Emerging concepts in pseudoenzyme classification, evolution, and signaling

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Science Signaling  13 Aug 2019:
Vol. 12, Issue 594, eaat9797
DOI: 10.1126/scisignal.aat9797
  • Fig. 1 Regulated assembly of the macromolecular BRISC-SHMT2 pseudoenzyme-containing complex.

    (Top) Schematic of the SHMT2 dimer-tetramer oligomerization transition, which is regulated by pyridoxal-5′-phosphate (PLP), the active cofactor form of vitamin B6. The SHMT2 dimer, which is inactive as a methyltransferase (left), specifically interacts with, and inhibits, the pseudoenzyme-containing BRISC complex (bottom), revealing a previously unknown moonlighting role for SHMT2. The PLP-bound SHMT2 tetramer, which is active as a methyltransferase, is unable to bind or inhibit the BRISC complex. (Bottom) Schematic of the multimeric BRISC-SHMT2 DUB complex, which contains the active DUB BRCC36 [MPN+ (MPR1, PAD1 N-terminal )] and the inactive Abraxas2 pseudo-DUB (MPN). BRCC45 contains three pseudo-E2 domains (UEVs) and is discussed further in Table 1 and in the text.

  • Fig. 2 Computational annotation of enzymes and pseudoenzymes in UniProt.

    A worked example of the inactive annotation for the C. elegans pseudophosphatase egg-4 (UniProtKB O01767), including the protein name, the protein-coding evidence (status), and the caution comment. The image is recreated and adapted from the UniProt website.

  • Fig. 3 Analyzing the (pseudo)kinome and (pseudo)phosphatome in a model worm.

    (A) Calculated percentage of pseudokinases in the C. elegans kinome (orange). (B) Percentage of pseudophosphatases in the C. elegans phosphatome (orange).

  • Fig. 4 Diversity in ATP-binding mode and the acquisition of noncanonical catalytic functions among bioinformatically annotated pseudokinases.

    (A) The catalytically inactive pseudokinase, FAM20A, still binds ATP (red sticks) but in an inverted conformation [Protein Data Bank (PDB): 5wrs] (35). Like the related FAM20B, the position of the αC helix (orange) differs from that in canonical protein kinases. (B) The highly atypical annotated pseudokinase SelO (PDB: 6eac) (38) can catalyze protein AMPylation via an unusual catalytic mechanism. Like FAM20A (A), SelO binds ATP in an inverted conformation but, in addition, it catalyzes AMP transfer to protein substrates. ATP analog AMP-PNP is shown as red sticks; Mg2+ and Ca2+ are shown as yellow and green spheres, respectively. (C) The predicted pseudokinase, FAM20B (PDB: 5xoo), is actually a catalytically active xylose kinase (37) involved in proteoglycan synthesis. The sugar substrate is shown as green sticks; an adenine (red sticks) is also modeled in the structure. (D) Like FAM20B, the predicted pseudokinase SgK169/protein O-mannose kinase (POMK; PDB: 5gza) is actually a sugar kinase (40). SgK169/POMK closely resembles a typical protein kinase fold with conventional αC helix (orange) position, nucleotide-binding mode (red sticks), and Mg2+ cofactors (yellow spheres), and excitingly, this structure captures the protein bound to its sugar substrate (green sticks).

  • Fig. 5 Estimating the ratio of pseudoenzymes in known enzyme families.

    Estimated proportion of pseudoenzymes within known enzyme families. A family is defined here as the group of SwissProt sequences that are homologous to one enzyme/entry in M-CSA. Each entry in M-CSA corresponds to one enzyme with a unique enzyme mechanism, so the same EC number can be represented more than once if it evolved independently with distinct mechanisms. Sequences are categorized as enzymes if they have a catalytic UniProt keyword and as pseudoenzymes otherwise. The orange bar corresponds to enzyme families that contain only enzymes; the blue bars correspond to enzyme families that contain a variable percentage of pseudoenzymes.

  • Fig. 6 Annotated phylogenetic tree for soybean β-amylase.

    Phylogenetic tree for homologous sequences of soybean β-amylase (P10538) annotated with the catalytic residues identified in M-CSA, their EC numbers, and protein families domains. The tree contains 20 homologs from plants (of which 6 are chloroplastic) and 5 from bacteria. The two inactive pseudoenzymes identified (Q9FM68 and Q8VYW2) belong to Thale cress (Arabidopsis thaliana), and one of these (Q9FM68) is known to have physiological regulatory functions in this organism.

  • Fig. 7 Analysis of the distribution of pseudoenzymes.

    (A) Sequences in CATH superfamilies are subclassified into functional families predicted to share similar structures and functions, and which can be used to understand protein function evolution. (B) Distribution of the number of enzyme superfamilies (containing catalytic domains) that have varying proportions of functional families with enzyme annotations. (C) The number of putative pseudoenzyme families in enzyme superfamilies (containing catalytic domains). These remain to be confirmed by further analysis and experimental testing.

  • Fig. 8 Pseudoenzymes in the NTF2 family.

    An example comparing the known pseudoenzyme family of NTF2 (blue) and the related enzyme family of scytalone hydratases (orange) in CATH superfamily 3.10.450.50. The established catalytic residues for the scytalone hydratase family (EC are shown in red. The structural alignment between the structures 1stdA00 and 1ounA00 from the enzyme and pseudoenzyme families was generated using CATH-superpose ( On the right-hand panel, the highly conserved residues in the two families are shown for structurally equivalent positions lying in the active site of the enzyme family. The height of the characters reflects the degree of conservation, and the colors change according to physicochemical characteristics.

  • Table 1 The remarkable diversity of pseudoenzymes.

    Examples of pseudoenzymes from across the kingdoms of life, organized by class and function. Pseudoenzymes are highlighted in blue, whereas relevant conventional enzymes are in black. A broad selection of well-studied pseudoenzymes are discussed; the list is not meant to be comprehensive. ADP, adenosine diphosphate; ROS, reactive oxygen species; IFN, interferon; GTPase, guanosine triphosphatase; RIPK3, receptor interacting serine/threonine protein kinase 3; COP1, constitutive photomorphogenic 1; TRIB1, Tribbles 1; DUSP4, dual specificity phosphatase 4; MTMR13, myotubularin-related protein 13; SP, serine protease; PPO, prophenoloxidases.

    PseudokinaseAllosteric regulation of
    conventional protein
    STRADα:LKB1, HER3:HER2, JAK JH2:JAK JH1, FAM20A:FAM20C, where a
    pseudokinase domain regulates the activity of a conventional kinase in a
    cognate pair; also a common mechanism in plant pseudokinases
    (including tandem pseudokinases) such as RKS1
    (2427, 35, 7779)
    Regulator of a
    phospholipid kinase
    Vps15 is a probable pseudokinase that forms part of a multimeric complex
    for regulation of the phosphatidylinositol 3-kinase Vps34
    Allosteric regulation of
    other enzymes
    Pseudokinase domains of guanylyl cyclase–A and guanylyl cyclase–B
    regulate activity of the tandem guanylyl cyclase domains
    Molecular switchPhosphorylation of the MLKL pseudokinase domain triggers exposure of
    the executioner four-helix bundle domain and cell death
    (47, 72)
    Protein interaction
    MLKL pseudokinase domain is regulated by binding to the RIPK3 kinase
    domain and HSP90:Cdc37 co-chaperones
    (73, 82)
    Scaffold for assembly of
    signaling complexes
    TRIB pseudokinases nucleate assembly of a complex between a substrate
    (such as C/EBPα) and the E3 ubiquitin ligase COP1, whose
    intrasubcellular localization is controlled by Tribbles 1 TRIB1
    (7476, 83)
    (73, 84)
    SgK223 (Pragmin)/SgK269 (PEAK1) forms higher-order signaling assemblies
    that include Src-family kinases
    (73, 8488)
    Fundamental metabolic
    regulators of
    isoprenoid lipid
    UbiB pseudoenzyme family adopts an (inactive?) atypical protein
    kinase-like fold found in bacteria, archaea, and eukaryotes; human
    mitochondrial ADCK3 pseudokinase binds nucleotides such as ADP but can
    be re-engineered into an ATP-dependent autophosphorylating enzyme;
    also relevant to the yeast Coq8p ATPase
    (69, 89)
    Pseudo-histidine kinaseProtein interaction
    Caulobacter DivL binds the response regulator, DivK to regulate asymmetric
    cell division
    PseudophosphataseOcclusion of conventional
    phosphatase access to
    substrate and
    subcellular localization
    STYX binds ERK1/2 kinase to occlude DUSP4 binding and anchors ERK1/2 in
    the nucleus
    Regulation of protein
    localization in a cell
    MTMR13 stabilizes lipid phosphatase MTMR2 and localizes MTMR2 to
    Regulation of signaling
    complex assembly
    STYX prevents substrate recruitment via FBXW7 to SCF E3 ligase complex(20)
    Redox sensor as part of
    A receptor protein tyrosine phosphatase alpha redox-active Cys residue in
    the D2 (pseudophosphatase) domain controls the catalytic output of the
    D1 (canonical phosphatase) domain after exposure to ROS
    PseudoproteaseAllosteric regulator of
    conventional protease
    cFLIP binds the cysteine protease, Caspase-8, to block apoptosis(93)
    Regulation of protein
    localization in a cell
    Mammalian iRhom proteins bind and regulate membrane trafficking of
    (94, 95)
    Multiple pseudo-orthocaspases disclosed (prokaryotic)(96)
    Activation of the
    Noncatalytic clip-domain SP family member PPAF-II from H. diomphalia
    binds and activates processed prophenoloxidases PPO1 and PPO2
    Prevention of zymogen
    Caspase-like pseudoproteases csp-2 and csp-3 prevent ced-3 autoactivation
    in C. elegans
    (98, 99)
    Allosteric regulator of
    conventional DUB
    Abraxas1/2 nucleate assembly of a higher-order heterotetramer with
    BRCC36 active DUB (see also Fig. 1)
    Allosteric regulator of
    conventional enzyme
    CSN6 supports the activity of CSN5 and acts as a scaffold for the Cop9
    signallosome complex
    Allosteric regulator of
    conventional DUB
    PSMD7 (Rpn8 in yeast) supports the activity of the active DUB PSMD14
    (Rpn11); part of the 19S regulatory particle of the proteasome
    (102, 103)
    Scaffold for assembly of
    enzyme complexes
    PRP8 (Prpf8 in yeast) is central to the assembly of the spliceosome complex
    required for mRNA splicing; Prp8 contains three additional
    pseudoenzyme domains (pseudo-endonuclease, pseudo-reverse
    transcriptase, pseudo-RNaseH)
    Scaffold for assembly of
    enzyme complexes
    USP39 (Sad1 in yeast) is a member of the spliceosome complex with
    presumed scaffolding roles
    Scaffold for assembly of
    enzyme complexes
    eIF3f and eIF3h are both inactive and integral for the assembly of eIF3
    complex required for protein synthesis
    Scaffold for assembly of
    enzyme complexes
    USP52 (Pan2 in yeast) serves as a scaffold for the Pan2-Pan3 deadenylating
    (16, 104)
    Unknown functionFAM105A is an OTU domain containing protein that is closest to OTULIN
    (also known as FAM105B or Gumby); likely functions as a protein
    interactin domain in the ER/Golgi; the MINDY4b pseudoenzyme is also
    part of the broader MINDY DUB family
    (16, 105)
    enzyme (pseudo-E2)
    Allosteric regulator of
    conventional E2 ligase
    The ubiquitin E2 variant (UEV) domain of Mms2 binds the active E2, Ubc13,
    to enable catalytic activity and dictate K63-linked ubiquitin linkages
    Regulation of protein
    localization in a cell
    The Tsg101 UEV domain exerts a chaperone function, including during viral
    Scaffold for assembly of
    signaling complexes
    BRCC45 contains three tandem UEV domains that support protein-protein
    interactions within the BRISC-SHMT2 complex required for IFN signaling
    and the BRCA1-A complex required for DNA damage repair
    PseudonucleaseAllosteric regulator of
    conventional nuclease
    CPSF-100 is a component of the pre-mRNA 3′ end processing complex
    containing the active counterpart, CPSF-73
    Nucleating and regulating
    assembly of
    protein:nucleic acid
    TEFM contributes to the elongation complex sliding clamp and sequesters
    ssDNA to maintain the mitochondrial transcription bubble
    Regulation of substrate
    Exuperantia pseudo-exonuclease domain regulates cellular localization of
    the bicoid mRNA in Drosophila cells
    PseudoATPaseAllosteric regulator of
    conventional ATPase
    EccC comprises two pseudoATPase domains that regulate the N-terminal
    conventional ATPase domain
    PseudoGTPaseAllosteric regulator of
    conventional GTPase
    GTP-bound Rnd1 or Rnd3/RhoE bind p190RhoGAP to regulate the catalytic
    activity of the GTPase, RhoA
    (112, 113)
    Allosteric control of
    conventional enzyme
    Three GTPase-like domains promote p190RhoGAP activity(114, 115)
    Scaffold for assembly of
    signaling complexes
    CENP-M cannot bind GTP or switch conformations but is essential for
    regulating kinetochore assembly
    Regulation of protein
    localization in a cell
    Yeast light intermediate domain (LIC) is a pseudoGTPase, devoid of nucleotide
    binding, which connects the dynein motor protein to cargo proteins
    PseudochitinaseSubstrate recruitment or
    YKL-39 binds chitooligosaccharides via 5 binding subsites, but does not
    process these as substrates
    (118, 119)
    PseudosialidaseScaffold for assembly of
    signaling complexes
    Plasmodium falciparum CyRPA nucleates assembly of the PfRh5-PfRipr
    complex that binds the host erythrocyte receptor, basigin, and mediates
    host cell invasion
    PseudoglycosidaseScaffold for assembly of
    signaling complexes
    β-Klotho tandem pseudoglycosidase domains act as a receptor for the
    hormone FGF21
    PseudolyaseAllosteric activation of
    conventional enzyme
    Dead paralog binding to S-adenosylmethionine decarboxylase displaces an
    N-terminal inhibitory segment to activate catalytic activity
    PseudotransferaseAllosteric activation of
    cellular enzyme
    Viral GAT recruits cellular phosphoribosyl-formylglycinamidine synthetase
    to deaminate RIG-I to block host antiviral defense
    Assembly of catalytically
    active enzyme via
    composite active site
    A composite active site is formed by dead and catalytic paralogs of T. brucei
    deoxyhypusine synthase heterotetramerize
    (126, 127)
    Assembly of
    mitochondrial fission
    MiD51 is an inactive nucloetidyltransferase dynamin receptor that binds
    nucleotide diphosphates and assembles Drp1, a regulator of
    mitochondrial fission
    Pantothenate kinaseEukaryotic “bifunctional” pantothenate kinase 4 (PANK4)
    phosphotransferases contain an N-terminal pantothenate pseudokinase
    domain fused to a phosphatase domain; human PANK domain of PANK4
    lacks catalytic Glu and Arg residues that are found in the PANK1-3 enzymes
    PseudosulfotransferaseUnknown neuronal
    Eukaryotic enzymatically inactive soluble sulfotransferase (SULT4A1) fails to
    bind in vitro to the universal sulfate donor PAPS or to sulfate a standard
    SULT substrate
    Pseudo-histone acetyl
    transferase (pseudoHAT)
    Possible scaffold for
    assembly of signaling
    Unlike bacterial Onychiuroides granulosus counterpart, human C-terminal
    domain of putative bifunctional glycoside hydrolase and histone
    acetyltransferase (AT) O-GlcNAcase (OGA) lacks catalytic residues and
    detectable acetyl CoA binding
    PseudophospholipaseScaffold for assembly of
    signaling complexes
    DUF1669 pseudophospholipase D-like domains in FAM83 family proteins
    bind and regulate the canonical kinase CK1
    (132, 133)
    Allosteric inactivation of
    conventional enzyme
    Viper phospholipase A2 inhibitor attenuates the toxicity of the active
    phospholipase A2 paralog via heterodimerization
    PseudooxidoreductaseAllosteric inactivation of
    conventional enzyme
    ALDH2*2 sequesters the active counterpart, ALDH2*1, to prevent its
    assembly into the tetrameric catalytic form
    PseudodismutaseCompetition for enzyme’s
    Poxviral superoxide dismutase (SOD)–like proteins bind and sequester
    Copper chaperone for superoxide dismutase to block activation of
    cellular SOD1
    PseudohydrolaseIntegrin activation by
    protein with cryptic
    diphosphate (Nudix) domain
    KRIT1 antagonizes ICAP1 (negative regulator of integrin function) through a
    competitive mechanism. N terminus of KRIT1 contains Nudix
    pseudoenzyme domain lacking canonical “Nudix box”
    Antiviral factor loss
    induces resistance to
    West Nile virus
    Enzymatically inactive Murine Oas1b (a 2′-5′-oligoadenylate synthetase 1
    paralog) has RNAse L-independent antiviral properties toward flavivirus
    in mice
    (138, 139)

Supplementary Materials

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    • Fig. S1. Estimating the ratio of pseudoenzymes in enzyme families.

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