Cloning, expression, purification, and characterization of Nocardia sp. GTP cyclohydrolase I

He, Aimin; Simpson, Dan; Daniels, Lacy; Rosazza, John P. N.

doi:10.1016/j.pep.2004.02.008

Cited by 10 publications

(7 citation statements)

References 42 publications

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“…Multiple sequence alignment of the M. alpina GTPCHs and other characterized GTPCH proteins reveals the presence of certain conserved motifs and residues (Supplementary Fig. S3), including five conserved motifs (Lys-Thr-Pro-Xaa-Arg-Xaa-Ala, Ser-Xaa-Cys-Glu-His-His, Gly-Leu-Ser-Lys-Leu-Ala-Arg, Gln-Val-Gln-Glu-Arg-Leu-Thr and Cys-Met-Val-Met-Arg-Gly-Val) that form a GTP-binding pocket (Du et al , 2009; He et al , 2004; Maita et al , 2004); a Lys-Thr-Arg-Glu motif at the C terminus involved in 7,8-dihydrobiopterin binding, resulting in inhibition of GTPCH activity (Du et al , 2009; Maita et al , 2004); four residues (Thr108, Ser153, Ser191 and Thr255) involved in multiple phosphorylation regulation, both positively and negatively (Du et al , 2009); and six residues (Ala205, Ala207, Pro214, Gly247, Arg250 and Lys254) involved in stimulatory or inhibitory effects on the GTPCH feedback regulatory protein (GFRP) (Maita et al , 2004). Compared with the highly conserved central and C-terminal amino acids of rat GTPCH, which are responsible for substrate binding and catalysis, the N-terminal parts of GTPCH differ depending on the species examined.…”

Section: Discussionmentioning

confidence: 99%

Biochemical characterization of the tetrahydrobiopterin synthesis pathway in the oleaginous fungus Mortierella alpina

et al. 2011

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We characterized the de novo biosynthetic pathway of tetrahydrobiopterin (BH 4 ) in the lipidproducing fungus Mortierella alpina. The BH 4 cofactor is essential for various cell processes, and is probably present in every cell or tissue of higher organisms. Genes encoding two copies of GTP cyclohydrolase I (GTPCH-1 and GTPCH-2) for the conversion of GTP to dihydroneopterin triphosphate (H 2 -NTP), 6-pyruvoyltetrahydropterin synthase (PTPS) for the conversion of H 2 -NTP to 6-pyruvoyltetrahydropterin (PPH 4 ), and sepiapterin reductase (SR) for the conversion of PPH 4 to BH 4 , were expressed heterologously in Escherichia coli. The recombinant enzymes were produced as His-tagged fusion proteins and were purified to homogeneity to investigate their enzymic activities. Enzyme products were analysed by HPLC and electrospray ionization-MS. Kinetic parameters and other properties of GTPCH, PTPS and SR were investigated. Physiological roles of BH 4 in M. alpina are discussed, and comparative analyses between GTPCH, PTPS and SR proteins and other homologous proteins were performed. The presence of two functional GTPCH enzymes has, as far as we are aware, not been reported previously, reflecting the unique ability of this fungus to synthesize both BH 4 and folate, using the GTPCH product as a common substrate. To our knowledge, this study is the first to report the comprehensive characterization of a BH 4 biosynthesis pathway in a fungus.

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Section: Discussionmentioning

confidence: 99%

Biochemical characterization of the tetrahydrobiopterin synthesis pathway in the oleaginous fungus Mortierella alpina

et al. 2011

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“…Guanosine triphosphate cyclohydrolase I GCH1 is reported in various microorganisms, including Escherichia coli ( Katzenmeier et al, 1990 ), Saccharomyces cerevisiae ( Nardese et al, 1996 ), Nocardia sp. ( He et al, 2004 ), and Rickettsia monacensis ( Bodnar et al, 2020 ). The comparison of sequences from different organisms has revealed that the enzyme was highly conserved throughout the evolutionary process.…”

Section: Introductionmentioning

confidence: 99%

A Putative Guanosine Triphosphate Cyclohydrolase I Named CaGCH1 Is Involved in Hyphal Branching and Fruiting Development in Cyclocybe aegerita

et al. 2022

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Guanosine triphosphate (GTP) cyclohydrolase I (GCH1) is the limiting enzyme of the tetrahydrobiopterin (BH4) synthesis pathway. The disruption of gch1 gene may cause conditional lethality due to folic acid auxotrophy in microorganisms, although the function of gch1 in basidiomycetes has not been deciphered so far. In the present study, gch1 expression in Cyclocybe aegerita (cagch1) was downregulated using the RNAi method, which resulted in growth retardation in both solid and liquid medium, with the hyphal tips exhibiting increased branching compared to that in the wild strain. The development of fruiting bodies in the mutant strains was significantly blocked, and there were short and bottle-shaped stipes. The transcriptional profile revealed that the genes of the MAPK pathway may be involved in the regulation of these effects caused by cagch1 knockdown, which provided an opportunity to study the role of gch1 in the development process of basidiomycetes.

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“…As parts of the NOS system, Nocardia sp. NRRL 5646 contains guanylate cyclase for cyclic GMP synthesis (Son & Rosazza, 2000), and GTP-cyclohydrolase I ( gch , GenBank accession number AY128670, He et al ., 2004b) for tetrahydrobiopterin biosynthesis.…”

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confidence: 99%

Nocardia iowensis sp. nov., an organism rich in biocatalytically important enzymes and nitric oxide synthase

Lamm

Khare

Conville

et al. 2009

INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLO

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Nocardia strain NRRL 5646, isolated from a garden soil sample in Osceola, Iowa, USA, was initially of interest as an antibiotic producer. It contained biocatalytically important enzymes and represented the first described nitric oxide synthase enzyme system in bacteria. The present polyphasic taxonomic study was undertaken to differentiate strain NRRL 5646T from related species of the genus Nocardia. Chemotaxonomic analyses included determinations of the fatty acid methyl ester profile (C 16 : 1 v6c/C 16 : 1 v7c, C 16 : 0 , C 18 : 1 v9c and C 18 : 0 10-methyl as major components), quinone [cyclo MK-8(H 4 ) as the major component], polar lipid (diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositol mannoside as major components) and mycolic acid. These results supported its placement within the genus Nocardia. Biochemical testing and 16S rRNA, 65-kDa heat-shock protein (hsp65) and preprotein translocase (secA1) gene sequence analyses differentiated strain NRRL 5646 T from recognized Nocardia species. Previous studies have demonstrated that other genetic sequences (carboxylic acid reductase, Nocardia phosphopantetheinyl transferase and GTP cyclohydrolase I) from strain NRRL 5646 T can also be used to substantiate its uniqueness. The level of 16S rRNA gene sequence similarity between strain NRRL 5646 T and the type strains of Nocardia tenerifensis and Nocardia brasiliensis was 98.8 %. However, strain NRRL 5646 T could be clearly distinguished from these Nocardia species based on DNA-DNA hybridization data. Consequently, strain NRRL 5646 T is considered to represent a novel species of the genus Nocardia, for which the name Nocardia iowensis sp. nov. is proposed. The type strain is NRRL 5646 T (5UI 122540Strain NRRL 5646 T was initially identified as an antibioticproducing bacterium isolated from a garden soil sample in Osceola, Iowa, USA (Hlavka & Bitha, 1977;Martin et al., 1977). This strain showed an extraordinary diversity of biotransforming enzymes and the first bacterial nitric oxide synthase (NOS) system with possible physiological importance, making its identification to the species level important. Strain NRRL 5646 T converts many natural and synthetic organic compounds into valuable products, including flavonoids (Herath et al., 2006;Maatooq & Rosazza, 2005), vanillic acid (Dhar et al., 2007) and 4-vinylphenol (Lee &Rosazza, 2002). A novel ATP/NADPHdependent carboxylic acid reductase was isolated from strain NRRL 5646T (Li & Rosazza, 1997) and subsequently cloned (car; GenBank accession number AY495697; He et al., 2004a). The reduction system required a phosphopantetheinyl transferase (npt, GenBank accession number DQ904035; Venkitasubramanian et al., 2007). Strain NRRL 5646 T contains the first NOS system characterized in prokaryotes (Chen & Rosazza, 1995). As part of the NOS system, strain NRRL 5646 T contains guanylate cyclase for cyclic GMP synthesis (Son & Rosazza, 2000), and GTP cyclohydrolase I (gch; GenBank accession number Abbreviation: NOS, nitric oxide ...

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Cloning, expression, purification, and characterization of Nocardia sp. GTP cyclohydrolase I

Cited by 10 publications

References 42 publications

Biochemical characterization of the tetrahydrobiopterin synthesis pathway in the oleaginous fungus Mortierella alpina

Biochemical characterization of the tetrahydrobiopterin synthesis pathway in the oleaginous fungus Mortierella alpina

A Putative Guanosine Triphosphate Cyclohydrolase I Named CaGCH1 Is Involved in Hyphal Branching and Fruiting Development in Cyclocybe aegerita

Nocardia iowensis sp. nov., an organism rich in biocatalytically important enzymes and nitric oxide synthase

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