Acyltransferases Families in L-Methionine Biosynthesis

Proposition of functional re-annotation of metA and metX genes

Experimental data Enzymatic screening of MetA and MetX proteins

Publication in Nature Chemical Biology, June 2017 - UMR Genomique Metabolique - Genoscope

To get a representative picture of the acyl-CoA usage in the first step of methionine biosynthesis, we selected enzymes from taxa representative of the prokaryotic biodiversity and then characterized their L homoserine O-acyltransferase activity. To collect enzymes encoded by metA and metX genes, sequences identified with the "Homoserine succinyltransferase" and the "Homoserine acetyltransferase" InterPro signatures (IPR005697 and IPR008220, respectively) were retrieved. Enzymes were then selected among the different taxonomy orders.

From 125 enzymes encoded by 73 metX and 52 metA selected for cloning, 104 could be purified for experimental assays. Formation of O-acyl-L-homoserine was monitored in the presence of acetyl-CoA or succinyl-CoA using a spectrophotometric assay. Results are summarized below are or can be download here. Previous published data related to experiment on MetA and MetX have been inventoried and added in this table. This table gives an overview of experimentally validated function of members of these two families of L-homoserine O-acyltransferases.

Kingdom Phylum Class Order Organism UniprotKB id  Gene Presence of cluster metXW  Cloning status  Homoserine O-acyl transferase activity   References & commentaries
BACTERIA                    
  Proteobacteria Gamma Enterobacteriales Escherichia coli K12 P07623 metA   cloned & purified HST   Enzyme activity determinded by Born & Blanchard, 1999 and in  this study
        Citrobacter koseri A0A078LMN5 metA   cloned & not purified nd Enzyme that belong to MetA-G3, predicted to be HST. Unstable protein,  activity not determined
        Salmonella typhimurium P37413 metA   not selected HST Gene identified by Mares et al., 1982 but no enzyme activity determination.
        Sodalis glossinidius A0A0I9QJG3 metA   cloned & not purified nd Enzyme that belong to MetA-G2, no prediction done. Unstable protein,  activity not determined
      Alteromonadales Shewanella baltica A6WLE9 metA   cloned & purified HST  
        Alteromonas macleodii B4RUL1 metA   cloned & purified HST  
        Pseudoalteromonas haloplanktis Q3IHM8 metA   cloned & purified HST  
        Pseudoalteromonas haloplanktis Q3IJY8 metX no cloned & purified no activity Marked by the absence of homoserine binding residues (belong to MetX-G4 group)
        Pseudoalteromonas atlantica Q15RG1 metA   cloned & purified HST  
        Colwellia psychrerythraea Q487C1 metA   cloned & not purified nd  Enzyme that belong to MetA-G2, predicted to be HST.
      Aeromonadales Succinatimonas hippei E8LIC0 metA   cloned & purified HAT  
      Chromatiales Nitrosococcus oceani Q3J7P0 metA   cloned & purified HST  
        Allochromatium vinosum D3RNP0 metA   cloned & purified HST  
        Thioalkalivibrio nitratireducens L0E1U3  metA   cloned & purified HST  
        Halothiobacillus neapolitanus D0L1T6 metX yes metXW cloned & purified HST  
      Cardiobacteriales Cardiobacterium hominis C8NA81 metA   cloned & purified HST  
      Pasteurellales Haemophilus influenzae P45131 metX no cloned & purified HAT HAT enzyme activity determined by Born et al., 2000 and this study
      Pseudomonadales Pseudomonas aeruginosa P57714 metX yes metXW cloned & purified HST HST activity detected on cell extract by Foglino et al. 1995. HST activity determinded in this study. No activity could be measured when MetX was cloned without MetW
        Pseudomonas syringae Q4ZZ78 metX yes metXW cloned & purified HST Gene identified by Andersen et al., 1998. predicted HAT function based by sequence similarity but no enzyme activity determination. HST Enzyme activity determinded in this study. No activity could be measured when MetX was cloned without MetW
        Pseudomonas putida Q88CT3 metX yes metXW cloned & purified HST Gene identified by Alaminos and Ramos, 2001. Predicted HAT function based on sequence similarity but without enzyme caracterization. HST Enzyme activity determinded in this study. No activity could be measured when MetX was cloned without MetW
        Acinetobacter baylyi ADP1 Q6FEQ3 metX yes metXW cloned & purified HST No activity could be measured when MetX was cloned without MetW
      Xanthomonadales Xanthomonas campestris Q8P6V8 metX1 no cloned & purified HST Enzyme that belong to MetX-G3b group
          Q8P8L2 metX2 no cloned & purified SST/HST Enzyme that belong to MetX-G3a group
        Frateuria aurantia H8L374 metX2 no cloned & purified SST/HST Enzyme that belong to MetX-G3a group
        Stenotrophomonas maltophilia A0A0I9RJ56 metX2 no cloned & purified SST/HST Enzyme that belong to MetX-G3a group
      Thiotricales Thiothrix nivea I3BN39  metA   cloned & purified HST  
      Methylococcales Methylobacter tundripaludum G3ISL7  metA   cloned & purified HST  
        Methylococcus capsulatus Q606Y5  metA   cloned & purified HST  
       Vibrionales Vibrio fischeri  Q5E338 metA   selected nd Technical failure in this study, activity not determined. Enzyme that belong to MetA-G3a, predicted to be SST/HST.
      Oceanospirillales Halomonas anticariensis S2L5R8  metX1 yes metX cloned alone & purified HST  
          S2KHP1 metX2 no cloned & purified SST/HST Enzyme that belong to MetX-G3a group
    Alpha Rhizobiales Agrobacterium tumefaciens Q7CWE8? metA   not selected HAT  Enzyme activity determined by Roten et al., 2013. Protein id is not given in the publication.
        Rhodopseudomonas palustris Q13C46 metA   cloned & purified HST  
        Methylobacterium extorquens C7C8V4 metA   cloned & purified HST  
        Bradyrhizobium japonicum Q89XR3 metA   selected nd Technical failure in this study, activity not determined.  Enzyme that belong to MetA-G4, predicted to be HST.
        Bradyrhizobium sp  A4Z2X7 metA   cloned & purified HST  
        Pelagibacterium halotolerans G4RES5  metA   cloned & purified HST weak activity HAT
      Rhodobacterales Silicibacter pomeroyi Q5LSN6 metA   cloned & purified HAT  
        Rhodobacter sphaeroïdes Q3J205 metA   cloned & purified HAT  
        Hyphomonas neptunium Q0BX37 metA   cloned & purified HAT  
      Sphingomonadales Novosphingobium aromaticirovans Q2GAJ2 metA   cloned & purified HST weak HAT activity. 
        Sphingobium yanoikuyae  K9CVD6 metX1 yes metX cloned alone & purified no activity Technical failure with the construct metXW
          K9CMJ3 metX2 no cloned & purified no activity “Unmodeled” sequences that could not be modeled due to low sequence homology with available reference 3D structures used as template in this study. Genomic context not related to methionine biosynthesis
          K9CLW2 metA no cloned & purified no activity “Unmodeled” sequences that could not be modeled due to low sequence homology with available reference 3D structures used as template in this study; Genomic context not related to methionine biosynthesis
      Caulobacter Caulobacter crescentus Q9AAS1 metX1 yes metX cloned alone & purified HAT  
          R0ED50 metX2 no cloned & purified no activity “Unmodeled” sequences that could not be modeled due to low sequence homology with available reference 3D structures used as template in this study; Genomic context not related to methionine biosynthesis
        Brevundimonas diminuta  F4QV02 metX no cloned & purified HST “Unmodeled” sequences that could not be modeled due to low sequence homology with available reference 3D structures used as template in this study. Genomic context related to methionine biosynthesis
      Rhodospirillales Gluconobacter oxydans Q5FUF4 metX yes selected nd Technical failure in this study, activity not determined.  Enzyme that belong to MetX-G1, predicted to be HAT.
    Beta Hydrogenophilales Thiobacillus denitrificans Q3SM51 metA   cloned & purified HST  
      Burkholderiales Bordetella petrii A9I0E6 metX yes metX cloned alone & purified HST  
        Burkholderia thailandensis  Q2T284 metX yes metXW cloned & purified HST Weaker activity when metX is cloned without metW
        Comamonas testosteroni B7X2B6 metX yes metXW cloned & purified HST  
      Neisseriales Neisseria gonorrhoeae A0A0I9QGZ7 metX yes metXW cloned & purified HST Weaker activity when metX is cloned without metW
    Delta Desulfovibrionales Desulfovibrio magneticus C4XNQ9 metX yes metX cloned alone & purified HAT  
      Desulfuromonadales Geobacter lovleyi B3E278 metX no cloned & purified HAT  
    Epsilon Campylobacterales Sulfurimonas autotrophica  E0UR96 metX no cloned & purified HAT  
        Campylobacter jejuni A3YN67 metA   selected nd Technical failure in this study, activity not determined.   Enzyme that belong to MetX-G1, predicted to be HAT.
      Nautiliales Nautilia profundicola B9L9I6 metX no cloned & purified HAT  
  Firmicutes Bacilli Bacillales Bacillus cereus Q814M3 metA   cloned & purified HAT   Ziegler et al., 2007;  Zubieta et al., 2008) and this study 
          Q72YZ7 metX no cloned & purified no activity marked by the absence of homoserine binding residues (belong to MetX-G4 group)
        Bacillus subtilis P54167 metA   not selected HAT  HAT activity determined on cell free extract by Brush & Paulus, 1971
        Bacillus polymyxa E3EFJ9 metA   not selected HAT Wyman et Paulus, 1975
        Bacillus clausii  Q5WG21 metA   cloned & purified HAT  
        Geobacillus kaustophilus A0A0D8BWP6 metA   cloned & purified HAT  
        Staphylococcus aureus A0A0J9X168 metX no not selected HAT Enzyme activity and 3D structure determined by Thangavelu et al., 2014. 
      Lactobacillale  Lactobacillus acidophilus Q5FJQ4 metA   cloned & purified SAT/HAT Enzyme that belong to MetA-G2.  
        Lactobacillus casei A0A120HUS7 metA   not selected SAT Bogicevic et al., 2016
        Lactobacillus helveticus C9LZ74 metA   selected nd Technical failure in this study, activity not determined
        Leuconostoc mesenteroides Q03V79 metA   cloned & purified HAT  
        Leuconostoc citreum A0A0I9QIC9 metA   selected nd Technical failure in this study, activity not determined. Belong to MetA-G2, predicted to be HAT
    Clostridia Clostridiales  Clostridium kluyveri A5N8M0 metA   cloned & purified HAT  
      Thermoanaerobacterales Caldanaerobacter subterraneus subsp. pacificus B7R821  metX no cloned & purified HAT  
    Erysipelotrichia Erysipelotrichales Catenibacterium mitsuokai E2NPN0 metA   cloned & purified HAT weak HST activity
    Negativicutes Selenomonadales Selenomonas noxia D4S5T0 metA   cloned & not purified nd Enzyme that belong to MetA-G1. Enzyme predicted to be HAT.
  Actinobacteria Actinobacteridae Actinomycetales Bifidobacterium adolescentis  A1A3D2 metA   cloned & purified HAT  
        Actinomyces urogenitalis C0W602 metX no selected nd Technical failure in this study, activity not determined. Enzyme that belong to MetX-G1. Enzyme predicted to be HAT.
        Brevibacterium flavum A0A0F6SQS0? metX no not selected HAT HAT activity determined on purified enzyme but no sequence was identified Ozaki & Shiio, 1982
        Corynebacterium glutamicum O68640 metX no cloned & purified HAT Enzyme activity determined by Park et al., 1998 and this study
        Propionibacterium avidum G4CZ70 metX yes selected nd Technical failure in this study, activity not determined. Enzyme that belong to MetX-G1. Enzyme predicted to be HAT.
       Streptomycetales Streptomyces lavendulae D2Z028 dcsE/metX no not selected SAT/HAT Oda et al., 2013
  Deinococcus/ thermus Deinococci Thermales Thermus thermophilus Q9RA51 metX no cloned & purified HAT   Kosuge et al, 2000. 
        Marinithermus hydrothermalis F2NLB2 metX no selected nd Technical failure in this study, activity not determined.  Enzyme that belong to MetX-G1. Enzyme predicted to be HAT.
       Deinococcales Deinococcus geothermalis Q1J115 metX1 no cloned & purified HAT   
          Q1J075 metX2 no selected nd Unmodeled
  Planctomycetes   Planctomycetia   Planctomycetales Rhodopirellula baltica Q7UG04 fused metXW fused metXW cloned & purified HAT  
  Spirochaetes  Spirochaetales Leptospiraceae Leptospira meyeri  P94891 metX yes not selected HAT Gene identified by Bouhry et al., 1997 but no  enzyme activity determination caracterization. 
        Leptospira interrogans Q8F4I0 metX yes metXW cloned & purified HAT  3D Structure determined by Wang et al., 2007 but no enzyme activity determination. Enzyme activity determined in this study. No activity when metX was cloned without metW
  Thermotogae Thermotogales  Thermotogaceae Thermotoga petrophila A5IIQ2 metA   cloned & purified HAT  
        Thermotoga maritima Q9WZY3 metA   not selected HAT Goudarzi & Born, 2006
  Chlorobi Chlorobia Chlorobiales Chlorobium ferrooxidans Q0YQC5 metX no selected nd Technical failure in this study, activity not determined. Enzyme that belong to MetX-G1. Enzyme predicted to be HAT.
  Chloroflexi Chloroflexia  Chloroflexales Chloroflexus aurantiacus A9WKM8 metX no cloned & purified HAT  
  Synergistetes Synergistia Synergistales Thermanaerovibrio acidaminovorans D1B6T6 metX no selected nd Technical failure in this study, activity not determined.  Enzyme that belong to MetX-G1. Enzyme predicted to be HAT.
  Fusobacterium Fusobacteriales Fusobacteriaceae  Ilyobacter polytropus E3H7X6 metA1   cloned & purified HAT  
          E3HDJ8 metA2   cloned & purified HAT  
        Fusobacterium nucleatum A5TWR5 metA   selected nd Technical failure in this study, activity not determined. Enzyme that belong to MetA-G1. Enzyme predicted to be HAT.
  Cyanobacteria Nostocales Nostocaceae  Anabaena variabilis Q3M5Q6 metX no cloned & purified HAT  
    Oscillatoriophycideae Chroococcales Synechococcus sp. Q5N2Y8 metA   selected nd Technical failure in this study, activity not determined
     Pleurocapsales Chroococcidiopsis Chroococcidiopsis thermalis K9TW70 metX no cloned & purified no activity Marked by the absence of homoserine binding residues (belong to MetX-G4 group). Genomic context not related to methionine biosynthesis
  Deferribacteres  Deferribacterales  Deferribacteraceae Deferribacter desulfuricans D3P9D1 metX no cloned & purified HAT  
  Bacteroidetes  Bacteroidia Bacteroidales  Bacteroïdes fragilis  Q5LHS7 metA   cloned & purified HAT  
        Parabacteroïdes distasonis A6LC32 metA   cloned & purified HAT  
        Alistipes putredinis B0MW14  metX no selected nd Technical failure in this study, activity not determined. Enzyme that belong to MetX-G1. Enzyme predicted to be HAT.
    Flavobacteriia  Flavobacteriales  Psychroflexus torquis K4ICC9 metX no cloned & purified HAT  
    Sphingobacteria  Rhodothermaceae  Salinibacter ruber Q2S5A6 metX no cloned & purified HAT  
    Cytophagia Cytophagales Cyclobacterium marinum G0J5N4 metX no cloned & purified HAT  
ARCHAE                    
  Euryarchaeota Methanomicrobia Methanosarcina Methanococcoides burtonii Q12XS2 metX no cloned & purified HAT  
        Methanohalobium evestigatum  D7E9E0 metX no cloned & purified HAT  
        Methanohalophilus mahii D5E9R7 metX no cloned & purified HAT  
        Methanosalsum zhilinae F7XKY8 metX no cloned & purified HAT  
        Methanosarcina acetivorans Q8TME4 metX no cloned & purified HAT  
      Methanocorpusculaceae Methanocorpusculum labreanum A2SQW3 metX no cloned & purified HAT  
      Methanoregulaceae Methanosphaerula palustris B8GHE0 metX no cloned & purified HAT  
    Methanococci   Methanococcus vannielii  A6UNL1 metX no cloned & purified HAT  
    Methanobacteria Methanobacteriales Methanobrevibacter smithii A5UJK2  metX no cloned & purified no activity  Enzyme that belong to MetX-G1. Enzyme predicted to be HAT.
        Methanosphaera stadtmanae  Q2NGH9 metX no cloned & purified HAT  
        Methanobrevibacter ruminantium D3E3E4 metX no cloned & purified HAT  
          D3E1Q1  metX no cloned & purified no activity Marked by the absence of homoserine binding residues (belong to MetX-G4 group)
        Methanobacterium formicicum K2QXP6  metX1 no cloned & purified HAT  
          K2RES5  metX2 no selected no activity predicted Technical failure in this study, activity not determined. Marked by the absence of homoserine binding residues (belong to MetX-G4 group)
        Methanothermobacter thermautotrophicus O27848 metX no cloned & purified no activity Marked by the absence of homoserine binding residues (belong to MetX-G4 group)
    Halobacteria Halobacteriales Haloquadratum walsbyi Q18E47 metX no cloned & purified no activity Enzyme that belong to MetX-G1 group predicted to be HAT
        Haloferax volcanii D4GY94 metX no cloned & purified HAT  
        Halorubrum lacusprofundi B9LTF0 metX no cloned & purified HAT  
        Haloferax gibbonsii  M0HG00 metX no cloned & purified HAT  
          M0HJP0 metA   cloned & purified no activity “Unmodeled” sequences that could not be modeled due to low sequence homology with available reference 3D structures used as template in this study
        Haloferax prahovense M0GNC6 metX no cloned & purified HAT  
          M0GQW6 metA   cloned & purified no activity “Unmodeled” sequences that could not be modeled due to low sequence homology with available reference 3D structures used as template in this study.  Genomic context not related to methionine biosynthesis
        Halogeometricum borinquense E4NSW7 metX1 no cloned & purified no activity  
          E4NRK0 metX2 no cloned & not purified nd “Unmodeled” sequences that could not be modeled due to low sequence homology with available reference 3D structures used as template in this study
        Natrialba taiwanensis L9ZNK3  metX no cloned & not purified nd Unstable protein,  activity not determined. Enzyme that belong to MetX-G1 group predicted to be HAT
        Natrinema pallidum L9YX67  metX no cloned & not purified nd Unstable protein,  activity not determined. Enzyme that belong to MetX-G1 group predicted to be HAT
        Natronomonas pharaonis Q3IUE8 metX no cloned & purified HAT  
     Thermoplasmata Thermoplasmatales Thermoplasmatales archaeon  M4YMP0 metA   cloned & purified HAT  
        Candidatus Methanomassiliicoccus intestinalis Issoire R9T6W8 metA   cloned & purified HAT  
        Candidatus Methanomethylophilus alvus  M9SGV8  metA   cloned & purified HAT  
EUKARYOTA                  
Fungi Ascomycota Saccharomycete Saccharomycetales Saccharomyces cerevisiae P08465 met2/metX no not selected HAT Langin et al., 1986. Gene identification but no biochemical data.Caracterized by Yamagata, S. 1987
        Pichia angusta ? met2/metX1 no not selected HAT Sohn et al., 2014. error: JN676930 do not correspond to a metX
          A0A067XHB3 sat1/metX2 no not selected No prediction Identified by Sohn et al., 2014 as probable serine-acetyltransferase (SAT) .  No prediction because the "Sequence status" in Uniprot is "fragment" 
    Eurotiomycetes Eurotiales Aspergillus nidulans  Q9Y875 metE/metX1 no not selected HAT Grynberg et al., 2001. Gene identification but no biochemical data.
          O13389 cysA/metX2 no cloned & purified SST Identified by Grynberg et al., 2000 as probable serine acetyltransferase. Enzyme that belong to MetX-G3c group. Experimentally caracterized as succinylserine transferase in this study.
    Schizosaccharomycetes Schizosaccharomycetales Schizosaccharomyces  pombe  O60062 met6/metX1 no not selected HAT Schweingruber et al., 1998 and Nazi et al., 2005.
          Q10341 cys2/metX2 no cloned & purified SST Identified by Ma et al., 2007  as probable serine acetyltransferase. Enzyme that belong to MetX-G3c group.Experimentally caracterized as succinylserine transferase in this study.
    Pezizomycetes Pezizales Ascobolus immersus P12917 met2/metX no not selected HAT Goyon et al., 1988
    Sordariomycetes Hypocreales Gibberella zeae I1RNS1? metE/metX1 no not selected HAT You-Kyoung Han et al., 2004. Methionine auxotroph mutants and complementation experiments. Gene identification but no biochemical. No UniprotKB id available in the article. 
  Basidiomycota Tremellomycetes Tremellales Cryptococcus neoformans J9VIY8 met2/metX no not selected HAT Nazi et al., 2007