Study of duplicated galU genes in Rhodococcus jostii and a putative new metabolic node for glucosamine-1P in rhodococci

https://doi.org/10.1016/j.bbagen.2020.129727Get rights and content

Highlights

  • Duplicated galU genes from Rhodococcus jostii were functionally characterized.

  • A specific pyrophosphorylase for glucosamine-1P (and UTP) was discovered.

  • Glucosamine-1P activity correlates with a trimeric GalU-type conformation.

  • In vitro enzyme kinetics for glucosamine-1P reaction showed in vivo feasibility.

  • A metabolic node could be postulated at the glucosamine-1P level in rhodococci.

Abstract

Backgound

Studying enzymes that determine glucose-1P fate in carbohydrate metabolism is important to better understand microorganisms as biotechnological tools. One example ripe for discovery is the UDP-glucose pyrophosphorylase enzyme from Rhodococcus spp. In the R. jostii genome, this gene is duplicated, whereas R. fascians contains only one copy.

Methods

We report the molecular cloning of galU genes from R. jostii and R. fascians to produce recombinant proteins RjoGalU1, RjoGalU2, and RfaGalU. Substrate saturation curves were conducted, kinetic parameters were obtained and the catalytic efficiency (kcat/Km) was used to analyze enzyme promiscuity. We also investigated the response of R. jostii GlmU pyrophosphorylase activity with different sugar-1Ps, which may compete for substrates with RjoGalU2.

Results

All enzymes were active as pyrophosphorylases and exhibited substrate promiscuity toward sugar-1Ps. Remarkably, RjoGalU2 exhibited one order of magnitude higher activity with glucosamine-1P than glucose-1P, the canonical substrate. Glucosamine-1P activity was also significant in RfaGalU. The efficient use of the phospho-amino-sugar suggests the feasibility of the reaction to occur in vivo. Also, RjoGalU2 and RfaGalU represent enzymatic tools for the production of (amino)glucosyl precursors for the putative synthesis of novel molecules.

Conclusions

Results support the hypothesis that partitioning of glucosamine-1P includes an uncharacterized metabolic node in Rhodococcus spp., which could be important for producing diverse alternatives for carbohydrate metabolism in biotechnological applications.

General significance

Results presented here provide a model to study evolutionary enzyme promiscuity, which could be used as a tool to expand an organism's metabolic repertoire by incorporating non-canonical substrates into novel metabolic pathways.

Introduction

A key feature of some organisms from the Rhodococcus genus is their ability to accumulate lipids, which is a property that can be exploited to engineer these organisms to become microbial factories for biofuel production [1,2]. During cultivation under nitrogen limitations and with gluconate as the sole carbon source, the oleaginous bacterium Rhodococcus jostii accumulates up to 70% of its dry cell weight as lipids [3]. In contrast, R. fascians does not show oleaginous behavior under the same conditions [4,5], except when grown in glycerol as a carbon supplier [1]. In addition to triacylglycerides, members of this bacterial genus can accumulate other storage compounds, such as glycogen or polyphosphates [3,6]. It has been hypothesized that glycogen functions as an intermediary molecule for temporal carbon allocation since the glucan is accumulated during exponential growth phase and later mobilized for lipid synthesis [3,6].

Recently, the biochemical characterization of the ADP-glucose (ADP-Glc) pyrophosphorylase (EC 2.7.7.27, ADP-GlcPPase) from R. jostii and R. fascians provided evidence for the above hypothesis from a kinetic and regulatory point of view [[7], [8], [9]]. This allosteric enzyme catalyzes the limiting step in glycogen synthesis by activating glucose-1P (Glc-1P) to form ADP-Glc, the specific glucosyl donor to the α-1,4-glucan chain elongation [10,11]. Indeed, the rhodococcal ADP-GlcPPase responds by allosteric regulation to specific intracellular signals of carbon availability, directing Glc-1P to glucan synthesis, via ADP-Glc. Thus, Glc-1P is the precursor for the temporal carbon allocation “metabolite” glycogen in these organisms.

The metabolic destiny of Glc-1P is dictated by actions of several NDP-GlcPPases, including ADP-GlcPPase and UDP-GlcPPase (EC 2.7.7.9, GalU) to produce ADP-Glc and UDP-Glc, respectively. The former is the specific substrate for glycogen elongation in bacteria [10,11] and in some Actinobacteria is related to trehalose synthesis [12,13]. UDP-Glc is generally ascribed to many metabolic fates, such as lipopolysaccharides [14,15], structural capsule/membrane oligosaccharides [16], or intracellular oligo- [17] or poly-saccharides [18]. Studying Glc-1P partitioning in rhodococci is critical to better understand carbon management for glycogen synthesis, carbohydrate metabolism, and other metabolic fates. Therefore, we performed a comparative study of two rhodococci with different metabolic behavior and focused on their UDP-GlcPPases, which are encoded by the gene galU. Interestingly, genomic analysis of rhodococci shows two galU genes (galU1 and galU2) are present in R. jostii [19], whereas R. fascians has only one galU gene (BioProject ID 286803).

Metabolic gene redundancy constitutes the foundation for rhodoccocal catabolic versatility, its functional robustness, and its adaptation to diverse environments, thereby providing enhanced competition capabilities [19,20]. Indeed, some examples of functional redundancy found in Rhodococcus spp. have been linked to mechanisms related to niche adaptation, especially aromatic compound degradative pathways [21]. The genetic redundancy in rhodococci may have originated either from gene duplication or horizontal gene transfer events [21]. Duplicated genes typically evolve to encode (iso)enzymes with different substrate preferences and/or expression profiles. Moreover, some duplications present functional redundancy if they perform the same cellular function or have overlapping substrate ranges [21]. These types of duplications are usually relatively recent or were under low evolutionary pressure. Nevertheless, gene duplication together with protein promiscuity is critical for offering a source of diversity and metabolic innovation in prokaryotes [22,23].

Given the plethora of functions derived from UDP-Glc synthesis, it is imperative to comprehend how UDP-GlcPPase gene duplications are distributed and affect metabolism in a variety of bacteria. Indeed, UDP-GlcPPase duplication and divergence has been briefly described in other organisms. For example, in some enterobacterial species a gene duplication of galU occurred and gave rise to an enzymatically nonfunctional galF gene. The proteins encoded by these genes have diverged and while GalF has lost enzymatic activity [24], it still interacts with GalU [25]. Additionally, in most cyanobacterial species a gene encoding a putative GDP-mannose PPase (EC 2.2.7.13) produces a protein with UDP-GlcPPase activity (named CugP). In some cyanobacteria, CugP occurs simultaneously with GalU proteins with redundant UDP-GlcPPase activities; however, the metabolic impact of such duplication was not analyzed [26]. A recent report presented the molecular cloning of duplicated galU genes in R. opacus [27], but the authors failed to obtain a soluble form of the recombinant GalU1 enzyme and thus it could not be further characterized. Herein we tackle this issue and report the comparative kinetic study of both GalU1 and GalU2 proteins encoded by the respective galU genes from R. jostii. We also characterized the single GalU enzyme present in the related bacterium R. fascians. Our results from the characterization of substrate utilization of rhodococcal GalUs also led us to characterize the critical bifunctional enzyme in amino sugar metabolism, GlmU [28], from R. jostii. Together, the results presented in this work with those recently obtained from studies on rhodococcal ADP-GlcPPases [[7], [8], [9]], strongly support an additional metabolic node for carbon allocation at the level of glucosamine-1P (GlcN-1P), similar to the node for Glc-1P.

Section snippets

Chemicals

Protein standards, antibiotics, IPTG, Glc-1P, GlcN-1P, galactose-1P (Gal-1P), N-acetylglucosamine-1P (GlcNAc-1P), mannose-1P (Man-1P), ATP, UTP, GTP, CTP, and oligonucleotides were from Sigma-Aldrich (Saint Louis, MO, USA). All other reagents were of the highest quality available.

Bacteria and plasmids

Escherichia coli Top 10 F′ cells (Invitrogen) and pGEM®-T Easy vector (Promega, ampicillin resistance) were used for cloning procedures. The galU gene from R. fascians (RfagalU) and glmU from R. jostii (RjoglmU) were

Recombinant expression and structural characterization

From the genomic analysis of R. jostii RHA1 [19], we identified two galU genes, RjogalU1 (903 bp) and RjogalU2 (921 bp). These genes encode putative UDP-GlcPPases, namely RjoGalU1 and RjoGalU2, with theoretical molecular masses of 31.75 kDa and 32.68 kDa, respectively. UDP-GlcPPases catalyze the reaction to transform Glc-1P and UTP to UDP-Glc and pyrophosphate. RjoGalU1 and RjoGalU2 sequences are 78% identical and share 95.7% and 98.4% identity compared tothe GalU1 and GalU2 enzymes recently

Kinetic/structural considerations

Nucleotide sugars are donors of carbohydrate moieties in the synthesis of many molecules, including oligo and polysaccharides, glycosylated secondary metabolites (i.e., antibiotics), and glycoproteins and glycolipids [49,50]. In bacteria, about 70 different nucleotide sugars have been identified. Almost all organisms reportedly produce UDP-Glc [51], but ADP–Glc is limited to bacteria, green algae, and plants [10,11]. Both are built from Glc-1P by the respective NDP-GlcPPases, depending upon the

Conclusion

The results of this study provide new insight into GlcN-1P metabolism and may impact the field at different levels. First, the rhodococcal GalU2-type enzyme constitutes a new enzymatic tool for producing a new molecule (UDP-GlcN) that would be suitable as a substrate for (amino)glucosyl transferases [85,86]. This widens the possibility of obtaining new glycosylated molecules, which could improve tools for glycorandomization [[58], [59], [60], [61]]. Secondly, this newly identified activity may

Author contributions

AEC, MAH and MDA performed the experiments. AEC, MDA, AAI, MAB and HMA conceived the study and designed the experiments. AEC, MLK, AAI, HMA and MDA refined the kinetic models and analyzed the data. AEC, MLK, AAI, HMA and MDA wrote the paper. All authors revised and approved the submitted manuscript.

Declaration of Competing Interest

The authors declare no conflict of interest.

Acknowledgements

This work was supported by grants from ANPCyT (PICT’17 1515 to AAI and PICT’15 0634 to MDAD) and CONICET (PIP2015-2016 0529, PUE 2018-033 to HMA and PUE 2016-0040 to IAL). AEC is a Fellow from CONICET. HMA, MAH, AAI and MDAD are Career Investigator members from the same Institution.

References (89)

  • M.B. Bosco et al.

    UDP-glucose pyrophosphorylase from Xanthomonas spp characterization of the enzyme kinetics, structure and inactivation related to oligomeric dissociation

    Biochimie

    (2009)
  • X. Lai et al.

    Expression, purification, and characterization of a functionally active Mycobacterium tuberculosis UDP-glucose pyrophosphorylase

    Protein Expr. Purif.

    (2008)
  • D. Kostrewa et al.

    Crystal structures of Streptococcus pneumoniae N-acetylglucosamine-1-phosphate Uridyltransferase, GlmU, in Apo form at 233Å resolution and in complex with UDP-N-acetylglucosamine and Mg2+ at 196Å resolution

    J. Mol. Biol.

    (2001)
  • T. Yang et al.

    In-microbe formation of nucleotide sugars in engineered Escherichia coli

    Anal. Biochem.

    (2012)
  • R. Moretti et al.

    Enhancing the latent nucleotide triphosphate flexibility of the glucose-1-phosphate thymidylyltransferase RmlA

    J. Biol. Chem.

    (2007)
  • R. Moretti et al.

    Expanding the nucleotide and sugar 1-phosphate promiscuity of nucleotidyltransferase RmlA via directed evolution

    J. Biol. Chem.

    (2011)
  • S. Blanchard et al.

    Enzymatic tools for engineering natural product glycosylation

    Curr. Opin. Chem. Biol.

    (2006)
  • D. Mengin-Lecreulx et al.

    Characterization of the essential gene glmM encoding phosphoglucosamine mutase in Escherichia coli

    J. Biol. Chem.

    (1996)
  • H.U. van der Heul et al.

    Regulation of antibiotic production in actinobacteria: new perspectives from the post-genomic era

    Nat. Prod. Rep.

    (2018)
  • G.-N. Wang et al.

    Synthesis and evaluation of glucosamine-6-phosphate analogues as activators of glmS riboswitch

    Tetrahedron

    (2012)
  • F. Vincent et al.

    Structure and kinetics of a monomeric glucosamine 6-phosphate deaminase: missing link of the NagB superfamily?

    J. Biol. Chem.

    (2005)
  • G.L. Moraes et al.

    Structural and functional features of enzymes of Mycobacterium tuberculosis peptidoglycan biosynthesis as targets for drug development

    Tuberculosis

    (2015)
  • A. Peracchi

    The limits of enzyme specificity and the evolution of metabolism

    Trends Biochem. Sci.

    (2018)
  • J. Rosenberg et al.

    Harnessing underground metabolism for pathway development

    Trends Biotechnol.

    (2019)
  • O.M. Herrero et al.

    Physiological and genetic differences amongst Rhodococcus species for using glycerol as a source for growth and triacylglycerol production

    Microbiology

    (2016)
  • O.M. Herrero et al.

    Rhodococcus bacteria as a promising source of oils from olive mill wastes

    World J. Microbiol. Biotechnol.

    (2018)
  • M.A. Hernandez et al.

    Biosynthesis of storage compounds by Rhodococcus jostii RHA1 and global identification of genes involved in their metabolism

    BMC Genomics

    (2008)
  • H.M. Alvarez et al.

    Accumulation of storage lipids in species of Rhodococcus and Nocardia and effect of inhibitors and polyethylene glycol

    Fett/Lipid

    (1997)
  • M.A. Hernandez et al.

    Glycogen formation by Rhodococcus species and the effect of inhibition of lipid biosynthesis on glycogen accumulation in Rhodococcus opacus PD630

    FEMS Microbiol. Lett.

    (2010)
  • A.E. Cereijo et al.

    On the kinetic and allosteric regulatory properties of the ADP-glucose pyrophosphorylase from Rhodococcus jostii: an approach to evaluate glycogen metabolism in oleaginous bacteria

    Front. Microbiol.

    (2016)
  • A.E. Cereijo et al.

    Glucosamine-6P and glucosamine-1P, respectively an activator and a substrate of rhodococcal ADP-glucose pyrophosphorylases, show a hint to ascertain (actino)bacterial glucosamine metabolism

    BioRxiv

    (2020)
  • M.A. Ballicora et al.

    ADP-glucose pyrophosphorylase, a regulatory enzyme for bacterial glycogen synthesis

    Microbiol. Mol. Biol. Rev.

    (2003)
  • M.A. Ballicora et al.

    ADP-glucose pyrophosphorylase: a regulatory enzyme for plant starch synthesis

    Photosynth. Res.

    (2004)
  • M. Mollerach et al.

    Characterization of the galU gene of Streptococcus pneumoniae encoding a uridine diphosphoglucose pyrophosphorylase: a gene essential for capsular polysaccharide biosynthesis

    J. Exp. Med.

    (1998)
  • L. Bonofiglio et al.

    Biochemical characterization of the pneumococcal glucose 1-phosphate uridylyltransferase (GalU) essential for capsule biosynthesis

    Curr. Microbiol.

    (2005)
  • L. Padilla et al.

    Impact of heterologous expression of Escherichia coli UDP-glucose pyrophosphorylase on trehalose and glycogen synthesis in Corynebacterium glutamicum

    Appl. Environ. Microbiol.

    (2004)
  • P. Ross et al.

    Cellulose biosynthesis and function in bacteria

    Microbiol. Rev.

    (1991)
  • M.P. McLeod et al.

    The complete genome of Rhodococcus sp RHA1 provides insights into a catabolic powerhouse

    PNAS

    (2006)
  • J.J. Díaz-Mejía et al.

    A network perspective on the evolution of metabolism by gene duplication

    Genome Biol.

    (2007)
  • M. Cappelletti et al.

    Genomics of Rhodococcus

  • M.E. Glasner et al.

    How enzyme promiscuity and horizontal gene transfer contribute to metabolic innovation

    FEBS J.

    (2020)
  • F.A. Kondrashov et al.

    Selection in the evolution of gene duplications

    Genome Biol.

    (2002)
  • A.C. Ebrecht et al.

    On the ancestral UDP-glucose pyrophosphorylase activity of GalF from Escherichia coli

    Front. Microbiol.

    (2015)
  • C.L. Marolda et al.

    The GalF protein of Escherichia coli is not a UDP-glucose pyrophosphorylase but interacts with the GalU protein possibly to regulate cellular levels of UDP-glucose

    Mol. Microbiol.

    (1996)
  • Cited by (4)

    • The ADP-glucose pyrophosphorylase from Melainabacteria: a comparative study between photosynthetic and non-photosynthetic bacterial sources

      2022, Biochimie
      Citation Excerpt :

      After running, the gels (containing between 5 and 50 μg of protein per well) were revealed with Coomassie Brilliant Blue. Protein molecular mass at the native state was determined by gel filtration using a Superdex 200 10/300 column (GE Healthcare), as previously reported [35,36]. The void volume of the column was determined using Dextran Blue (Promega).

    View full text