Characterization of 4-guanidinobutyrase from Aspergillus niger

Saragadam, Tejaswani; Kumar, Sunil; Punekar, N. S.

doi:10.1099/mic.0.000782

Cited by 4 publications

(14 citation statements)

References 43 publications

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“…Notable exceptions are the Co 2+ -preferring arginase of Helicobacter pylori and the guanidinobutyrases of Aspergillus niger and Arthrobacter sp. KUJ 8602 that depend on Zn 2+ ions [33][34][35] . For the arginase of Bacillus anthracis, Mn 2+ was proposed as the native metal cofactor, but the isolated enzyme was more active when Mn 2+ was replaced by Ni 2+ (refs.…”

Section: Discussionmentioning

confidence: 99%

Discovery of a Ni2+-dependent guanidine hydrolase in bacteria

Funck

Sinn

Fleming

et al. 2022

Nature

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Nitrogen availability is a growth-limiting factor in many habitats 1 , and the global nitrogen cycle involves prokaryotes and eukaryotes competing for this precious resource. Only some bacteria and archaea can fix elementary nitrogen; all other organisms depend on the assimilation of mineral or organic nitrogen. The nitrogenrich compound guanidine occurs widely in nature 2-4 , but its utilization is impeded by pronounced resonance stabilization 5 , and enzymes catalysing hydrolysis of free guanidine have not been identified. Here we describe the arginase family protein GdmH (Sll1077) from Synechocystis sp. PCC 6803 as a Ni 2+ -dependent guanidine hydrolase. GdmH is highly specific for free guanidine. Its activity depends on two accessory proteins that load Ni 2+ instead of the typical Mn 2+ ions into the active site. Crystal structures of GdmH show coordination of the dinuclear metal cluster in a geometry typical for arginase family enzymes and allow modelling of the bound substrate. A unique amino-terminal extension and a tryptophan residue narrow the substrate-binding pocket and identify homologous proteins in further cyanobacteria, several other bacterial taxa and heterokont algae as probable guanidine hydrolases. This broad distribution suggests notable ecological relevance of guanidine hydrolysis in aquatic habitats.

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Section: Discussionmentioning

confidence: 99%

Discovery of a Ni2+-dependent guanidine hydrolase in bacteria

Funck

Sinn

Fleming

et al. 2022

Nature

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“…The yeast strains were maintained on yeast nitrogen base (YNB) agar supplemented with 5 mM ammonium sulfate (AS) and 2% glucose. Saccharomyces cerevisiae 12T7cI ( Δcar1::kanMX4, ura3 ), an arginase-negative yeast mutant, was used to screen error-prone PCR products of A. niger GBase cDNA (Saragadam et al 2019 ). Synthetic minimal medium [glucose 2.0 g, yeast nitrogen base (YNB and AS in 1:3 ratio) 0.66 g, distilled water 100 ml, agar 2.0 g] was used for this screening and the transformants were later replica plated on synthetic minimal medium supplemented with either 5 mM GB or GP instead of AS.…”

Section: Methodsmentioning

confidence: 99%

“…The resultant p426GPD-CparGB plasmid was transformed into S. cerevisiae 12T7cI strain ( Δcar1::kanMX4, ura3 ); the p426GPD-GB (for expressing A. niger GBase) was also transformed in parallel, for comparison. The AnGBase cDNA was moved as an Eco RI- Xho I fragment into p426GPD to obtain p426GPD-GB (Saragadam et al 2019 ). Yeast transformed with empty p426GPD vector served as control.…”

Section: Methodsmentioning

confidence: 99%

“…Interestingly, GB is efficiently utilized by A. niger , but GP is not. The A. niger GBase acts 25 times more efficiently on GB than on GP, and the ∆gbu (GBase knockout) strain was unable to grow on GP (Saragadam et al 2019 ). This suggested the absence of a specific GPase in this fungus.…”

Section: Introductionmentioning

confidence: 99%

“…Not many GBase structures are available (Nakada and Itoh 2002 , 2005 , Lee et al 2011 ), and hence rational substrate specificity engineering through site-directed mutagenesis is difficult. At first, we attempted to improve AnGBase specificity towards GP through EMS mutagenesis and screening of A. niger conidia, albeit with little success (Saragadam et al 2019 ). A directed evolution approach (using error-prone PCR) was also undertaken; for this, in vitro , mutated AnGBase ORFs were transformed into Saccharomyces cerevisiae 12T7cI ( Δcar1::kanMX4, ura3 ).…”

Section: Introductionmentioning

confidence: 99%

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Characterization of a novel 4-guanidinobutyrase from Candida parapsilosis

Gaikwad,

Punekar,

Pathan

2024

FEMS Yeast Research

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Enzymes of the ureohydrolase superfamily are specific in recognizing their substrates. While looking to broaden the substrate specificity of 4-guanidinobutyrase (GBase), we isolated a yeast, typed as Candida parapsilosis (NCIM 3689), that efficiently utilized both 4-guanidinobutyrate (GB) and 3-guanidinopropionate (GP) as a sole source of nitrogen. A putative GBase sequence was identified from its genome upon pBLAST query using the GBase sequence from Aspergillus niger (AnGBase). The C. parapsilosis GBase (CpGBase) ORF was PCR amplified, cloned and sequenced. Further, the functional CpGBase protein expressed in Saccharomyces cerevisiae functioned as GBase and 3-guanidinopropionase (GPase). S. cerevisiae cannot grow on GB or GP. However, the transformants expressing CpGBase acquired the ability to utilize and grow on both GB and GP. The expressed CpGBase protein was enriched and analyzed for substrate saturation and product inhibition by γ-aminobutyric acid and β-alanine. In contrast to the well-characterized AnGBase, CpGBase from C. parapsilosis is a novel ureohydrolase and showed hyperbolic saturation for GB and GP with comparable efficiency (Vmax/KM values of 3.4 and 2.0, respectively). With the paucity of structural information and limited active site data available on ureohydrolases, CpGBase offers an excellent paradigm to explore this class of enzymes.

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Discovery of a Ni2+-dependent heterohexameric metformin hydrolase

Li,

Xu,

Zhang

et al. 2024

Nat Commun

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The biguanide drug metformin is a first-line blood glucose-lowering medication for type 2 diabetes, leading to its presence in the global environment. However, little is known about the fate of metformin by microbial catabolism. Here, we characterize a Ni2+-dependent heterohexameric enzyme (MetCaCb) from the ureohydrolase superfamily, catalyzing the hydrolysis of metformin into guanylurea and dimethylamine. Either subunit alone is catalytically inactive, but together they work as an active enzyme highly specific for metformin. The crystal structure of the MetCaCb complex shows the coordination of the binuclear metal cluster only in MetCa, with MetCb as a protein binder of its active cognate. An in-silico search and functional assay discover a group of MetCaCb-like protein pairs exhibiting metformin hydrolase activity in the environment. Our findings not only establish the genetic and biochemical foundation for metformin catabolism but also provide additional insights into the adaption of the ancient enzymes toward newly occurred substrate.

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Characterization of 4-guanidinobutyrase from Aspergillus niger

Cited by 4 publications

References 43 publications

Discovery of a Ni2+-dependent guanidine hydrolase in bacteria

Discovery of a Ni2+-dependent guanidine hydrolase in bacteria

Characterization of a novel 4-guanidinobutyrase from Candida parapsilosis

Discovery of a Ni2+-dependent heterohexameric metformin hydrolase

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