency toward tigecycline when compared to Tet(X2), resulting in the expression of phenotypic tigecycline resistance in E. coli strains bearing the tet(X4) gene. Comparison between the structures of Tet(X4) and Tet(X4)-tigecycline complex and those of Tet(X2) showed that they shared an identical FAD-binding site and that the FAD and tigecycline adopted similar conformation in the catalytic pocket. Although the amino acid changes in Tet(X4) are not pivotal residues for FAD binding and substrate recognition, such substitutions caused the refolding of several alpha helixes and beta sheets in the secondary structure of the substrate-binding domain of Tet(X4), resulting in the formation of a larger number of loops in the structure. These changes in turn render the substrate-binding domain of Tet(X4) more flexible and efficient in capturing substrate molecules, thereby improving catalytic efficiency. Conclusions Our works provide a better understanding of the molecular recognition of tigecycline by the TetX enzymes; these findings can help guide the rational design of the next-generation tetracycline antibiotics that can resist inactivation of the TetX variants.

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The Peptide-Substrate-binding Domain of Collagen Prolyl 4-Hydroxylases Is a Tetratricopeptide Repeat Domain with Functional Aromatic Residues

Journal of Biological Chemistry ◽

10.1074/jbc.m410007200 ◽

2004 ◽

Vol 279 (50) ◽

pp. 52255-52261 ◽

Cited By ~ 24

Author(s):

Mira Pekkala ◽

Reija Hieta ◽

Ulrich Bergmann ◽

Kari I. Kivirikko ◽

Rik K. Wierenga ◽

...

Keyword(s):

Substrate Binding ◽

Binding Domain ◽

Tetratricopeptide Repeat ◽

Side Chains ◽

Type I ◽

Type Ii ◽

Peptide Substrate ◽

Deep Groove ◽

Peptide Substrate Binding ◽

Substrate Binding Domain

Collagen prolyl 4-hydroxylases catalyze the formation of 4-hydroxyproline in -X-Pro-Gly-sequences and have an essential role in collagen synthesis. The vertebrate enzymes are α2β2tetramers in which the catalytic α-subunits contain separate peptide-substrate-binding and catalytic domains. We report on the crystal structure of the peptide-substrate-binding domain of the human type I enzyme refined at 2.3 Å resolution. It was found to belong to a family of tetratricopeptide repeat domains that are involved in many protein-protein interactions and consist of five α-helices forming two tetratricopeptide repeat motifs plus the solvating helix. A prominent feature of its concave surface is a deep groove lined by tyrosines, a putative binding site for proline-rich Tripeptides. Solvent-exposed side chains of three of the tyrosines have a repeat distance similar to that of a poly-l-proline type II helix. The aromatic surface ends at one of the tyrosines, where the groove curves almost 90° away from the linear arrangement of the three tyrosine side chains, possibly inducing a bent conformation in the bound peptide. This finding is consistent with previous suggestions by others that a minimal structural requirement for proline 4-hydroxylation may be a sequence in the poly-l-proline type II conformation followed by a β-turn in the Pro-Gly segment. Site-directed mutagenesis indicated that none of the tyrosines was critical for tetramer assembly, whereas most of them were critical for the binding of a peptide substrate and inhibitor both to the domain and the α2β2enzyme tetramer.

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Plant cyclopropylsterol-cycloisomerase: key amino acids affecting activity and substrate specificity

Biochemical Journal ◽

10.1042/bj20131239 ◽

2014 ◽

Vol 459 (2) ◽

pp. 289-299 ◽

Cited By ~ 4

Author(s):

Alain Rahier ◽

Francis Karst

Keyword(s):

Amino Acids ◽

Substrate Specificity ◽

Active Site ◽

Plant Sterol ◽

Substrate Binding ◽

Binding Domain ◽

Substrate Binding Domain

The present study identifies six specific amino acids of the cyclopropylsterol-cycloisomerase from the plant sterol pathway that control its activity or substrate specificity, and are likely to be located in the substrate-binding domain of the active site.

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