scholarly journals Sequence and Structural Analysis of AA9 and AA10 LPMOs: An Insight into the Basis of Substrate Specificity and Regioselectivity

2019 ◽  
Vol 20 (18) ◽  
pp. 4594 ◽  
Author(s):  
Xiaoli Zhou ◽  
Xiaohua Qi ◽  
Hongxia Huang ◽  
Honghui Zhu
Keyword(s):  
Structural Analysis ◽  
Substrate Specificity ◽  
Molecular Basis ◽  
Substrate Binding ◽  
Binding Modes ◽  
Substrate Specificities ◽  
Lytic Polysaccharide Monooxygenases ◽  
Polysaccharide Monooxygenases ◽  
Natural Carbon ◽  
Insight Into

Lytic polysaccharide monooxygenases (LPMOs) are key enzymes in both the natural carbon cycle and the biorefinery industry. Understanding the molecular basis of LPMOs acting on polysaccharide substrates is helpful for improving industrial cellulase cocktails. Here we analyzed the sequences, structures, and substrate binding modes of LPMOs to uncover the factors that influence substrate specificity and regioselectivity. Our results showed that the different compositions of a motif located on L2 affect the electrostatic potentials of substrate binding surfaces, which in turn affect substrate specificities of AA10 LPMOs. A conserved Asn at a distance of 7 Å from the active center Cu might, together with the conserved Ser immediately before the second catalytic His, determine the localization of LPMOs on substrate, and thus contribute to C4-oxidizing regioselectivity. The findings in this work provide an insight into the molecular basis of substrate specificity and regioselectivity of LPMOs.

scholarly journals Identification of the molecular determinants driving the substrate specificity of fungal lytic polysaccharide monooxygenases (LPMOs)

2020 ◽  
pp. jbc.RA120.015545
Author(s):  
Kristian E. H. Frandsen ◽  
Mireille Haon ◽  
Sacha Grisel ◽  
Bernard Henrissat ◽  
Leila Lo Leggio ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Time Course ◽  
Plant Cell Wall ◽  
Plant Biomass ◽  
Substrate Binding ◽  
Glycoside Hydrolases ◽  
Sequence Alignments ◽  
Lytic Polysaccharide Monooxygenases ◽  
Molecular Determinants ◽  
Polysaccharide Monooxygenases

Understanding enzymatic breakdown of plant biomass is crucial to develop nature-inspired biotechnological processes. Lytic polysaccharide monooxygenases (LPMOs) are microbial enzymes secreted by fungal saprotrophs involved in carbon recycling. LPMOs modify biomass by oxidatively cleaving polysaccharides thereby enhancing the efficiency of glycoside hydrolases. Fungal AA9 LPMOs are active on cellulose but some members also display activity on hemicelluloses and/or oligosaccharides. Although the active site subsites are well defined for a few model LPMOs, the molecular determinants driving broad substrate specificity are still not easily predictable. Based on bioinformatic clustering and sequence alignments, we selected seven fungal AA9 LPMOs that differ in the amino-acid residues constituting their subsites. Investigation of their substrate specificities revealed that all these LPMOs are active on cellulose and cello-oligosaccharides, as well as plant cell wall-derived hemicellulosic polysaccharides and carry out C4 oxidative cleavage. The product profiles from cello-oligosaccharides degradation suggests that the subtle differences in amino acids sequence within the substrate-binding loop regions lead to different preferred binding modes. Our functional analyses allowed us to probe the molecular determinants of substrate binding within two AA9 LPMO sub-clusters. Many wood-degrading fungal species rich in AA9 genes have at least one AA9 enzyme with structural loop features that allow recognition of short β-(1,4)-linked glucan chains. Time-course monitoring of these AA9 LPMOs on cello-oligosaccharides also provides a useful model system for mechanistic studies of LPMO catalysis. These results are valuable for the understanding of LPMO contribution to wood decaying process in nature and for the development of sustainable biorefineries.

scholarly journals Kinetic insights into the peroxygenase activity of cellulose-active lytic polysaccharide monooxygenases (LPMOs)

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Riin Kont ◽  
Bastien Bissaro ◽  
Vincent G. H. Eijsink ◽  
Priit Väljamäe
Keyword(s):  
Catalytic Properties ◽  
Biomass Conversion ◽  
Side Reactions ◽  
Cellulose Oxidation ◽  
Multiple Substrates ◽  
Glycosidic Bonds ◽  
Lytic Polysaccharide Monooxygenases ◽  
Polysaccharide Monooxygenases ◽  
Global Carbon ◽  
Insight Into

AbstractLytic polysaccharide monooxygenases (LPMOs) are widely distributed in Nature, where they catalyze the hydroxylation of glycosidic bonds in polysaccharides. Despite the importance of LPMOs in the global carbon cycle and in industrial biomass conversion, the catalytic properties of these monocopper enzymes remain enigmatic. Strikingly, there is a remarkable lack of kinetic data, likely due to a multitude of experimental challenges related to the insoluble nature of LPMO substrates, like cellulose and chitin, and to the occurrence of multiple side reactions. Here, we employed competition between well characterized reference enzymes and LPMOs for the H2O2 co-substrate to kinetically characterize LPMO-catalyzed cellulose oxidation. LPMOs of both bacterial and fungal origin showed high peroxygenase efficiencies, with kcat/KmH2O2 values in the order of 105–106 M−1 s−1. Besides providing crucial insight into the cellulolytic peroxygenase reaction, these results show that LPMOs belonging to multiple families and active on multiple substrates are true peroxygenases.

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 Insight into the Binding and Hydrolytic Preferences of hNudt16 Based on Nucleotide Diphosphate Substrates

2021 ◽  
Vol 22 (20) ◽  
pp. 10929
Author(s):  
Magdalena Chrabąszczewska ◽  
Maria Winiewska-Szajewska ◽  
Natalia Ostrowska ◽  
Elżbieta Bojarska ◽  
Janusz Stępiński ◽  
...  
Keyword(s):  
Molecular Docking ◽  
Substrate Specificity ◽  
Ligand Binding ◽  
Regulatory Role ◽  
Stacking Interactions ◽  
Binding Modes ◽  
Biological Functions ◽  
Insight Into ◽  
Potential Regulatory Role

Nudt16 is a member of the NUDIX family of hydrolases that show specificity towards substrates consisting of a nucleoside diphosphate linked to another moiety X. Several substrates for hNudt16 and various possible biological functions have been reported. However, some of these reports contradict each other and studies comparing the substrate specificity of the hNudt16 protein are limited. Therefore, we quantitatively compared the affinity of hNudt16 towards a set of previously published substrates, as well as identified novel potential substrates. Here, we show that hNudt16 has the highest affinity towards IDP and GppG, with Kd below 100 nM. Other tested ligands exhibited a weaker affinity of several orders of magnitude. Among the investigated compounds, only IDP, GppG, m7GppG, AppA, dpCoA, and NADH were hydrolyzed by hNudt16 with a strong substrate preference for inosine or guanosine containing compounds. A new identified substrate for hNudt16, GppG, which binds the enzyme with an affinity comparable to that of IDP, suggests another potential regulatory role of this protein. Molecular docking of hNudt16-ligand binding inside the hNudt16 pocket revealed two binding modes for representative substrates. Nucleobase stabilization by Π stacking interactions with His24 has been associated with strong binding of hNudt16 substrates.

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.

scholarly journals Towards a better understanding of the substrate specificity of the UDP-N-acetylglucosamine C4 epimerase WbpP

2005 ◽  
Vol 389 (1) ◽  
pp. 173-180 ◽  
Author(s):  
Melinda DEMENDI ◽  
Noboru ISHIYAMA ◽  
Joseph S. LAM ◽  
Albert M. BERGHUIS ◽  
Carole CREUZENET
Keyword(s):  
Enzyme Activity ◽  
Substrate Specificity ◽  
Molecular Basis ◽  
Substrate Binding ◽  
Structural Data ◽  
Binding Pocket ◽  
Substrate Binding Pocket ◽  
Expected Effect ◽  
Biochemical Assays ◽  
Golden Opportunity

WbpP is the only genuine UDP-GlcNAc (UDP-N-acetylglucosamine) C4 epimerase for which both biochemical and structural data are available. This represents a golden opportunity to elucidate the molecular basis for its specificity for N-acetylated substrates. Based on the comparison of the substrate binding site of WbpP with that of other C4 epimerases that convert preferentially non-acetylated substrates, or that are able to convert both acetylated and non-acetylated substrates equally well, specific residues of WbpP were mutated, and the substrate specificity of the mutants was determined by direct biochemical assays and kinetic analyses. Most of the mutations tested were anticipated to trigger a significant switch in substrate specificity, mostly towards a preference for non-acetylated substrates. However, only one of the mutations (A209H) had the expected effect, and most others resulted in enhanced specificity of WbpP for N-acetylated substrates (Q201E, G102K, Q201E/G102K, A209N and S143A). One mutation (S144K) totally abolished enzyme activity. These data indicate that, although all residues targeted in the present study turned out to be important for catalysis, determinants of substrate specificity are not confined to the substrate-binding pocket and that longer range interactions are essential in allowing proper positioning of various ligands in the binding pocket. Hence prediction or engineering of substrate specificity solely based on sequence analysis, or even on modelling of the binding pocket, might lead to incorrect functional assignments.

scholarly journals Evolution of substrate specificity in bacterial AA10 lytic polysaccharide monooxygenases

2014 ◽  
Vol 7 (1) ◽  
Author(s):  
Adam J Book ◽  
Ragothaman M Yennamalli ◽  
Taichi E Takasuka ◽  
Cameron R Currie ◽  
George N Phillips ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Lytic Polysaccharide Monooxygenases ◽  
Polysaccharide Monooxygenases
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