The crystal structure of Escherichia coli spermidine synthase SpeE reveals a unique substrate-binding pocket

2010 ◽  
Vol 169 (3) ◽  
pp. 277-285 ◽  
Author(s):  
Xingding Zhou ◽  
Teck Khiang Chua ◽  
Karolina L. Tkaczuk ◽  
Janusz M. Bujnicki ◽  
J. Sivaraman
Keyword(s):  
Crystal Structure ◽  
Escherichia Coli ◽  
Substrate Binding ◽  
Binding Pocket ◽  
Spermidine Synthase ◽  
Substrate Binding Pocket

scholarly journals Crystal structure of steroid reductase SRD5A reveals conserved steroid reduction mechanism

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Yufei Han ◽  
Qian Zhuang ◽  
Bo Sun ◽  
Wenping Lv ◽  
Sheng Wang ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Biochemical Characterization ◽  
Substrate Binding ◽  
Binding Pocket ◽  
Substrate Binding Pocket ◽  
Transmembrane Segments ◽  
Therapeutic Molecules ◽  
Binding Cavity ◽  
Immune System Regulation ◽  
Mediated Reduction

AbstractSteroid hormones are essential in stress response, immune system regulation, and reproduction in mammals. Steroids with 3-oxo-Δ4 structure, such as testosterone or progesterone, are catalyzed by steroid 5α-reductases (SRD5As) to generate their corresponding 3-oxo-5α steroids, which are essential for multiple physiological and pathological processes. SRD5A2 is already a target of clinically relevant drugs. However, the detailed mechanism of SRD5A-mediated reduction remains elusive. Here we report the crystal structure of PbSRD5A from Proteobacteria bacterium, a homolog of both SRD5A1 and SRD5A2, in complex with the cofactor NADPH at 2.0 Å resolution. PbSRD5A exists as a monomer comprised of seven transmembrane segments (TMs). The TM1-4 enclose a hydrophobic substrate binding cavity, whereas TM5-7 coordinate cofactor NADPH through extensive hydrogen bonds network. Homology-based structural models of HsSRD5A1 and -2, together with biochemical characterization, define the substrate binding pocket of SRD5As, explain the properties of disease-related mutants and provide an important framework for further understanding of the mechanism of NADPH mediated steroids 3-oxo-Δ4 reduction. Based on these analyses, the design of therapeutic molecules targeting SRD5As with improved specificity and therapeutic efficacy would be possible.

scholarly journals Crystal structure and mechanism of human carboxypeptidase O: Insights into its specific activity for acidic residues

2018 ◽  
Vol 115 (17) ◽  
pp. E3932-E3939 ◽  
Author(s):  
Maria C. Garcia-Guerrero ◽  
Javier Garcia-Pardo ◽  
Esther Berenguer ◽  
Roberto Fernandez-Alvarez ◽  
Gifty B. Barfi ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Specific Activity ◽  
Dominant Role ◽  
Substrate Binding ◽  
Digestive Enzyme ◽  
Binding Pocket ◽  
Site Directed Mutagenesis ◽  
Enzyme Specificity ◽  
Substrate Binding Pocket ◽  
Acidic Residues

Human metallocarboxypeptidase O (hCPO) is a recently discovered digestive enzyme localized to the apical membrane of intestinal epithelial cells. Unlike pancreatic metallocarboxypeptidases, hCPO is glycosylated and produced as an active enzyme with distinctive substrate specificity toward C-terminal (C-t) acidic residues. Here we present the crystal structure of hCPO at 1.85-Å resolution, both alone and in complex with a carboxypeptidase inhibitor (NvCI) from the marine snail Nerita versicolor. The structure provides detailed information regarding determinants of enzyme specificity, in particular Arg275, placed at the bottom of the substrate-binding pocket. This residue, located at “canonical” position 255, where it is Ile in human pancreatic carboxypeptidases A1 (hCPA1) and A2 (hCPA2) and Asp in B (hCPB), plays a dominant role in determining the preference of hCPO for acidic C-t residues. Site-directed mutagenesis to Asp and Ala changes the specificity to C-t basic and hydrophobic residues, respectively. The single-site mutants thus faithfully mimic the enzymatic properties of CPB and CPA, respectively. hCPO also shows a preference for Glu over Asp, probably as a consequence of a tighter fitting of the Glu side chain in its S1′ substrate-binding pocket. This unique preference of hCPO, together with hCPA1, hCPA2, and hCPB, completes the array of C-t cleavages enabling the digestion of the dietary proteins within the intestine. Finally, in addition to activity toward small synthetic substrates and peptides, hCPO can also trim C-t extensions of proteins, such as epidermal growth factor, suggesting a role in the maturation and degradation of growth factors and bioactive peptides.

scholarly journals Crystal structure of TchmY from Actinoplanes teichomyceticus

2019 ◽  
Vol 75 (9) ◽  
pp. 570-575
Author(s):  
Zhenzhen Yang ◽  
Lilan Zhang ◽  
Xuejing Yu ◽  
Shan Wu ◽  
Yong Yang ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Escherichia Coli ◽  
Gene Clusters ◽  
Binding Pocket ◽  
Animal Nutrition ◽  
Biosynthetic Pathways ◽  
Substrate Binding Pocket ◽  
Actinoplanes Teichomyceticus ◽  
Unknown Sequence ◽  
Potential Substrate

Moenomycin-type antibiotics are phosphoglycolipids that are notable for their unique modes of action and have proven to be useful in animal nutrition. The gene clusters tchm from Actinoplanes teichomyceticus and moe from Streptomyces are among a limited number of known moenomycin-biosynthetic pathways. Most genes in tchm have counterparts in the moe cluster, except for tchmy and tchmz, the functions of which remain unknown. Sequence analysis indicates that TchmY belongs to the isoprenoid enzyme C2-like superfamily and may serve as a prenylcyclase. The enzyme was proposed to be involved in terminal cyclization of the moenocinyl chain in teichomycin, leading to the diumycinol chain of moenomycin isomers. Here, recombinant TchmY protein was expressed in Escherichia coli and its crystal structure was solved by SIRAS. Structural analysis and comparison with other prenylcyclases were performed. The overall fold of TchmY consists of an (α/α)6-barrel, and a potential substrate-binding pocket is found in the central chamber. These results should provide important information regarding the biosynthetic basis of moenomycin antibiotics.

scholarly journals The structure of the hexameric atrazine chlorohydrolase AtzA

2015 ◽  
Vol 71 (3) ◽  
pp. 710-720 ◽  
Author(s):  
T. S. Peat ◽  
J. Newman ◽  
S. Balotra ◽  
D. Lucent ◽  
A. C. Warden ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Substrate Binding ◽  
Physiological Role ◽  
Binding Pocket ◽  
Soil Organism ◽  
Substrate Binding Pocket ◽  
Herbicide Atrazine ◽  
Hexameric Form ◽  
Considerable Strain

Atrazine chlorohydrolase (AtzA) was discovered and purified in the early 1990s from soil that had been exposed to the widely used herbicide atrazine. It was subsequently found that this enzyme catalyzes the first and necessary step in the breakdown of atrazine by the soil organismPseudomonassp. strain ADP. Although it has taken 20 years, a crystal structure of the full hexameric form of AtzA has now been obtained. AtzA is less well adapted to its physiological role (i.e.atrazine dechlorination) than the alternative metal-dependent atrazine chlorohydrolase (TrzN), with a substrate-binding pocket that is under considerable strain and for which the substrate is a poor fit.

scholarly journals Crystal structure of the dimethylsulfide monooxygenase DmoA from Hyphomicrobium sulfonivorans

2018 ◽  
Vol 74 (12) ◽  
pp. 781-786 ◽  
Author(s):  
Hai-Yan Cao ◽  
Peng Wang ◽  
Ming Peng ◽  
Xuan Shao ◽  
Xiu-Lan Chen ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Sequence Similarity ◽  
Substrate Binding ◽  
Detailed Comparison ◽  
Binding Pocket ◽  
Flavin Mononucleotide ◽  
Substrate Binding Pocket ◽  
High Sequence Similarity ◽  
Tim Barrel

DmoA is a monooxygenase which uses dioxygen (O2) and reduced flavin mononucleotide (FMNH2) to catalyze the oxidation of dimethylsulfide (DMS). Although it has been characterized, the structure of DmoA remains unknown. Here, the crystal structure of DmoA was determined to a resolution of 2.28 Å and was compared with those of its homologues LadA and BdsA. The results showed that their overall structures are similar: they all share a conserved TIM-barrel fold which is composed of eight α-helices and eight β-strands. In addition, they all have five additional insertions. Detailed comparison showed that the structures have notable differences despite their high sequence similarity. The substrate-binding pocket of DmoA is smaller compared with those of LadA and BdsA.

scholarly journals Molecular mimicry in deoxy-nucleotide catalysis: the structure of Escherichia coli dGTPase reveals the molecular basis of dGTP selectivity: New structural methods offer insight on dGTPases

2018 ◽  
Author(s):  
Christopher O. Barnes ◽  
Ying Wu ◽  
Jinhu Song ◽  
Guowu Lin ◽  
Elizabeth L. Baxter ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Conformational Changes ◽  
Reverse Transcription ◽  
Sequence Homology ◽  
Active Site ◽  
Substrate Binding ◽  
Critical Role ◽  
Binding Pocket ◽  
Substrate Binding Pocket ◽  
Cellular Survival

AbstractDeoxynucleotide triphosphate triphosphyohydrolyases (dNTPases) play a critical role in cellular survival and DNA replication through the proper maintenance of cellular dNTP pools by hydrolyzing dNTPs into deoxynucleosides and inorganic triphosphate (PPPi). While the vast majority of these enzymes display broad activity towards canonical dNTPs, exemplified by Sterile Alpha Motif (SAM) and Histidine-aspartate (HD) domain-containing protein 1 (SAMHD1), which blocks reverse transcription of retroviruses in macrophages by maintaining dNTP pools at low levels, Escherichia coli (Ec)-dGTPase is the only known enzyme that specifically hydrolyzes dGTP. However, the mechanism behind dGTP selectivity is unclear. Here we present the free-, ligand (dGTP)- and inhibitor (GTP)-bound structures of hexameric E. coli dGTPase. To obtain these structures, we applied UV-fluorescence microscopy, video analysis and highly automated goniometer-based instrumentation to map and rapidly position individual crystals randomly-located on fixed target holders, resulting in the highest indexing-rates observed for a serial femtosecond crystallography (SFX) experiment. The structure features a highly dynamic active site where conformational changes are coupled to substrate (dGTP), but not inhibitor binding, since GTP locks dGTPase in its apo form. Moreover, despite no sequence homology, dGTPase and SAMHD1 share similar active site and HD motif architectures; however, dGTPase residues at the end of the substrate-binding pocket mimic Watson Crick interactions providing Guanine base specificity, while a 7 Å cleft separates SAMHD1 residues from dNTP bases, abolishing nucleotide-type discrimination. Furthermore, the structures sheds light into the mechanism by which long distance binding (25 Å) of single stranded DNA in an allosteric site primes the active site by conformationally “opening” a tyrosine gate allowing enhanced substrate binding.Significance StatementdNTPases play a critical role in cellular survival through maintenance of cellular dNTP. While dNTPases display activity towards dNTPs, such as SAMHD1 –which blocks reverse transcription of HIV-1 in macrophages– Escherichia coli (Ec)-dGTPase is the only known enzyme that specifically hydrolyzes dGTP. Here we use novel free electron laser data collection to shed light into the mechanisms of (Ec)-dGTPase selectivity. The structure features a dynamic active site where conformational changes are coupled to dGTP binding. Moreover, despite no sequence homology between (Ec)-dGTPase and SAMHD1, both enzymes share similar active site architectures; however, dGTPase residues at the end of the substrate-binding pocket provide dGTP specificity, while a 7 Å cleft separates SAMHD1 residues from dNTP.

Characterization of the Acyl Substrate Binding Pocket of Acetyl-CoA Synthetase†

Biochemistry ◽  
2006 ◽  
Vol 45 (38) ◽  
pp. 11482-11490 ◽  
Author(s):  
Cheryl Ingram-Smith ◽  
Barrett I. Woods ◽  
Kerry S. Smith
Keyword(s):  
Substrate Binding ◽  
Binding Pocket ◽  
Substrate Binding Pocket ◽  
Acetyl Coa
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