Skip to main content
SpringerLink
Log in

Genetic and biochemical characterization of a 4-hydroxybenzoate hydroxylase from Corynebacterium glutamicum

  • Biotechnologically Relevant Enzymes and Proteins
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

Corynebacterium glutamicum uses 4-hydroxybenzoic acid (4HBA) as sole carbon source for growth. Previous studies showed that 4HBA was taken up into cells via PcaK, and the aromatic ring was cleaved via protocatechuate 3,4-dioxygenase. In this study, the gene pobA Cg (ncgl1032) involved in the conversion of 4HBA into 3,4-dihydroxybenzoate (protocatechuate) was identified, and the gene product PobA Cg was characterized as a 4HBA 3-hydroxylase, which is a homodimer of PobACg. The pobA Cg is physically associated with pcaK and formed a putative operon, but the two genes were located distantly to the pca cluster, which encode other enzymes for 4HBA/protocatechuate degradation. This new 4HBA 3-hydroxylase is unique in that it prefers NADPH to NADH as a cosubstrate, although its sequence is similar to other 4HBA 3-hydroxylases that prefer NADH as a cosubstrate. Sited-directed mutagenesis on putative NADPH-binding sites, D38 and T42, further improved its affinity to NADPH as well as its catalytic efficiency.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  • Bertani I, Kojic M, Venturi V (2001) Regulation of the p-hydroxybenzoic acid hydroxylase gene (pobA) in plant-growth-promoting Pseudomonas putida WCS358. Microbiology (SGM) 147:1611–1620

    Article  CAS  Google Scholar 

  • Biegert T, Altenschmidt U, Eckerskorn C, Fuchs G (1993) Enzymes of anaerobic metabolism of phenolic compounds. 4-Hydroxybenzoate-CoA ligase from a denitrifying Pseudomonas species. Eur J Biochem 213:555–561

    Article  CAS  PubMed  Google Scholar 

  • Brzostowicz PC, Reams AB, Clark TJ, Neidle EL (2003) Transcriptional cross-regulation of the catechol and protocatechuate branches of the beta-ketoadipate pathway contributes to carbon source-dependent expression of the Acinetobacter sp. strain ADP1 pobA gene. Appl Environ Microbiol 69:1598–1606

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Chaudhry MT, Huang Y, Shen XH, Poetsch A, Jiang CY, Liu SJ (2007) Genome-wide investigation of aromatic acid transporters in Corynebacterium glutamicum. Microbiology (SGM) 153:857–865

    Article  CAS  Google Scholar 

  • Eggink G, Engel H, Vriend G, Terpstra P, Witholt B (1990) Rubredoxin reductase of Pseudomonas oleovorans. Structural relationship to other flavoprotein oxidoreductases based on one NAD and two FAD fingerprints. J Mol Biol 212:135–142

    Article  CAS  PubMed  Google Scholar 

  • Entsch B, Ballou DP (1989) Purification, properties, and oxygen reactivity of p-hydroxybenzoate hydroxylase from Pseudomonas aeruginosa. Biochim Biophys Acta 999:313–322

    Article  CAS  PubMed  Google Scholar 

  • Entsch B (1990) Hydroxybenzoate hydroxylase. Methods Enzymol 188:138–147

    Article  CAS  PubMed  Google Scholar 

  • Eppink MH, Boeren SA, Vervoort J, van Berkel WJ (1997a) Purification and properties of 4-hydroxybenzoate 1-hydroxylase (decarboxylating), a novel flavin adenine dinucleotide-dependent monooxygenase from Candida parapsilosis CBS604. J Bacteriol 179:6680–6687

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Eppink MH, Schreuder HA, van Berkel WJ (1997b) Identification of a novel conserved sequence motif in flavoprotein hydroxylases with a putative dual function in FAD/NAD(P)H binding. Protein Sci 6:2454–2458

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Eppink MH, Schreuder HA, van Berkel WJ (1998) Lys42 and Ser42 variants of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens reveal that Arg42 is essential for NADPH binding. Euro J Biochem 253:194–201

    Article  CAS  Google Scholar 

  • Eppink MH, Overkamp KM, Schreuder HA, Van Berkel WJ (1999) Switch of coenzyme specificity of p-hydroxybenzoate hydroxylase. J Mol Biol 292:87–96

    Article  CAS  PubMed  Google Scholar 

  • Eschrich K, van der Bolt FJ, de Kok A, van Berkel WJ (1993) Role of Tyr201 and Tyr385 in substrate activation by p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens. Eur J Biochem 216:137–146

    Article  CAS  PubMed  Google Scholar 

  • Gatti DL, Palfey BA, Lah MS, Entsch B, Massey V, Ballou DP, Ludwig ML (1994) The mobile flavin of 4-OH benzoate hydroxylase. Science 266:110–114

    Article  CAS  PubMed  Google Scholar 

  • Hosokawa K, Stanier RY (1966) Crystallization and properties of p-hydroxybenzoate hydroxylase from Pseudomonas putida. J Biol Chem 241:2453–2460

    Article  CAS  PubMed  Google Scholar 

  • Huang K, Lin YG, Winter HH (1992) p-Hydroxybenzoate/ethylene terephthalate copolyester: structure of high-melting crystals formed during partially molten state annealing. Polymer 33:4533–4537

    Article  CAS  Google Scholar 

  • Ikeda M, Nakagawa S (2003) The Corynebacterium glutamicum genome: features and impacts on biotechnological processes. Appl Microbiol Biotechnol 62:99–109

    Article  CAS  PubMed  Google Scholar 

  • Iwaki H, Saji H, Abe K, Hasegawa Y (2005) Cloning and sequence analysis of the 4-hydroxybenzoate 3-hydroxylase gene from a cyclohexanecarboxylate-degrading gram-positive bacterium, “Corynebacterium cyclohexanicum” strain ATCC 51369. Microbes Envion 20:144–150

    Article  Google Scholar 

  • Jadan AP, van Berkel WJ, Golovleva LA, Golovlev EL (2001) Purification and properties of p-hydroxybenzoate hydroxylases from Rhodococcus strains. Biochemistry (Mosc) 66:898–903

    Article  CAS  Google Scholar 

  • Jakoby M, Ngouoto-Nkili CE, Burkovski A (1999) Construction and application of new Corynebacterium glutamicum vectors. Biotechnol Tech 13:437–441

    Article  CAS  Google Scholar 

  • DiMarco AA, Ornston LN (1994) Regulation of p-hydroxybenzoate hydroxylase synthesis by PobR bound to an operator in Acinetobacter calcoaceticus. J Bacteriol 176:4277–4284

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ortiz-Maldonado M, Entsch B, Ballou DP (2004) Oxygen reactions in p-hydroxybenzoate hydroxylase utilize the H-bond network during catalysis. Biochemistry 43:15246–15257

    Article  CAS  PubMed  Google Scholar 

  • Overhage J, Kresse AU, Priefert H, Sommer H, Krammer G, Rabenhorst J, Steinbüchel A (1999) Molecular characterization of the genes pcaG and pcaH, encoding protocatechuate 3,4-dioxygenase, which are essential for vanillin catabolism in Pseudomonas sp. strain HR199. Appl Environ Microbiol 65:951–960

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Peng X, Misawa N, Harayama S (2003) Isolation and characterization of thermophilic bacilli degrading cinnamic, 4-coumaric, and ferulic acids. Appl Environ Microbiol 69:1417–1427

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Peng X, Adachi K, Chen C, Kasai H, Kanoh K, Shizuri Y, Misawa N (2006) Discovery of a marine bacterium producing 4-hydroxybenzoate and its alkyl esters, parabens. Appl Environ Microbiol 72:5556–5561

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Qi S-W, Chaudhry MT, Zhang Y, Meng B, Huang Y, Zhao K-X, Poetsch A, Jiang C-Y, Liu S, Liu S-J (2007) Comparative proteomes of Corynebacterium glutamicum grown on aromatic compounds revealed novel proteins involved in aromatic degradation and a clear link between aromatic catabolism and gluconeogenesis via fructose-1,6-bisphosphatase. Proteomics 7:3775–3787

    Article  CAS  PubMed  Google Scholar 

  • Quinn JA, McKay DB, Entsch B (2001) Analysis of the pobA and pobR genes controlling expression of p-hydroxybenzoate hydroxylase in Azotobacter chroococcum. Gene 264:77–85

    Article  CAS  PubMed  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

    Google Scholar 

  • Sambrook J, Russell DW (2001) Molecular cloning, 3rd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp 1319–1325

    Google Scholar 

  • Schäfer A, Tauch A, Jager W, Kalinowski J, Thierbach G, Pühler A (1994) Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum. Gene 145:69–73

    Article  PubMed  Google Scholar 

  • Seibold B, Matthes M, Eppink MH, Lingens F, van Berkel WJ, Mueller R (1996) 4-Hydroxybenzoate hydroxylase from Pseudomonas sp. CSB3 purification, characterization, gene cloning, sequence analysis and assignment of structural features determining the cofactor specificity. Eur J Biochem 239:469–478

    Article  CAS  PubMed  Google Scholar 

  • Shen X-H, Huang Y, Liu S-J (2005) Genomic analysis and identification of catabolic pathways for aromatic compounds in Corynebacterium glutamicum. Microbes Environ 20:160–167

    Article  Google Scholar 

  • Soni MG, Taylor SL, Greenberg NA, Burdock GA (2002) Evaluation of the health aspects of methyl paraben: a review of the published literature. Food Chem Toxicol 40:1335–1373

    Article  CAS  PubMed  Google Scholar 

  • Sterjiades R (1993) Properties of NADH/NADPH-dependent p-hydroxybenzoate hydroxylase from Moraxella sp. Biotechnol Appl Biochem 17:77–90

    CAS  Google Scholar 

  • Sznitowska M, Janicki S, Dabrowska EA, Gajewska M (2002) Physicochemical screening of antimicrobial agents as potential preservatives for submicron emulsions. Eur J Pharm Sci 15:489–495

    Article  CAS  PubMed  Google Scholar 

  • Tauch A, Kassing F, Kalinowski J, Puhler A (1995) The Corynebacterium xerosis composite transposon Tn5432 consists of two identical insertion sequences, designated IS1249, flanking the erythromycin resistance gene ermCX. Plasmid 34:119–131

    Article  CAS  PubMed  Google Scholar 

  • Tauch A, Kirchner O, Loffler B, Gotker S, Puhler A, Kalinowski J (2002) Efficient electrotransformation of Corynebacterium diphtheriae with a mini-replicon derived from the Corynebacterium glutamicum plasmid pGA1. Curr Microbiol 45:362–367

    Article  CAS  PubMed  Google Scholar 

  • Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Tsuji H, Oka T, Kimoto M, Hong YM, Natori Y, Ogawa T (1996) Cloning and sequencing of cDNA encoding 4-aminobenzoate hydroxylase from Agaricus bisporus. Biochim Biophys Acta 1309:31–36

    Article  PubMed  Google Scholar 

  • Walker KD, Klettke K, Akiyama T, Croteau R (2004) Cloning, heterologous expression, and characterization of a phenylalanine aminomutase involved in Taxol biosynthesis. J Biol Chem 279:53947–53954

    Article  CAS  PubMed  Google Scholar 

  • Wang J, Ortiz-Maldonado M, Entsch B, Massey V, Ballou D, Gatti DL (2002) Protein and ligand dynamics in 4-hydroxybenzoate hydroxylase. Proc Nat Acad Sci USA 99:608–613

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wierenga RK, de Jong RJ, Kalk KH, Hol WG, Drenth J (1979) Crystal structure of p-hydroxybenzoate hydroxylase. J Mol Biol 131:55–73

    Article  CAS  PubMed  Google Scholar 

  • Wierenga RK, Terpstra P, Hol WG (1986) Prediction of the occurrence of the ADP-binding beta alpha beta-fold in proteins, using an amino acid sequence fingerprint. J Mol Biol 187:101–107

    Article  CAS  PubMed  Google Scholar 

  • Wong CM, Dilworth MJ, Glenn AR (1994) Cloning and sequencing show that 4-hydroxybenzoate hydroxylase (PobA) is required for uptake of 4-hydroxybenzoate in Rhizobium leguminosarum. Microbiology (SGM) 140:2775–2786

    Article  CAS  Google Scholar 

  • Xiang L, Moore BS (2003) Characterization of benzoyl coenzyme A biosynthesis genes in the enterocin-producing bacterium “Streptomyces maritimus”. J Bacteriol 185:399–404

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

This work was supported by grants from the National Natural Science Foundation of China (30725001, 20577067) and Chinese Academy of Sciences (KSCX2-YW-G-009). Careful reading and corrections by Prof. Joseph Zeyer at Institute for Biogeochemistry and Pollutant Dynamics, Federal Institute of Technology, Switzerland, are highly acknowledged.

Author information

Authors and Affiliations

  1. State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Datun Road Jia-3#, Chaoyang District, Beijing, 100101, People’s Republic of China

    Yan Huang, Ke-xin Zhao, Xi-Hui Shen, Chen-Ying Jiang & Shuang-Jiang Liu

Authors
  1. Yan Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ke-xin Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Xi-Hui Shen
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Chen-Ying Jiang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Shuang-Jiang Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Shuang-Jiang Liu.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Huang, Y., Zhao, Kx., Shen, XH. et al. Genetic and biochemical characterization of a 4-hydroxybenzoate hydroxylase from Corynebacterium glutamicum . Appl Microbiol Biotechnol 78, 75–83 (2008). https://doi.org/10.1007/s00253-007-1286-0

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-007-1286-0

Keywords

Search

Navigation