scholarly journals Biochemical and Kinetic Characterization of the Eukaryotic Phosphotransacetylase Class IIa Enzyme from Phytophthora ramorum

2015 ◽  
Vol 14 (7) ◽  
pp. 652-660
Author(s):  
Tonya Taylor ◽  
Cheryl Ingram-Smith ◽  
Kerry S. Smith
Keyword(s):  
Phytophthora Ramorum ◽  
Regulatory Domain ◽  
Kinetic Characterization ◽  
Acetyl Phosphate ◽  
Acetyl Coa ◽  
Content Type ◽  
Regulatory Domains ◽  
Key Enzyme ◽  
Acetyl Group

ABSTRACT Phosphotransacetylase (Pta), a key enzyme in bacterial metabolism, catalyzes the reversible transfer of an acetyl group from acetyl phosphate to coenzyme A (CoA) to produce acetyl-CoA and P i . Two classes of Pta have been identified based on the absence (Pta I ) or presence (Pta II ) of an N-terminal regulatory domain. Pta I has been fairly well studied in bacteria and one genus of archaea; however, only the Escherichia coli and Salmonella enterica Pta II enzymes have been biochemically characterized, and they are allosterically regulated. Here, we describe the first biochemical and kinetic characterization of a eukaryotic Pta from the oomycete Phytophthora ramorum . The two Ptas from P. ramorum , designated PrPta II 1 and PrPta II 2, both belong to class II. PrPta II 1 displayed positive cooperativity for both acetyl phosphate and CoA and is allosterically regulated. We compared the effects of different metabolites on PrPta II 1 and the S. enterica Pta II and found that, although the N-terminal regulatory domains share only 19% identity, both enzymes are inhibited by ATP, NADP, NADH, phosphoenolpyruvate (PEP), and pyruvate in the acetyl-CoA/P i -forming direction but are differentially regulated by AMP. Phylogenetic analysis of bacterial, archaeal, and eukaryotic sequences identified four subtypes of Pta II based on the presence or absence of the P-loop and DRTGG subdomains within the N-terminal regulatory domain. Although the E. coli , S. enterica , and P. ramorum enzymes all belong to the IIa subclass, our kinetic analysis has indicated that enzymes within a subclass can still display differences in their allosteric regulation.

scholarly journals Contributions of Environmental Signals and Conserved Residues to the Functions of Carbon Storage Regulator A of Borrelia burgdorferi

2013 ◽  
Vol 81 (8) ◽  
pp. 2972-2985 ◽  
Author(s):  
S. L. Rajasekhar Karna ◽  
Rajesh G. Prabhu ◽  
Ying-Han Lin ◽  
Christine L. Miller ◽  
J. Seshu
Keyword(s):  
Borrelia Burgdorferi ◽  
Carbon Storage ◽  
Vertebrate Host ◽  
Acetyl Phosphate ◽  
Mobility Shift ◽  
Acetyl Coa ◽  
Site Specific ◽  
Content Type ◽  
Environmental Signals ◽  
Carbon Storage Regulator

ABSTRACTCarbon storage regulator A ofBorrelia burgdorferi(CsrABb) contributes to vertebrate host-specific adaptation by modulating activation of the Rrp2-RpoN-RpoS pathway and is critical for infectivity. We hypothesized that the functions of CsrABbare dependent on environmental signals and on select residues. We analyzed the phenotype ofcsrABbdeletion and site-specific mutants to determine the conserved and pathogen-specific attributes of CsrABb. Levels of phosphate acetyltransferase (Pta) involved in conversion of acetyl phosphate to acetyl-coenzyme A (acetyl-CoA) and posttranscriptionally regulated by CsrABbin thecsrABbmutant were reduced from or similar to those in the control strains under unfed- or fed-tick conditions, respectively. Increased levels of supplemental acetate restored vertebrate host-responsive determinants in thecsrABbmutant to parental levels, indicating that both the levels of CsrABband the acetyl phosphate and acetyl-CoA balance contribute to the activation of the Rrp2-RpoN-RpoS pathway. Site-specific replacement of 8 key residues of CsrABb(8S) with alanines resulted in increased levels of CsrABband reduced levels of Pta and acetyl-CoA, while levels of RpoS, BosR, and other members ofrpoSregulon were elevated. Truncation of 7 amino acids at the C terminus of CsrABb(7D) resulted in reducedcsrABbtranscripts and posttranscriptionally reduced levels of FliW located upstream of CsrABb. Electrophoretic mobility shift assays revealed increased binding of 8S mutant protein to the CsrA binding box upstream ofptacompared to the parental and 7D truncated protein. Two CsrABbbinding sites were also identified upstream offliWwithin theflgKcoding sequence. These observations reveal conserved and unique functions of CsrABbthat regulate adaptive gene expression inB. burgdorferi.

scholarly journals A Pathway for Degradation of Uracil to Acetyl Coenzyme A in Bacillus megaterium

2020 ◽  
Vol 86 (7) ◽  
Author(s):  
Di Zhu ◽  
Yifeng Wei ◽  
Jinyu Yin ◽  
Dazhi Liu ◽  
Ee Lui Ang ◽  
...  
Keyword(s):  
Energy Source ◽  
Bacillus Megaterium ◽  
Coenzyme A ◽  
Catabolic Pathway ◽  
Acetyl Coa ◽  
Acetyl Coenzyme A ◽  
Content Type ◽  
Biochemical Pathways ◽  
Acetyl Coenzyme

ABSTRACT Bacteria utilize diverse biochemical pathways for the degradation of the pyrimidine ring. The function of the pathways studied to date has been the release of nitrogen for assimilation. The most widespread of these pathways is the reductive pyrimidine catabolic pathway, which converts uracil into ammonia, carbon dioxide, and β-alanine. Here, we report the characterization of a β-alanine:pyruvate aminotransferase (PydD2) and an NAD+-dependent malonic semialdehyde dehydrogenase (MSDH) from a reductive pyrimidine catabolism gene cluster in Bacillus megaterium. Together, these enzymes convert β-alanine into acetyl coenzyme A (acetyl-CoA), a key intermediate in carbon and energy metabolism. We demonstrate the growth of B. megaterium in defined medium with uracil as its sole carbon and energy source. Homologs of PydD2 and MSDH are found in association with reductive pyrimidine pathway genes in many Gram-positive bacteria in the order Bacillales. Our study provides a basis for further investigations of the utilization of pyrimidines as a carbon and energy source by bacteria. IMPORTANCE Pyrimidine has wide occurrence in natural environments, where bacteria use it as a nitrogen and carbon source for growth. Detailed biochemical pathways have been investigated with focus mainly on nitrogen assimilation in the past decades. Here, we report the discovery and characterization of two important enzymes, PydD2 and MSDH, which constitute an extension for the reductive pyrimidine catabolic pathway. These two enzymes, prevalent in Bacillales based on our bioinformatics studies, allow stepwise conversion of β-alanine, a previous “end product” of the reductive pyrimidine degradation pathway, to acetyl-CoA as carbon and energy source.

scholarly journals Characterization of the Highly Active Polyhydroxyalkanoate Synthase of Chromobacterium sp. Strain USM2

2011 ◽  
Vol 77 (9) ◽  
pp. 2926-2933 ◽  
Author(s):  
Kesaven Bhubalan ◽  
Jo-Ann Chuah ◽  
Fumi Shozui ◽  
Christopher J. Brigham ◽  
Seiichi Taguchi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Specific Activity ◽  
Weight Percent ◽  
Pha Synthase ◽  
Content Type ◽  
Highly Active ◽  
Key Enzyme ◽  
Polyhydroxyalkanoate Synthase

ABSTRACTThe synthesis of bacterial polyhydroxyalkanoates (PHA) is very much dependent on the expression and activity of a key enzyme, PHA synthase (PhaC). Many efforts are being pursued to enhance the activity and broaden the substrate specificity of PhaC. Here, we report the identification of a highly active wild-type PhaC belonging to the recently isolatedChromobacteriumsp. USM2 (PhaCCs). PhaCCsshowed the ability to utilize 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), and 3-hydroxyhexanoate (3HHx) monomers in PHA biosynthesis. Anin vitroassay of recombinant PhaCCsexpressed inEscherichia colishowed that its polymerization of 3-hydroxybutyryl-coenzyme A activity was nearly 8-fold higher (2,462 ± 80 U/g) than that of the synthase from the model strainC. necator(307 ± 24 U/g). Specific activity using a Strep2-tagged, purified PhaCCswas 238 ± 98 U/mg, almost 5-fold higher than findings of previous studies using purified PhaC fromC. necator. Efficient poly(3-hydroxybutyrate) [P(3HB)] accumulation inEscherichia coliexpressing PhaCCsof up to 76 ± 2 weight percent was observed within 24 h of cultivation. To date, this is the highest activity reported for a purified PHA synthase. PhaCCsis a naturally occurring, highly active PHA synthase with superior polymerizing ability.

scholarly journals Biochemical and Kinetic Characterization of Xylulose 5-Phosphate/Fructose 6-Phosphate Phosphoketolase 2 (Xfp2) from Cryptococcus neoformans

2014 ◽  
Vol 13 (5) ◽  
pp. 657-663 ◽  
Author(s):  
Katie Glenn ◽  
Cheryl Ingram-Smith ◽  
Kerry S. Smith
Keyword(s):  
Cryptococcus Neoformans ◽  
Fungal Pathogen ◽  
Allosteric Regulation ◽  
Valuable Insight ◽  
Kinetic Characterization ◽  
Content Type ◽  
Metabolic Role ◽  
Separate Site ◽  
Opportunistic Fungal Pathogen

ABSTRACTXylulose 5-phosphate/fructose 6-phosphate phosphoketolase (Xfp), previously thought to be present only in bacteria but recently found in fungi, catalyzes the formation of acetyl phosphate from xylulose 5-phosphate or fructose 6-phosphate. Here, we describe the first biochemical and kinetic characterization of a eukaryotic Xfp, from the opportunistic fungal pathogenCryptococcus neoformans, which has twoXFPgenes (designatedXFP1andXFP2). Our kinetic characterization ofC. neoformansXfp2 indicated the existence of both substrate cooperativity for all three substrates and allosteric regulation through the binding of effector molecules at sites separate from the active site. Prior to this study, Xfp enzymes from two bacterial genera had been characterized and were determined to follow Michaelis-Menten kinetics.C. neoformansXfp2 is inhibited by ATP, phosphoenolpyruvate (PEP), and oxaloacetic acid (OAA) and activated by AMP. ATP is the strongest inhibitor, with a half-maximal inhibitory concentration (IC50) of 0.6 mM. PEP and OAA were found to share the same or have overlapping allosteric binding sites, while ATP binds at a separate site. AMP acts as a very potent activator; as little as 20 μM AMP is capable of increasing Xfp2 activity by 24.8% ± 1.0% (mean ± standard error of the mean), while 50 μM prevented inhibition caused by 0.6 mM ATP. AMP and PEP/OAA operated independently, with AMP activating Xfp2 and PEP/OAA inhibiting the activated enzyme. This study provides valuable insight into the metabolic role of Xfp within fungi, specifically the fungal pathogenCryptococcus neoformans, and suggests that at least some Xfps display substrate cooperative binding and allosteric regulation.

scholarly journals Kinetic Characterization of the Inhibition of Plant Acetyl-CoA Carboxylase by the Grass Herbicides, Quizalofop and Sethoxydim

1990 ◽  
Vol 15 (2) ◽  
pp. 245-247 ◽  
Author(s):  
Kunimitu NAKAHIRA ◽  
Osamu HAYASHI ◽  
Masaaki UCHIYAMA ◽  
Koichi SUZUKI
Keyword(s):  
Kinetic Characterization ◽  
Acetyl Coa Carboxylase ◽  
Acetyl Coa

scholarly journals Molecular Characterization of a NovelN-Acetyltransferase from Chryseobacterium sp.

2013 ◽  
Vol 80 (5) ◽  
pp. 1770-1776 ◽  
Author(s):  
Shinji Takenaka ◽  
Kenji Yoshida ◽  
Kosei Tanaka ◽  
Ken-ichi Yoshida
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Hypothetical Protein ◽  
Relative Activity ◽  
Acyl Donor ◽  
Aminosalicylic Acid ◽  
Acetyl Coa ◽  
Content Type ◽  
Acetyl Group ◽  
Distinct Lineage

ABSTRACTN-Acetyltransferase fromChryseobacteriumsp. strain 5-3B is an acetyl coenzyme A (acetyl-CoA)-dependent enzyme that catalyzes the enantioselective transfer of an acetyl group from acetyl-CoA to the amino group ofl-2-phenylglycine to produce (2S)-2-acetylamino-2-phenylacetic acid. We purified the enzyme from strain 5-3B and deduced the N-terminal amino acid sequence. The gene, designatednatA, was cloned with two other hypothetical protein genes; the three genes probably form a 2.5-kb operon. The deduced amino acid sequence of NatA showed high levels of identity to sequences of putativeN-acetyltransferases ofChryseobacteriumspp. but not to other known arylamine and arylalkylamineN-acetyltransferases. Phylogenetic analysis indicated that NatA forms a distinct lineage from knownN-acetyltransferases. We heterologously expressed recombinant NatA (rNatA) inEscherichia coliand purified it. rNatA showed high activity forl-2-phenylglycine and its chloro- and hydroxyl-derivatives. TheKmandVmaxvalues forl-2-phenylglycine were 0.145 ± 0.026 mM and 43.6 ± 2.39 μmol · min−1· mg protein−1, respectively. The enzyme showed low activity for 5-aminosalicylic acid and 5-hydroxytryptamine, which are reported as good substrates of a known arylamineN-acetyltransferase and an arylalkylamineN-acetyltransferase. rNatA had a comparatively broad acyl donor specificity, transferring acyl groups tol-2-phenylglycine and producing the corresponding 2-acetylamino-2-phenylacetic acids (relative activity with acetyl donors acetyl-CoA, propanoyl-CoA, butanoyl-CoA, pentanoyl-CoA, and hexanoyl-CoA, 100:108:122:10:<1).

scholarly journals Structural characterization of a GNAT family acetyltransferase from Elizabethkingia anophelis bound to acetyl-CoA reveals a new dimeric interface

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
P. Shirmast ◽  
S. M. Ghafoori ◽  
R. M. Irwin ◽  
J. Abendroth ◽  
S. J. Mayclin ◽  
...  
Keyword(s):  
High Resolution ◽  
Tertiary Structure ◽  
Crystallographic Structure ◽  
Acetyl Coa ◽  
Diverse Range ◽  
In Silico Docking ◽  
Dimeric Interface ◽  
High Resolution Structure ◽  
Acetyl Group

AbstractGeneral control non-repressible 5 (GCN5)-related N-acetyltransferases (GNATs) catalyse the acetylation of a diverse range of substrates, thereby orchestrating a variety of biological processes within prokaryotes and eukaryotes. GNAT enzymes can catalyze the transfer of an acetyl group from acetyl coenzyme A to substrates such as aminoglycoside antibiotics, amino acids, polyamines, peptides, vitamins, catecholamines, and large macromolecules including proteins. Although GNATs generally exhibit low to moderate sequence identity, they share a conserved catalytic fold and conserved structural motifs. In this current study we characterize the high-resolution X-ray crystallographic structure of a GNAT enzyme bound with acetyl-CoA from Elizabethkingia anophelis, an important multi-drug resistant bacterium. The tertiary structure is comprised of six α-helices and nine β-strands, and is similar with other GNATs. We identify a new and uncharacterized GNAT dimer interface, which is conserved in at least two other unpublished GNAT structures. This suggests that GNAT enzymes can form at least five different types of dimers, in addition to a range of other oligomers including trimer, tetramer, hexamer, and dodecamer assemblies. The high-resolution structure presented in this study is suitable for future in-silico docking and structure–activity relationship studies.

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