Role of the malic enzyme in metabolism of the halotolerant methanotroph Methylotuvimicrobium alcaliphilum 20Z

Autoři: Olga N. Rozova aff001;  Ildar I. Mustakhimov aff001;  Sergei Y. But aff001;  Aleksandr S. Reshetnikov aff001;  Valentina N. Khmelenina aff001
Působiště autorů: Federal Research Center “Pushchino Scientific Center for Biological Research of the Russian Academy of Sciences”, G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino, Moscow Region, Russia aff001
Vyšlo v časopise: PLoS ONE 14(11)
Kategorie: Research Article
doi: 10.1371/journal.pone.0225054


The bacteria utilizing methane as a growth substrate (methanotrophs) are important constituents of the biosphere. Methanotrophs mitigate the emission of anthropogenic and natural greenhouse gas methane to the environment and are the promising agents for future biotechnologies. Many aspects of CH4 bioconversion by methanotrophs require further clarification. This study was aimed at characterizing the biochemical properties of the malic enzyme (Mae) from the halotolerant obligate methanotroph Methylotuvimicrobium alcaliphilum 20Z. The His6-tagged Mae was obtained by heterologous expression in Escherichia coli BL21 (DE3) and purified by affinity metal chelating chromatography. As determined by gel filtration and non-denaturating gradient gel electrophoresis, the molecular mass of the native enzyme is 260 kDa. The homotetrameric Mae (65x4 kDa) catalyzed an irreversible NAD+-dependent reaction of L-malate decarboxylation into pyruvate with a specific activity of 32 ± 2 units mg-1 and Km value of 5.5 ± 0.8 mM for malate and 57 ± 5 μM for NAD+. The disruption of the mae gene by insertion mutagenesis resulted in a 20-fold increase in intracellular malate level in the mutant compared to the wild type strain. Based on both enzyme and mutant properties, we conclude that the malic enzyme is involved in the control of intracellular L-malate level in Mtm. alcaliphilum 20Z. Genomic analysis has revealed that Maes present in methanotrophs fall into two different clades in the amino acid-based phylogenetic tree, but no correlation of the division with taxonomic affiliations of the host bacteria was observed.

Klíčová slova:

Enzyme metabolism – Enzymes – Methane – Molecular mass – Phosphates – Pyruvate – Citric acid cycle – Dehydrogenases


1. Hanson RS, Hanson TE. Methanotrophic bacteria. Microbiol Rev. 1996; 60(2):439–471. 8801441.

2. Henard CA, Smith H, Dowe N, Kalyuzhnaya MG, Pienkos PT, Guarnieri MT. Bioconversion of methane to lactate by an obligate methanotrophic bacterium. Sci. Rep. 2016; 6:21585. doi: 10.1038/srep21585 26902345.

3. Strong PJ, Kalyuzhnaya M, Silverman J, Clarke WP. A methanotroph-based biorefinery: potential scenarios for generating multiple products from a single fermentation. Bioresour Technol. 2016; 215:314–323. doi: 10.1016/j.biortech.2016.04.099 27146469.

4. de la Torre A, Metivier A, Chu F, Laurens LM, Beck DA, Pienkos PT, Lidstrom ME, Kalyuzhnaya MG. Genome-scale metabolic reconstructions and theoretical investigation of methane conversion in Methylomicrobium buryatense strain 5G(B1). Microb Cell Fact. 2015; 14:188. doi: 10.1186/s12934-015-0377-3 26607880.

5. Kalyuzhnaya MG, Puri AW, Lidstrom ME. Metabolic engineering in methanotrophic bacteria. Metab Eng. 2015; 29:142–152. doi: 10.1016/j.ymben.2015.03.010 25825038.

6. Kalyuzhnaya MG. Methane biocatalysis: selecting the right microbe. In Eckert CA, Trinh CT, editors. Biotechnology for biofuel production and optimization. Amsterdam: Elsevier; 2016. p. 353–383.

7. Dedysh SN, Naumoff DG, Vorobev AV, Kyrpides N, Woyke T, Shapiro N, Crombie AT, Murrell JC, Kalyuzhnaya MG, Smirnova AV, Dunfield PF. Draft genome sequence of Methyloferula stellata AR4, an obligate methanotroph possessing only a soluble methane monooxygenase. Genome Announc. 2015; 3(2):e01555–14. doi: 10.1128/genomeA.01555-14 25745010.

8. Chistoserdova L, Kalyuzhnaya MG, Lidstrom ME. The expanding world of methylotrophic metabolism. Annu Rev Microbiol. 2009; 63:477–499. doi: 10.1146/annurev.micro.091208.073600 19514844.

9. But SY, Egorova SV, Khmelenina VN, Trotsenko YA. Serine-glyoxylate aminotranferases from methanotrophs using different C1-assimilation pathways. Antonie Van Leeuwenhoek. 2019; 112(5):741–751. doi: 10.1007/s10482-018-1208-4 30511326.

10. Anvar SY, Frank J, Pol A, Schmitz A, Kraaijeveld K, den Dunnen JT, Op den Camp HJ. The genomic landscape of the verrucomicrobial methanotroph Methylacidiphilum fumariolicum SolV. BMC Genomics. 2014; 15:914. doi: 10.1186/1471-2164-15-914 25331649.

11. Erikstad HA, Birkeland NK. Draft genome sequence of "Candidatus Methylacidiphilum kamchatkense" Strain Kam1, a thermoacidophilic methanotrophic Verrucomicrobium. Genome Announc. 2015; 3(2). pii: e00065-15. doi: 10.1128/genomeA.00065-15 25745002.

12. Khmelenina VN, Murrell JC, Smith T, Trotsenko YA. Physiology and Biochemistry of the Aerobic Methanotrophs. In Rojo F, editor. Aerobic Utilization of Hydrocarbons, Oils and Lipids, Handbook of Hydrocarbon and Lipid Microbiology. Springer International Publishing AG, part of Springer Nature. 2018.

13. Mustakhimov II, But SY, Reshetnikov AS, Khmelenina VN, Trotsenko YA. Homo- and heterologous reporter proteins for evaluation of promoter activity in Methylomicrobium alcaliphilum 20Z. Prikl Biokhim Mikrobiol. 2016; 52(3):279–86. 29509383.

14. Fu Y, Li Y, Lidstrom M. The oxidative TCA cycle operates during methanotrophic growth of the Type I methanotroph Methylomicrobium buryatense 5GB1. Metab Eng. 2017; 42:43–51. doi: 10.1016/j.ymben.2017.05.003 28552747.

15. Garg S, Wu H, Clomburg JM, Bennett GN. Bioconversion of methane to C-4 carboxylic acids using carbon flux through acetyl-CoA in engineered Methylomicrobium buryatense 5GB1C. Metab Eng. 2018; 48:175–183. doi: 10.1016/j.ymben.2018.06.001 29883803.

16. Akberdin IR, Thompson M, Hamilton R, Desai N, Alexander D, Henard CA, Guarnieri MT, Kalyuzhnaya MG. Methane utilization in Methylomicrobium alcaliphilum 20ZR: a systems approach. Sci Rep. 2018; 8(1):4753. doi: 10.1038/s41598-018-23088-w 29540803.

17. Vuilleumier S, Khmelenina VN, Bringel F, Reshetnikov AS, Lajus A, Mangenot S, Rouy Z, Op den Camp HJ, Jetten MS, Dispirito AA, Dunfield P, Klotz MG, Semrau JD, Stein LY, Barbe V, Médigue C, Trotsenko YA, Kalyuzhnaya MG. Genome sequence of the haloalkaliphilic methanotrophic bacterium Methylomicrobium alcaliphilum 20Z. J Bacteriol. 2012; 194(2):551–2. doi: 10.1128/JB.06392-11 22207753.

18. Kalyuzhnaya MG, Yang S, Rozova ON, Smalley NE, Clubb J, Lamb A, Gowda GA, Raftery D, Fu Y, Bringel F, Vuilleumier S, Trotsenko YA, Beck D, Khmelenina VN, Lidstrom ME. Highly efficient methane biocatalysis revealed in methanotrophic bacterium. Nature Commun. 2013; 4:2785. doi: 10.1038/ncomms3785 24302011.

19. Rozova ON, Khmelenina VN, Gavletdinova JZ, Mustakhimov II, Trotsenko YA. Acetate kinase—an enzyme of the postulated phosphoketolase pathway in Methylomicrobium alcaliphilum 20Z. Antonie van Leeuwenhoek. 2015; 108(4):965–74. doi: 10.1007/s10482-015-0549-5 26275877.

20. Henard CA, Smith HK, Guarnieri MT. Phosphoketolase overexpression increases biomass and lipid yield from methane in an obligate methanotrophic biocatalyst. Metab Eng. 2017; 41:152–158. doi: 10.1016/j.ymben.2017.03.007 28377275.

21. Khmelenina VN, Rozova ON, Akberdin IR, Kalyuzhnaya MG, Trotsenko YA. Pyrophosphate-dependent enzymes in methanotrophs: new findings and views. In Kalyuzhnaya MG, Xing X.H., editors. Methane Biocatalysis: Paving the Way to Sustainability. Switzerland: Springer, International Publishing AG; 2018. pp. 83–98.

22. Chiba Y, Kamikawa R, Nakada-Tsukui K, Saito-Nakano Y, Nozaki T. Discovery of PPi-type phosphoenolpyruvate carboxykinase genes in eukaryotes and bacteria. J Biol Chem. 2015; 290(39):23960–23970. doi: 10.1074/jbc.M115.672907 26269598.

23. Rozova ON, Khmelenina VN, Bocharova KA, Mustakhimov II, Trotsenko YA. Role of NAD+-dependent malate dehydrogenase in the metabolism of Methylomicrobium alcaliphilum 20Z and Methylosinus trichosporium OB3b. Microorganisms. 2015; 3(1):47–59; doi: 10.3390/microorganisms3010047 27682078.

24. Choi PH, Jo J, Lin YC, Lin MH, Chou CY, Dietrich LEP, Tong L. A distinct holoenzyme organization for two-subunit pyruvate carboxylase. Nat Commun. 2016; 7:12713. doi: 10.1038/ncomms12713 27708276.

25. Dahinden P, Auchli Y, Granjon T, Taralczak M, Wild M, Dimroth P. Oxaloacetate decarboxylase of Vibrio cholerae: purification, characterization, and expression of the genes in Escherichia coli.Arch Microbiol. 2005; 183:121–129. doi: 10.1007/s00203-004-0754-5 15647905.

26. Dimroth P, Jockel P, Schmid M. Coupling mechanism of the oxaloacetate decarboxylase Na(+) pump. Biochim Biophys Acta. Rev. 2001; 1505(1):1–14. doi: 10.1016/s0005-2728(00)00272-3 11248184.

27. Lietzan AD, St Maurice M. Functionally diverse biotin-dependent enzymes with oxaloacetate decarboxylase activity. Arch Biochem Biophys. 2014; 15;544:75–86. doi: 10.1016/ 24184447.

28. Rozova ON, Khmelenina VN, Mustakhimov II, But SY, Trotsenko YA. Properties of malic enzyme from the aerobic methanotroph Methylosinus trichosporium. Biochemistry (Moscow) 2019; 84(4):390–397. doi: 10.1134/S0006297919040060 31228930.

29. Espariz M, Repizo G, Blancato V, Mortera P, Alarcon S, Magni C. Identification of malic and soluble oxaloacetate decarboxylase enzymes in Enterococcus faecalis. FEBS J. 2011; 278(12):2140–2151. doi: 10.1111/j.1742-4658.2011.08131.x 21518252.

30. Bologna FP, Andreo CS, Drincovich MF. Escherichia coli malic enzymes: two isoforms with substantial differences in kinetic properties, metabolic regulation, and structure. J Bac. 2007; 189(16):5937–5946. doi: 10.1128/JB.00428-07 17557829.

31. Mitsch MJ, Voegele RT, Cowie A, Osteras M, Finan TM. Chimeric structure of the NAD(P)+- and NADP+-dependent malic enzymes of Rhizobium (Sinorhizobium) meliloti. J Biol Chem. 1998; 273(15):9330–9336. doi: 10.1074/jbc.273.15.9330 9535928.

32. Sauer U, Eikmanns BJ. The PEP–pyruvate–oxaloacetate node as the switch point for carbon flux distribution in bacteria. FEMS Microbiol Rev. 2005; 29(4):765–794. doi: 10.1016/j.femsre.2004.11.002 16102602.

33. Lerondel G, Doan T, Zamboni N, Sauer U, Aymerich S. YtsJ has the major physiological role of the four paralogous malic enzyme isoforms in Bacillus subtilis. J Bacteriol. 2006 Jul;188(13):4727–36. doi: 10.1128/JB.00167-06 16788182.

34. Zhang Y, Smallbone LA, diCenzo GC, Morton R, Finan TM. Loss of malic enzymes leads to metabolic imbalance and altered levels of trehalose and putrescine in the bacterium Sinorhizobium meliloti. BMC Microbiol. 2016; 16(1):163. doi: 10.1186/s12866-016-0780-x 27456220.

35. Doan T, Servant P, Tojo S, Yamaguchi H, Lerondel G, Yoshida K, Fujita Y, Aymerich S. The Bacillus subtilis ywkA gene encodes a malic enzyme and its transcription is activated by the YufL/YufM two-component system in response to malate. Microbiology. 2003; 149:2331–43. doi: 10.1099/mic.0.26256-0 12949160.

36. Murai T, Tokushige M, Nagai J, Katsuki H. Physiological functions of NAD- and NADP-linked malic enzymes in Escherichia coli. Biochem Biophys Res Commun. 1971; 43:875–881. doi: 10.1016/0006-291x(71)90698-x 4397922.

37. Khmelenina VN, Kalyuzhnaya MG, Sakharovsky VG, Suzina NE, Trotsenko YA, Gottschalk G. Osmoadaptation in halophilic and alkaliphilic methanotrophs. Arch Microbiol 1999; 172(5):321–329. doi: 10.1007/s002030050786 10550474.

38. Sambrook J, Russell DW. Molecular Cloning: a Laboratory Manual, 3rd Edn. New York: Cold Spring Harbor Laboratory; 2001.

39. Reshetnikov AS, Rozova ON, Khmelenina VN, Mustakhimov II, Beschastny AP, Murrell JC, Trotsenko YA. Characterization of the pyrophosphate-dependent 6-phosphofructokinase from Methylococcus capsulatus Bath. FEMS Microbiol Lett. 2008; 288(2): 202–210. doi: 10.1111/j.1574-6968.2008.01366.x 19054082.

40. Ye W, Huo G, Chen J, Liu F, Yin J, Yang L, Ma X. Heterologous expression of the Bacillus subtilis (natto) alanine dehydrogenase in Escherichia coli and Lactococcus lactis. Microbiol Res. 2010; 165(4):268–275. doi: 10.1016/j.micres.2009.05.008 19720515.

41. Sender PD, Martin MG, Peiru S, Magni C. Characterization of an oxaloacetate decarboxylase that belongs to the malic enzyme family. FEBS Lett. 2004; 570(1–3): 217–222. doi: 10.1016/j.febslet.2004.06.038 15251467.

42. Shacterle GR, Pollack RL. A simplified method for quantitative assay of small amounts of protein in biological material. Anal Biochem. 1973; 51(2):654–657. doi: 10.1016/0003-2697(73)90523-x 4735559.

43. Tamura K, Dudley J, Nei M, Kumar S. MEGA4: Molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol. 2007; 24(8):1596–1599. doi: 10.1093/molbev/msm092 17488738.

44. Groisillier A, Lonvaud-Funel A. Comparison of partial malolactic enzyme gene sequences for phylogenetic analysis of some lactic acid bacteria species and relationships with the malic enzyme. Int J Syst Bacteriol. 1999; 49:1417–1428. doi: 10.1099/00207713-49-4-1417 10555321.

45. Chen F, Okabe Y, Osano K, Tajima S. Purification and characterization of an NAD-malic enzyme from Bradyrhizobium japonicum A1017. Appl Environ Microbiol. 1998; 64(10):4073–4075. 9758846.

46. Kawai S, Suzuki H, Yamamoto K, Inui M, Yukawa H, Kumagai H. Purification and characterization of a malic enzyme from the ruminal bacterium Streptococcus bovis ATCC 15352 and cloning and sequencing of its gene. Appl Environ Microbiol. 1996; 62(8):2692–2700. 8702261.

47. Taillefer M, Rydzak T, Levin DB, Oresnik IJ, Sparling R. Reassessment of the transhydrogenase/malate shunt pathway in Clostridium thermocellum ATCC 27405 through kinetic characterization of malic enzyme and malate dehydrogenase. Appl Environ Microbiol. 2015; 81(7):2423–32. doi: 10.1128/AEM.03360-14 25616802.

48. Voegele RT, Mitsch MJ, Finan TM. Characterization of two members of a novel malic enzyme class. Biochim Biophys Acta. 1999; 1432(2):275–85. doi: 10.1016/s0167-4838(99)00112-0 10407149.

49. Kobayashi K, Doi S, Negoro S, Urabe I, Okada H. Structure and properties of malic enzyme from Bacillus stearothermophilus. J Biol Chem. 1989; 264(6):3200–5. 2644282.

50. Chen F, Okabe Y, Osano K, Tajima S. Purification and characterization of the NADP-malic enzyme from Bradyrhizobium japonicum A1017. Biosci Biotech Biochem. 1997; 61(2):384–386. doi: 10.1271/bbb.61.384 9058984.

51. Gourdon P, Baucher M-F, Lindley ND, Guyonvarch A. Cloning of the malic enzyme gene from Corynebacterium glutamicum and role of the enzyme in lactate metabolism. Appl Environ Microbiol. 2000; 66(7):2981–2987. doi: 10.1128/aem.66.7.2981-2987.2000 10877795.

52. Bartolucci S, Rella R, Guagliardi A, Raia CA, Gambacorta A, De Rosa M, Rossi M. Malic enzyme from archaebacterium Sulfolobus solfataricus. Purification, structure, and kinetic properties. J Biol Chem. 1987; 262(16):7725–31. 3108257.

53. Mallick S, Harris BG, Cook PF. Kinetic mechanism of NAD:malic enzyme from Ascaris suum in the direction of reductive carboxylation. J Biol Chem. 1991; 266(5):2732–2738. 1993653.

54. Wierenga RK, Terpstra P, Hol WG. Prediction of the occurrence of the ADP-binding βαβ-fold in proteins, using an amino acid sequence fingerprint. J Mol Biol. 1986; 187(1):101–107. doi: 10.1016/0022-2836(86)90409-2 3959077.

55. Chang GG, Tong L. Structure and function of malic enzymes, a new class of oxidative decarboxylases. Biochemistry. 2003; 42(44):12721–12733. doi: 10.1021/bi035251+ 14596586.

56. Ratledge C. The role of malic enzyme as the provider of NADPH in oleaginous microorganisms: a reappraisal and unsolved problems. Biotechnol Lett. 2014; 36:1557–1568. doi: 10.1007/s10529-014-1532-3 24752812.

57. Chistoserdova L. Wide Distribution of genes for tetrahydromethanopterin/methanofuran-linked C1 transfer reactions argues for their presence in the common ancestor of bacteria and archaea. Front Microbiol. 2016; 7:1425. doi: 10.3389/fmicb.2016.01425 27679616.

58. Mustakhimov II, Reshetnikov AS, Khmelenina VN. The ectoine degradation pathway in halotolerant methanotrophs. Absracts of 12th International conference Biocatalysis-2019: fundamentals and application. Innovations and High Technologies MSU Ltd. 2019; 119.

59. Lea PJ, Chen Z-H, Leegood RC, Walke, RP. Does phosphoenolpyruvate carboxykinase have a role in both amino acid and carbohydrate metabolism? Amino Acids. 2001; 20: 225–241. doi: 10.1007/s007260170041 11354601.

60. Fu Y, He L, Reeve J, Beck DAC, Lidstrom ME. Core metabolism shifts during growth on methanol versus methane in the methanotroph Methylomicrobium buryatense 5GB1. M Bio. 2019; 10(2). pii: e00406–19. doi: 10.1128/mBio.00406-19 30967465.

61. Nguyen AD, Park JY, Hwang IY, Hamilton R, Kalyuzhnaya MG, Kim D, Lee EY. Genome-scale evaluation of core one-carbon metabolism in gammaproteobacterial methanotrophs grown on methane and methanol. Metab Eng. 2019; pii: S1096-7176(19)30307-6. doi: 10.1016/j.ymben.2019.10.004 31626985.

Článek vyšel v časopise


2019 Číslo 11