scholarly journals Structural and evolutionary relationships of “AT-less” type I polyketide synthase ketosynthases

2015 ◽  
Vol 112 (41) ◽  
pp. 12693-12698 ◽  
Author(s):  
Jeremy R. Lohman ◽  
Ming Ma ◽  
Jerzy Osipiuk ◽  
Boguslaw Nocek ◽  
Youngchang Kim ◽  
...  
Keyword(s):  
Amino Acid ◽  
Substrate Specificity ◽  
Polyketide Synthase ◽  
Acyl Carrier Protein ◽  
Structural Diversity ◽  
Type I ◽  
Substrate Specificities ◽  
In Trans ◽  
Canonical Type ◽  
Insight Into

Acyltransferase (AT)-less type I polyketide synthases (PKSs) break the type I PKS paradigm. They lack the integrated AT domains within their modules and instead use a discrete AT that acts in trans, whereas a type I PKS module minimally contains AT, acyl carrier protein (ACP), and ketosynthase (KS) domains. Structures of canonical type I PKS KS-AT didomains reveal structured linkers that connect the two domains. AT-less type I PKS KSs have remnants of these linkers, which have been hypothesized to be AT docking domains. Natural products produced by AT-less type I PKSs are very complex because of an increased representation of unique modifying domains. AT-less type I PKS KSs possess substrate specificity and fall into phylogenetic clades that correlate with their substrates, whereas canonical type I PKS KSs are monophyletic. We have solved crystal structures of seven AT-less type I PKS KS domains that represent various sequence clusters, revealing insight into the large structural and subtle amino acid residue differences that lead to unique active site topologies and substrate specificities. One set of structures represents a larger group of KS domains from both canonical and AT-less type I PKSs that accept amino acid-containing substrates. One structure has a partial AT-domain, revealing the structural consequences of a type I PKS KS evolving into an AT-less type I PKS KS. These structures highlight the structural diversity within the AT-less type I PKS KS family, and most important, provide a unique opportunity to study the molecular evolution of substrate specificity within the type I PKSs.

scholarly journals Structure based engineering for substrate specificity of pinoresinol-lariciresinol reductases

2020 ◽  
Author(s):  
Ying Xiao ◽  
Kai Shao ◽  
Jingwen Zhou ◽  
Lian Wang ◽  
Di Wu ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Molecular Mechanism ◽  
Large Scale ◽  
Structural Diversity ◽  
Commercial Production ◽  
Structural Elements ◽  
Substrate Specificities ◽  
Lignan Biosynthesis ◽  
Binding Forms ◽  
Insight Into

Abstract Pinoresinol–lariciresinol reductases (PLR) are enzymes involved in the lignan biosynthesis after the initial dimerization of two monolignols, which represents the entry point for the synthesis of 8-8′ lignans and contributes greatly to their structural diversity. Of particular interest has been in determining how differing substrate specificities are achieved with these enzymes. Here, we present crystal structures of IiPLR1/AtPrR1/AtPrR2 in the apo, substrate-binding and product-binding forms. Each structure contains a head-to-tail homodimer, and the catalytic pocket comprises structural elements from both monomers. A loop β4 covers the top of the pocket, and residue 98 from the loop governs catalytic specificity. Structure-guided mutagenesis could switch the substrate specificity of IiPLR1 and AtPrR2, respectively. Our study provides new insight into the molecular mechanism underlying the substrate specificity of PLR/PrRs and suggests an efficient strategy for the large-scale commercial production of the pharmaceutically valuable compound lariciresinol.

scholarly journals Structure-based engineering of substrate specificity for pinoresinol-lariciresinol reductases

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Ying Xiao ◽  
Kai Shao ◽  
Jingwen Zhou ◽  
Lian Wang ◽  
Xueqi Ma ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Bound States ◽  
Large Scale ◽  
Structural Diversity ◽  
Structural Elements ◽  
Isatis Indigotica ◽  
Substrate Specificities ◽  
Lignan Biosynthesis ◽  
Insight Into

AbstractPinoresinol–lariciresinol reductases (PLRs) are enzymes involved in the lignan biosynthesis after the initial dimerization of two monolignols, and this represents the entry point for the synthesis of 8-8′ lignans and contributes greatly to their structural diversity. Of particular interest has been the determination of how differing substrate specificities are achieved with these enzymes. Here, we present crystal structures of IiPLR1 from Isatis indigotica and pinoresinol reductases (PrRs) AtPrR1 and AtPrR2 from Arabidopsis thaliana, in the apo, substrate-bound and product-bound states. Each structure contains a head-to-tail homodimer, and the catalytic pocket comprises structural elements from both monomers. β4 loop covers the top of the pocket, and residue 98 from the loop governs catalytic specificity. The substrate specificities of IiPLR1 and AtPrR2 can be switched via structure-guided mutagenesis. Our study provides insight into the molecular mechanism underlying the substrate specificity of PLRs/PrRs and suggests an efficient strategy for the large-scale commercial production of the pharmaceutically valuable compound lariciresinol.

scholarly journals Modular type I polyketide synthase acyl carrier protein domains share a common N-terminally extended fold

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Luisa Moretto ◽  
Rachel Heylen ◽  
Natalie Holroyd ◽  
Steven Vance ◽  
R. William Broadhurst
Keyword(s):  
Polyketide Synthase ◽  
Acyl Carrier Protein ◽  
Protein Domains ◽  
Carrier Protein ◽  
Type I

An Evolutionary Model Encompassing Substrate Specificity and Reactivity of Type I Polyketide Synthase Thioesterases

ChemBioChem ◽  
2014 ◽  
Vol 15 (18) ◽  
pp. 2656-2661 ◽  
Author(s):  
Taylor P. A. Hari ◽  
Puneet Labana ◽  
Meaghan Boileau ◽  
Christopher N. Boddy
Keyword(s):  
Substrate Specificity ◽  
Polyketide Synthase ◽  
Evolutionary Model ◽  
Type I

Solution Structure of an Acyl Carrier Protein Domain from a Fungal Type I Polyketide Synthase,

Biochemistry ◽  
2010 ◽  
Vol 49 (10) ◽  
pp. 2186-2193 ◽  
Author(s):  
Pakorn Wattana-amorn ◽  
Christopher Williams ◽  
Eliza Płoskoń ◽  
Russell J. Cox ◽  
Thomas J. Simpson ◽  
...  
Keyword(s):  
Solution Structure ◽  
Polyketide Synthase ◽  
Acyl Carrier Protein ◽  
Protein Domain ◽  
Carrier Protein ◽  
Type I

scholarly journals Extracellular-matrix degradation at acid pH. Avian osteoclast acid collagenase isolation and characterization

1993 ◽  
Vol 290 (3) ◽  
pp. 873-884 ◽  
Author(s):  
H C Blair ◽  
S L Teitelbaum ◽  
L E Grosso ◽  
D L Lacey ◽  
H L Tan ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Substrate Specificity ◽  
Cathepsin B ◽  
Type I Collagen ◽  
Sequence Similarity ◽  
Bone Matrix ◽  
Collagenolytic Activity ◽  
Type I ◽  
Isolation And Characterization

Osteoclasts degrade bone matrix, which is mainly type I collagen and hydroxyapatite, in an acidic extracellular compartment. Thus we reasoned that osteoclasts must produce an acid collagenase. We purified this enzyme, a 31 kDa protein, from avian osteoclast lysates (in 100 mM acetate/1 mM CHAPS/1 mM dithiothreitol, pH 4.4), fractionated by (NH2)2SO4 precipitation, gelatin-affinity, cation exchange, and gel filtration. Fraction activity was measured using diazotized collagen or 3H-labelled cross-linked collagen (decalcified and trypsin-treated metabolically L-[4,5-3H]proline-labelled bone) as substrates. Iodoacetate, leupeptin, antipain, pepstatin and mercurials inhibited collagenolysis by the isolated proteinase; mercurial derivatives could not be re-activated by dithiothreitol. Collagen degradation was maximal at pH 4.4; purified proteinase reproduced the collagenolytic activity of cell lysates. The N-terminal amino acid sequence from the isolated protein and its CNBr degradation fragments showed sequence similarity to mammalian cathepsin Bs, and near-identity with avian liver cathepsin B. Peptide substrate specificity of the osteoclastic enzyme resembled those of mammalian cathepsin B and its avian liver counterpart, but degradation of low-molecular-mass substrates by the osteoclastic enzyme was slower, reflecting generally lower kcat. values. Further, kcat/Km varied less between arginine-containing substrates than for previously reported cathepsin Bs, indicating different substrate specificity of the osteoclast enzyme. Polyclonal antibody raised to a 25 kDa fragment of the enzyme recognized a single 31 kDa band in SDS/PAGE of osteoclast lysates blotted to poly(vinylidene difluoride), adsorbed collagenolytic activity of osteoclast lysates, and stained avian osteoclasts in tissue sections. Degenerate sense- and antisense-oligonucleotide primers, predicted from segments of primary amino acid sequence, amplified a 486 bp DNA fragment; this was cloned and sequenced. Of 162 amino acids encoded, 77% are identical with those of human cathepsin B; hybridization identified a 2.4 kb RNA in osteoclast lysates. We conclude that the major avian osteoclast collagenolytic enzyme is a cathepsin B, whose activity varies from other enzymes of its class.

scholarly journals The Molecular Mass and Isoelectric Point of Plant Proteomes

2019 ◽  
Author(s):  
Tapan Kumar Kumar Mohanta ◽  
Abdulatif Khan ◽  
Abeer Hashem ◽  
Elsayed Fathi Abd_Allah ◽  
Ahmed Al-Harrasi
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Amino Acid ◽  
Molecular Mass ◽  
Plant Species ◽  
Isoelectric Point ◽  
Polyketide Synthase ◽  
Type I ◽  
Plant Proteins ◽  
Higher Plant

Abstract Background Cell contain diverse array of proteins with different molecular weight and isoelectric point (pI). The molecular weight and pI of protein play important role in determining the molecular biochemical function. Therefore, it was important to understand the detail regarding the molecular weight and pI of the plant proteins. Results A proteome-wide analysis of plant proteomes from 145 species revealed a pI range of 1.99 (epsin) to 13.96 (hypothetical protein). The spectrum of molecular mass of the plant proteins varied from 0.54 to 2236.8 kDa. A putative Type-I polyketide synthase (22244 amino acids) in Volvox carteri was found to be the largest protein in the plant kingdom. However, Type-I polyketide synthase was not found in higher plant species. Titin (806.46 kDa) and misin/midasin (730.02 kDa) were the largest proteins identified in higher plant species. The pI and molecular weight of the plant proteins showed a trimodal distribution. An acidic pI (56.44% of proteins) was found to be predominant over a basic pI (43.34% of proteins) and the abundance of acidic pI proteins was higher in unicellular algae species relative to multicellular higher plants. In contrast, the seaweed, Porphyra umbilicalis, possesses a higher proportion of basic pI proteins (70.09%). Plant proteomes were also found to contain selenocysteine (Sec), amino acid that was found only in lower eukaryotic aquatic plant lineage. Amino acid composition analysis showed Leu was high and Trp was low abundant amino acids in the plant proteome. Additionally, the plant proteomes also possess ambiguous amino acids Xaa (unknown), Asx (asparagine or aspartic acid), Glx (glutamine or glutamic acid), and Xle (leucine or isoleucine) as well. Conclusion The diverse molecular weight and isoelectric point range of plant proteome will be helpful to understand their biochemical and functional aspects. The presence of selenocysteine proteins in lower eukaryotic organism is of interest and their expression in higher plant system can help us to understand their functional role.

scholarly journals Identification of a starter unit acyl-carrier protein transacylase domain in an iterative type I polyketide synthase

2006 ◽  
Vol 103 (45) ◽  
pp. 16728-16733 ◽  
Author(s):  
J. M. Crawford ◽  
B. C. R. Dancy ◽  
E. A. Hill ◽  
D. W. Udwary ◽  
C. A. Townsend
Keyword(s):  
Polyketide Synthase ◽  
Acyl Carrier Protein ◽  
Carrier Protein ◽  
Type I ◽  
Starter Unit

scholarly journals Inside Back Cover: An Evolutionary Model Encompassing Substrate Specificity and Reactivity of Type I Polyketide Synthase Thioesterases (ChemBioChem 18/2014)

ChemBioChem ◽  
2014 ◽  
Vol 15 (18) ◽  
pp. 2791-2791
Author(s):  
Taylor P. A. Hari ◽  
Puneet Labana ◽  
Meaghan Boileau ◽  
Christopher N. Boddy
Keyword(s):  
Substrate Specificity ◽  
Polyketide Synthase ◽  
Evolutionary Model ◽  
Type I ◽  
Back Cover

scholarly journals Insights into Substrate Specificity of NlpC/P60 Cell Wall Hydrolases Containing Bacterial SH3 Domains

mBio ◽  
2015 ◽  
Vol 6 (5) ◽  
Author(s):  
Qingping Xu ◽  
Dominique Mengin-Lecreulx ◽  
Xueqian W. Liu ◽  
Delphine Patin ◽  
Carol L. Farr ◽  
...  
Keyword(s):  
Cell Wall ◽  
Amino Acid ◽  
Substrate Specificity ◽  
Catalytic Domain ◽  
Substrate Binding ◽  
Diaminopimelic Acid ◽  
Single Mutation ◽  
Molecular Architecture ◽  
Substrate Specificities ◽  
Cell Wall Hydrolases

ABSTRACTBacterial SH3 (SH3b) domains are commonly fused with papain-like Nlp/P60 cell wall hydrolase domains. To understand how the modular architecture of SH3b and NlpC/P60 affects the activity of the catalytic domain, three putative NlpC/P60 cell wall hydrolases were biochemically and structurally characterized. These enzymes all have γ-d-Glu-A2pm (A2pm is diaminopimelic acid) cysteine amidase (ordl-endopeptidase) activities but with different substrate specificities. One enzyme is a cell wall lysin that cleaves peptidoglycan (PG), while the other two are cell wall recycling enzymes that only cleave stem peptides with an N-terminall-Ala. Their crystal structures revealed a highly conserved structure consisting of two SH3b domains and a C-terminal NlpC/P60 catalytic domain, despite very low sequence identity. Interestingly, loops from the first SH3b domain dock into the ends of the active site groove of the catalytic domain, remodel the substrate binding site, and modulate substrate specificity. Two amino acid differences at the domain interface alter the substrate binding specificity in favor of stem peptides in recycling enzymes, whereas the SH3b domain may extend the peptidoglycan binding surface in the cell wall lysins. Remarkably, the cell wall lysin can be converted into a recycling enzyme with a single mutation.IMPORTANCEPeptidoglycan is a meshlike polymer that envelops the bacterial plasma membrane and bestows structural integrity. Cell wall lysins and recycling enzymes are part of a set of lytic enzymes that target covalent bonds connecting the amino acid and amino sugar building blocks of the PG network. These hydrolases are involved in processes such as cell growth and division, autolysis, invasion, and PG turnover and recycling. To avoid cleavage of unintended substrates, these enzymes have very selective substrate specificities. Our biochemical and structural analysis of three modular NlpC/P60 hydrolases, one lysin, and two recycling enzymes, show that they may have evolved from a common molecular architecture, where the substrate preference is modulated by local changes. These results also suggest that new pathways for recycling PG turnover products, such as tracheal cytotoxin, may have evolved in bacteria in the human gut microbiome that involve NlpC/P60 cell wall hydrolases.

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