scholarly journals Structural insights into the LCIB protein family reveals a new group of β-carbonic anhydrases

2016 ◽  
Vol 113 (51) ◽  
pp. 14716-14721 ◽  
Author(s):  
Shengyang Jin ◽  
Jian Sun ◽  
Tobias Wunder ◽  
Desong Tang ◽  
Asaph B. Cousins ◽  
...  
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Active Site ◽  
Phaeodactylum Tricornutum ◽  
Protein Family ◽  
Carbonic Anhydrases ◽  
Uncharacterized Protein ◽  
C Protein ◽  
Functional Homolog ◽  
Slow Kinetics ◽  
Structural Insights

Aquatic microalgae have evolved diverse CO2-concentrating mechanisms (CCMs) to saturate the carboxylase with its substrate, to compensate for the slow kinetics and competing oxygenation reaction of the key photosynthetic CO2-fixing enzyme rubisco. The limiting CO2-inducible B protein (LCIB) is known to be essential for CCM function inChlamydomonas reinhardtii. To assign a function to this previously uncharacterized protein family, we purified and characterized a phylogenetically diverse set of LCIB homologs. Three of the six homologs are functional carbonic anhydrases (CAs). We determined the crystal structures of LCIB and limiting CO2-inducible C protein (LCIC) fromC. reinhardtiiand a CA-functional homolog fromPhaeodactylum tricornutum, all of which harbor motifs bearing close resemblance to the active site of canonical β-CAs. Our results identify the LCIB family as a previously unidentified group of β-CAs, and provide a biochemical foundation for their function in the microalgal CCMs.

Structural and biochemical characterization of novel carbonic anhydrases from Phaeodactylum tricornutum

2020 ◽  
Vol 76 (7) ◽  
pp. 676-686
Author(s):  
Shengyang Jin ◽  
Daniela Vullo ◽  
Silvia Bua ◽  
Alessio Nocentini ◽  
Claudiu T. Supuran ◽  
...  
Keyword(s):  
Phaeodactylum Tricornutum ◽  
Catalytic Mechanism ◽  
Biochemical Characterization ◽  
Protein Family ◽  
Carbonic Anhydrases ◽  
Highly Active ◽  
Potential Alternative ◽  
Photosynthetic Microorganisms ◽  
Inducible Protein

Carbonic anhydrases (CAs) are a well characterized family of metalloenzymes that are highly efficient in facilitating the interconversion between carbon dioxide and bicarbonate. Recently, CA activity has been associated with the LCIB (limiting CO2-inducible protein B) protein family, which has been an interesting target in aquatic photosynthetic microorganisms. To gain further insight into the catalytic mechanism of this new group of CAs, the X-ray structure of a highly active LCIB homolog (PtLCIB3) from the diatom Phaeodactylum tricornutum was determined. The CA activities of PtLCIB3, its paralog PtLCIB4 and a variety of their mutants were also measured. It was discovered that PtLCIB3 has a classic β-CA fold and its overall structure is highly similar to that of its homolog PtLCIB4. Subtle structural alterations between PtLCIB3 and PtLCIB4 indicate that an alternative proton-shuttle cavity could perhaps be one reason for their remarkable difference in CA activity. A potential alternative proton-shuttle route in the LCIB protein family is suggested based on these results.

scholarly journals Characterization of a novel type of carbonic anhydrase that acts without metal cofactors

BMC Biology ◽  
2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Yoshihisa Hirakawa ◽  
Miki Senda ◽  
Kodai Fukuda ◽  
Hong Yang Yu ◽  
Masaki Ishida ◽  
...  
Keyword(s):  
Active Site ◽  
Ph Control ◽  
Sequence Motif ◽  
Carbonic Anhydrases ◽  
Uncharacterized Protein ◽  
Metal Free ◽  
X Ray Crystallography ◽  
Control Respiration ◽  
Metal Cofactor ◽  
Protein Group

Abstract Background Carbonic anhydrases (CAs) are universal metalloenzymes that catalyze the reversible conversion of carbon dioxide (CO2) and bicarbonate (HCO3-). They are involved in various biological processes, including pH control, respiration, and photosynthesis. To date, eight evolutionarily unrelated classes of CA families (α, β, γ, δ, ζ, η, θ, and ι) have been identified. All are characterized by an active site accommodating the binding of a metal cofactor, which is assumed to play a central role in catalysis. This feature is thought to be the result of convergent evolution. Results Here, we report that a previously uncharacterized protein group, named “COG4337,” constitutes metal-independent CAs from the newly discovered ι-class. Genes coding for COG4337 proteins are found in various bacteria and photosynthetic eukaryotic algae. Biochemical assays demonstrated that recombinant COG4337 proteins from a cyanobacterium (Anabaena sp. PCC7120) and a chlorarachniophyte alga (Bigelowiella natans) accelerated CO2 hydration. Unexpectedly, these proteins exhibited their activity under metal-free conditions. Based on X-ray crystallography and point mutation analysis, we identified a metal-free active site within the cone-shaped α+β barrel structure. Furthermore, subcellular localization experiments revealed that COG4337 proteins are targeted into plastids and mitochondria of B. natans, implicating their involvement in CO2 metabolism in these organelles. Conclusions COG4337 proteins shared a short sequence motif and overall structure with ι-class CAs, whereas they were characterized by metal independence, unlike any known CAs. Therefore, COG4337 proteins could be treated as a variant type of ι-class CAs. Our findings suggested that this novel type of ι-CAs can function even in metal-poor environments (e.g., the open ocean) without competition with other metalloproteins for trace metals. Considering the widespread prevalence of ι-CAs across microalgae, this class of CAs may play a role in the global carbon cycle.

Investigating the functional link between TMEM165 and SPCA1

2019 ◽  
Vol 476 (21) ◽  
pp. 3281-3293 ◽  
Author(s):  
Elodie Lebredonchel ◽  
Marine Houdou ◽  
Hans-Heinrich Hoffmann ◽  
Kateryna Kondratska ◽  
Marie-Ange Krzewinski ◽  
...  
Keyword(s):  
Subcellular Localization ◽  
Complete Loss ◽  
Protein Family ◽  
Uncharacterized Protein ◽  
Functional Link ◽  
P Type

TMEM165 was highlighted in 2012 as the first member of the Uncharacterized Protein Family 0016 (UPF0016) related to human glycosylation diseases. Defects in TMEM165 are associated with strong Golgi glycosylation abnormalities. Our previous work has shown that TMEM165 rapidly degrades with supraphysiological manganese supplementation. In this paper, we establish a functional link between TMEM165 and SPCA1, the Golgi Ca2+/Mn2+ P-type ATPase pump. A nearly complete loss of TMEM165 was observed in SPCA1-deficient Hap1 cells. We demonstrate that TMEM165 was constitutively degraded in lysosomes in the absence of SPCA1. Complementation studies showed that TMEM165 abundance was directly dependent on SPCA1's function and more specifically its capacity to pump Mn2+ from the cytosol into the Golgi lumen. Among SPCA1 mutants that differentially impair Mn2+ and Ca2+ transport, only the Q747A mutant that favors Mn2+ pumping rescues the abundance and Golgi subcellular localization of TMEM165. Interestingly, the overexpression of SERCA2b also rescues the expression of TMEM165. Finally, this paper highlights that TMEM165 expression is linked to the function of SPCA1.

scholarly journals HPF1 remodels the active site of PARP1 to enable the serine ADP-ribosylation of histones

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Fa-Hui Sun ◽  
Peng Zhao ◽  
Nan Zhang ◽  
Lu-Lu Kong ◽  
Catherine C. L. Wong ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Active Site ◽  
Negative Charge ◽  
Structural Data ◽  
Dna Breaks ◽  
Adp Ribosylation ◽  
Local Conformation ◽  
Key Residues ◽  
Structural Insights

AbstractUpon binding to DNA breaks, poly(ADP-ribose) polymerase 1 (PARP1) ADP-ribosylates itself and other factors to initiate DNA repair. Serine is the major residue for ADP-ribosylation upon DNA damage, which strictly depends on HPF1. Here, we report the crystal structures of human HPF1/PARP1-CAT ΔHD complex at 1.98 Å resolution, and mouse and human HPF1 at 1.71 Å and 1.57 Å resolution, respectively. Our structures and mutagenesis data confirm that the structural insights obtained in a recent HPF1/PARP2 study by Suskiewicz et al. apply to PARP1. Moreover, we quantitatively characterize the key residues necessary for HPF1/PARP1 binding. Our data show that through salt-bridging to Glu284/Asp286, Arg239 positions Glu284 to catalyze serine ADP-ribosylation, maintains the local conformation of HPF1 to limit PARP1 automodification, and facilitates HPF1/PARP1 binding by neutralizing the negative charge of Glu284. These findings, along with the high-resolution structural data, may facilitate drug discovery targeting PARP1.

scholarly journals Structural Insights into the Molecular Evolution of the Archaeal Exo-β-d-Glucosaminidase

2019 ◽  
Vol 20 (10) ◽  
pp. 2460
Author(s):  
Shouhei Mine ◽  
Masahiro Watanabe
Keyword(s):  
Molecular Evolution ◽  
Primary Structure ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Thermostable Enzyme ◽  
Crystallographic Analysis ◽  
Evolutionary Pathway ◽  
Chitosan Oligosaccharides ◽  
Tim Barrel ◽  
Structural Insights

The archaeal exo-β-d-glucosaminidase (GlmA), a thermostable enzyme belonging to the glycosidase hydrolase (GH) 35 family, hydrolyzes chitosan oligosaccharides into monomer glucosamines. GlmA is a novel enzyme in terms of its primary structure, as it is homologous to both GH35 and GH42 β-galactosidases. The catalytic mechanism of GlmA is not known. Here, we summarize the recent reports on the crystallographic analysis of GlmA. GlmA is a homodimer, with each subunit comprising three distinct domains: a catalytic TIM-barrel domain, an α/β domain, and a β1 domain. Surprisingly, the structure of GlmA presents features common to GH35 and GH42 β-galactosidases, with the domain organization resembling that of GH42 β-galactosidases and the active-site architecture resembling that of GH35 β-galactosidases. Additionally, the GlmA structure also provides critical information about its catalytic mechanism, in particular, on how the enzyme can recognize glucosamine. Finally, we postulate an evolutionary pathway based on the structure of an ancestor GlmA to extant GH35 and GH42 β-galactosidases.

scholarly journals Structural insights into lipoprotein N-acylation byEscherichia coliapolipoprotein N-acyltransferase

2017 ◽  
Vol 114 (30) ◽  
pp. E6044-E6053 ◽  
Author(s):  
Cameron L. Noland ◽  
Michele D. Kattke ◽  
Jingyu Diao ◽  
Susan L. Gloor ◽  
Homer Pantua ◽  
...  
Keyword(s):  
Active Site ◽  
Signal Sequence ◽  
Acyl Chain ◽  
Transmembrane Helix ◽  
Chloride Ion ◽  
Catalytic Triad ◽  
Signal Peptidase ◽  
Membrane Protein Folding ◽  
E Coli ◽  
Structural Insights

Gram-negative bacteria express a diverse array of lipoproteins that are essential for various aspects of cell growth and virulence, including nutrient uptake, signal transduction, adhesion, conjugation, sporulation, and outer membrane protein folding. Lipoprotein maturation requires the sequential activity of three enzymes that are embedded in the cytoplasmic membrane. First, phosphatidylglycerol:prolipoprotein diacylglyceryl transferase (Lgt) recognizes a conserved lipobox motif within the prolipoprotein signal sequence and catalyzes the addition of diacylglycerol to an invariant cysteine. The signal sequence is then cleaved by signal peptidase II (LspA) to give an N-terminal S-diacylglyceryl cysteine. Finally, apolipoproteinN-acyltransferase (Lnt) catalyzes the transfer of thesn-1-acyl chain of phosphatidylethanolamine to this N-terminal cysteine, generating a mature, triacylated lipoprotein. Although structural studies of Lgt and LspA have yielded significant mechanistic insights into this essential biosynthetic pathway, the structure of Lnt has remained elusive. Here, we present crystal structures of wild-type and an active-site mutant ofEscherichia coliLnt. The structures reveal a monomeric eight-transmembrane helix fold that supports a periplasmic carbon–nitrogen hydrolase domain containing a Cys–Glu–Lys catalytic triad. Two lipids are bound at the active site in the structures, and we propose a putative phosphate recognition site where a chloride ion is coordinated near the active site. Based on these structures and complementary cell-based, biochemical, and molecular dynamics approaches, we propose a mechanism for substrate engagement and catalysis byE. coliLnt.

scholarly journals Characterization of CamH from Methanosarcina thermophila, Founding Member of a Subclass of the γ Class of Carbonic Anhydrases

2009 ◽  
Vol 192 (5) ◽  
pp. 1353-1360 ◽  
Author(s):  
Sabrina A. Zimmerman ◽  
Jean-Francois Tomb ◽  
James G. Ferry
Keyword(s):  
Active Site ◽  
Carbonic Anhydrases ◽  
Methanosarcina Thermophila ◽  
E Coli ◽  
Founding Member ◽  
Supplemental Zinc ◽  
Intermolecular Proton Transfer ◽  
Ping Pong ◽  
Iron Form

ABSTRACT The homotrimeric enzyme Mt-Cam from Methanosarcina thermophila is the archetype of the γ class of carbonic anhydrases. A search of databases queried with Mt-Cam revealed that a majority of the homologs comprise a putative subclass (CamH) in which there is major conservation of all of the residues essential for the archetype Mt-Cam except Glu62 and an acidic loop containing the essential proton shuttle residue Glu84. The CamH homolog from M. thermophila (Mt-CamH) was overproduced in Escherichia coli and characterized to validate its activity and initiate an investigation of the CamH subclass. The Mt-CamH homotrimer purified from E. coli cultured with supplemental zinc (Zn-Mt-CamH) contained 0.71 zinc and 0.15 iron per monomer and had k cat and kcat /Km values that were substantially lower than those for the zinc form of Mt-Cam (Zn-Mt-Cam). Mt-CamH purified from E. coli cultured with supplemental iron (Fe-Mt-CamH) was also a trimer containing 0.15 iron per monomer and only a trace amount of zinc and had an effective k cat (k cat eff) value normalized for iron that was 6-fold less than that for the iron form of Mt-Cam, whereas the k cat/Km eff was similar to that for Fe-Mt-Cam. Addition of 50 mM imidazole to the assay buffer increased the k cat eff of Fe-Mt-CamH more than 4-fold. Fe-Mt-CamH lost activity when it was exposed to air or 3% H2O2, which supports the hypothesis that Fe2+ has a role in the active site. The k cat for Fe-Mt-CamH was dependent on the concentration of buffer in a way that indicates that it acts as a second substrate in a “ping-pong” mechanism accepting a proton. The k cat/Km was not dependent on the buffer, consistent with the mechanism for all carbonic anhydrases in which the interconversion of CO2 and HCO3 − is separate from intermolecular proton transfer.

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