ID NANA_STAA8 Reviewed; 293 AA. AC Q2G160; DT 05-FEB-2008, integrated into UniProtKB/Swiss-Prot. DT 21-MAR-2006, sequence version 1. DT 27-MAR-2024, entry version 103. DE RecName: Full=N-acetylneuraminate lyase {ECO:0000255|HAMAP-Rule:MF_01237, ECO:0000303|PubMed:23418011}; DE Short=NAL {ECO:0000255|HAMAP-Rule:MF_01237, ECO:0000303|PubMed:23418011}; DE Short=Neu5Ac lyase {ECO:0000255|HAMAP-Rule:MF_01237, ECO:0000303|PubMed:23418011}; DE EC=4.1.3.3 {ECO:0000255|HAMAP-Rule:MF_01237, ECO:0000269|PubMed:23418011, ECO:0000269|PubMed:27943302}; DE AltName: Full=N-acetylneuraminate pyruvate-lyase {ECO:0000255|HAMAP-Rule:MF_01237}; DE AltName: Full=N-acetylneuraminic acid aldolase {ECO:0000255|HAMAP-Rule:MF_01237}; DE AltName: Full=Sialate lyase {ECO:0000255|HAMAP-Rule:MF_01237}; DE AltName: Full=Sialic acid aldolase {ECO:0000255|HAMAP-Rule:MF_01237}; DE AltName: Full=Sialic acid lyase {ECO:0000255|HAMAP-Rule:MF_01237}; GN Name=nanA {ECO:0000255|HAMAP-Rule:MF_01237, GN ECO:0000303|PubMed:23418011}; OrderedLocusNames=SAOUHSC_00295; OS Staphylococcus aureus (strain NCTC 8325 / PS 47). OC Bacteria; Bacillota; Bacilli; Bacillales; Staphylococcaceae; OC Staphylococcus. OX NCBI_TaxID=93061; RN [1] RP NUCLEOTIDE SEQUENCE [LARGE SCALE GENOMIC DNA]. RC STRAIN=NCTC 8325 / PS 47; RA Gillaspy A.F., Worrell V., Orvis J., Roe B.A., Dyer D.W., Iandolo J.J.; RT "The Staphylococcus aureus NCTC 8325 genome."; RL (In) Fischetti V., Novick R., Ferretti J., Portnoy D., Rood J. (eds.); RL Gram positive pathogens, 2nd edition, pp.381-412, ASM Press, Washington RL D.C. (2006). RN [2] {ECO:0007744|PDB:4AH7, ECO:0007744|PDB:4AHO, ECO:0007744|PDB:4AHP, ECO:0007744|PDB:4AHQ, ECO:0007744|PDB:4AMA} RP X-RAY CRYSTALLOGRAPHY (1.95 ANGSTROMS) OF APOENZYME, WILD-TYPE AND MUTANTS RP CYS-165 AND GAMMA-THIALYSINE-165 IN COMPLEX WITH PYRUVATE, FUNCTION, RP CATALYTIC ACTIVITY, BIOPHYSICOCHEMICAL PROPERTIES, SUBUNIT, MUTAGENESIS OF RP LYS-165, AND ACTIVE SITE. RC STRAIN=NCTC 8325 / PS 47; RX PubMed=23418011; DOI=10.1002/cbic.201200714; RA Timms N., Windle C.L., Polyakova A., Ault J.R., Trinh C.H., Pearson A.R., RA Nelson A., Berry A.; RT "Structural insights into the recovery of aldolase activity in N- RT acetylneuraminic acid lyase by replacement of the catalytically active RT lysine with gamma-thialysine by using a chemical mutagenesis strategy."; RL ChemBioChem 14:474-481(2013). RN [3] {ECO:0007744|PDB:5KZD, ECO:0007744|PDB:5KZE} RP X-RAY CRYSTALLOGRAPHY (1.74 ANGSTROMS) OF APOENZYME AND IN COMPLEX WITH RP SIALIC ACID ALDITOL, FUNCTION, CATALYTIC ACTIVITY, ACTIVITY REGULATION, RP BIOPHYSICOCHEMICAL PROPERTIES, AND SUBUNIT. RC STRAIN=MRSA; RX PubMed=27943302; DOI=10.1002/1873-3468.12462; RA North R.A., Watson A.J., Pearce F.G., Muscroft-Taylor A.C., Friemann R., RA Fairbanks A.J., Dobson R.C.; RT "Structure and inhibition of N-acetylneuraminate lyase from methicillin- RT resistant Staphylococcus aureus."; RL FEBS Lett. 590:4414-4428(2016). RN [4] {ECO:0007744|PDB:5A8G} RP X-RAY CRYSTALLOGRAPHY (1.72 ANGSTROMS) OF 2-293 IN COMPLEX WITH RP FLUOROPYRUVATE, FUNCTION, BIOPHYSICOCHEMICAL PROPERTIES, AND MUTAGENESIS OF RP GLU-192. RX PubMed=26537532; DOI=10.1039/c5ob02037a; RA Stockwell J., Daniels A.D., Windle C.L., Harman T.A., Woodhall T., Lebl T., RA Trinh C.H., Mulholland K., Pearson A.R., Berry A., Nelson A.; RT "Evaluation of fluoropyruvate as nucleophile in reactions catalysed by N- RT acetyl neuraminic acid lyase variants: scope, limitations and RT stereoselectivity."; RL Org. Biomol. Chem. 14:105-112(2016). RN [5] {ECO:0007744|PDB:5LKY} RP X-RAY CRYSTALLOGRAPHY (1.70 ANGSTROMS) OF 2-293 OF MUTANT RP 2,3-DIHYDROXYPROPYL CYSTEINE-190 IN COMPLEX WITH FLUOROPYRUVATE, RP BIOPHYSICOCHEMICAL PROPERTIES, AND BIOTECHNOLOGY. RX PubMed=28196894; DOI=10.1073/pnas.1616816114; RA Windle C.L., Simmons K.J., Ault J.R., Trinh C.H., Nelson A., Pearson A.R., RA Berry A.; RT "Extending enzyme molecular recognition with an expanded amino acid RT alphabet."; RL Proc. Natl. Acad. Sci. U.S.A. 114:2610-2615(2017). CC -!- FUNCTION: Catalyzes the reversible aldol cleavage of N-acetylneuraminic CC acid (sialic acid; Neu5Ac) to form pyruvate and N-acetylmannosamine CC (ManNAc) via a Schiff base intermediate (PubMed:23418011, CC PubMed:27943302). In vitro, catalyzes the reaction between CC fluoropyruvate and ManNAc, albeit much less efficiently than with CC pyruvate as donor, leading to the synthesis of fluorinated analogs of CC N-acetylneuraminic acid (PubMed:26537532). Variants are useful CC catalysts of reactions between fluoropyruvate and unnatural aldehyde CC substrates (PubMed:26537532). {ECO:0000269|PubMed:23418011, CC ECO:0000269|PubMed:26537532, ECO:0000269|PubMed:27943302}. CC -!- CATALYTIC ACTIVITY: CC Reaction=aceneuramate = N-acetyl-D-mannosamine + pyruvate; CC Xref=Rhea:RHEA:23296, ChEBI:CHEBI:15361, ChEBI:CHEBI:17122, CC ChEBI:CHEBI:173083; EC=4.1.3.3; Evidence={ECO:0000255|HAMAP- CC Rule:MF_01237, ECO:0000269|PubMed:23418011, CC ECO:0000269|PubMed:27943302}; CC -!- ACTIVITY REGULATION: Strongly inhibited by sialic acid alditol. CC {ECO:0000269|PubMed:27943302}. CC -!- BIOPHYSICOCHEMICAL PROPERTIES: CC Kinetic parameters: CC KM=2.2 mM for N-acetylneuraminate (at pH 7.4) CC {ECO:0000269|PubMed:23418011}; CC KM=2.4 mM for N-acetylneuraminate (at pH 6.8) CC {ECO:0000269|PubMed:23418011}; CC KM=3.2 mM for N-acetylneuraminate {ECO:0000269|PubMed:27943302}; CC KM=2 mM for N-acetylneuraminate {ECO:0000269|PubMed:26537532}; CC KM=3.1 mM for ManNAc (for the aldol condensation of ManNAc and CC pyruvate) {ECO:0000269|PubMed:28196894}; CC KM=3 mM for erythrose (for the aldol condensation of erythrose and CC pyruvate) {ECO:0000269|PubMed:28196894}; CC Vmax=40 umol/min/mg enzyme {ECO:0000269|PubMed:27943302}; CC Note=kcat is 250 min(-1) for the cleavage of Neu5Ac at pH 7.4, and is CC 260 min(-1) at pH 6.8 (PubMed:23418011). kcat is 22.1 sec(-1) with CC N-acetylneuraminate as substrate (PubMed:27943302). kcat is 510 CC min(-1) with N-acetylneuraminate as substrate (PubMed:26537532). kcat CC is 0.8 min(-1) with ManNAc as substrate (for the aldol condensation CC of ManNAc and pyruvate) (PubMed:28196894). kcat is 0.5 min(-1) with CC erythrose as substrate (for the aldol condensation of erythrose and CC pyruvate) (PubMed:28196894). {ECO:0000269|PubMed:23418011, CC ECO:0000269|PubMed:26537532, ECO:0000269|PubMed:27943302, CC ECO:0000269|PubMed:28196894}; CC pH dependence: CC Optimum pH is 7.4. {ECO:0000269|PubMed:23418011}; CC -!- PATHWAY: Amino-sugar metabolism; N-acetylneuraminate degradation; D- CC fructose 6-phosphate from N-acetylneuraminate: step 1/5. CC {ECO:0000255|HAMAP-Rule:MF_01237}. CC -!- SUBUNIT: Homotetramer. {ECO:0000255|HAMAP-Rule:MF_01237, CC ECO:0000269|PubMed:23418011, ECO:0000269|PubMed:27943302}. CC -!- SUBCELLULAR LOCATION: Cytoplasm {ECO:0000255|HAMAP-Rule:MF_01237}. CC -!- BIOTECHNOLOGY: Introduction of a non-canonical amino acid into the CC protein alters the substrate specificity of the enzyme and achieves CC levels of activity toward a new substrate that are otherwise CC unattainable by mutagenesis to any of the canonical amino acids CC (PubMed:28196894). By modifying Phe-190 to a 2,3-dihydroxypropyl CC cysteine, the formation of 3-deoxy-2-heptulosonic acid (DHA) from CC erythrose and pyruvate is significantly increased (PubMed:28196894). CC {ECO:0000269|PubMed:28196894}. CC -!- MISCELLANEOUS: When the catalytic Lys-165 residue is replaced by the CC unnatural amino acid gamma-thialysine, the enzyme regains significant CC activity compared with the Cys-165 mutant; regains of approximately 17% CC of the wild-type catalytic efficiency. {ECO:0000269|PubMed:23418011}. CC -!- SIMILARITY: Belongs to the DapA family. NanA subfamily. CC {ECO:0000255|HAMAP-Rule:MF_01237}. CC --------------------------------------------------------------------------- CC Copyrighted by the UniProt Consortium, see https://www.uniprot.org/terms CC Distributed under the Creative Commons Attribution (CC BY 4.0) License CC --------------------------------------------------------------------------- DR EMBL; CP000253; ABD29464.1; -; Genomic_DNA. DR RefSeq; WP_001030738.1; NZ_LS483365.1. DR RefSeq; YP_498885.1; NC_007795.1. DR PDB; 4AH7; X-ray; 2.30 A; A/B/C/D=2-293. DR PDB; 4AHO; X-ray; 2.00 A; A/B/C/D=1-293. DR PDB; 4AHP; X-ray; 2.10 A; A/B/C/D=2-293. DR PDB; 4AHQ; X-ray; 1.95 A; A/B/C/D=2-293. DR PDB; 4AMA; X-ray; 2.35 A; A/B/C/D=2-293. DR PDB; 5A8G; X-ray; 1.72 A; A/B=2-293. DR PDB; 5KZD; X-ray; 2.33 A; A/B/C/D=1-293. DR PDB; 5KZE; X-ray; 1.74 A; A/B/C/D/E/F/G/H=1-293. DR PDB; 5LKY; X-ray; 1.70 A; A/B/C/D=2-293. DR PDBsum; 4AH7; -. DR PDBsum; 4AHO; -. DR PDBsum; 4AHP; -. DR PDBsum; 4AHQ; -. DR PDBsum; 4AMA; -. DR PDBsum; 5A8G; -. DR PDBsum; 5KZD; -. DR PDBsum; 5KZE; -. DR PDBsum; 5LKY; -. DR AlphaFoldDB; Q2G160; -. DR SMR; Q2G160; -. DR STRING; 93061.SAOUHSC_00295; -. DR PaxDb; 1280-SAXN108_0299; -. DR GeneID; 3918976; -. DR KEGG; sao:SAOUHSC_00295; -. DR PATRIC; fig|93061.5.peg.268; -. DR eggNOG; COG0329; Bacteria. DR HOGENOM; CLU_049343_5_1_9; -. DR OrthoDB; 9782828at2; -. DR BRENDA; 4.1.3.3; 3352. DR UniPathway; UPA00629; UER00680. DR PRO; PR:Q2G160; -. DR Proteomes; UP000008816; Chromosome. DR GO; GO:0005829; C:cytosol; IBA:GO_Central. DR GO; GO:0008747; F:N-acetylneuraminate lyase activity; IBA:GO_Central. DR GO; GO:0005975; P:carbohydrate metabolic process; IEA:UniProtKB-UniRule. DR GO; GO:0019262; P:N-acetylneuraminate catabolic process; IBA:GO_Central. DR CDD; cd00954; NAL; 1. DR Gene3D; 3.20.20.70; Aldolase class I; 1. DR HAMAP; MF_01237; N_acetylneuram_lyase; 1. DR InterPro; IPR013785; Aldolase_TIM. DR InterPro; IPR002220; DapA-like. DR InterPro; IPR005264; NanA. DR InterPro; IPR020625; Schiff_base-form_aldolases_AS. DR PANTHER; PTHR42849; N-ACETYLNEURAMINATE LYASE; 1. DR PANTHER; PTHR42849:SF1; N-ACETYLNEURAMINATE LYASE; 1. DR Pfam; PF00701; DHDPS; 1. DR PIRSF; PIRSF001365; DHDPS; 1. DR PRINTS; PR00146; DHPICSNTHASE. DR SMART; SM01130; DHDPS; 1. DR SUPFAM; SSF51569; Aldolase; 1. DR PROSITE; PS00666; DHDPS_2; 1. PE 1: Evidence at protein level; KW 3D-structure; Carbohydrate metabolism; Cytoplasm; Lyase; KW Reference proteome; Schiff base. FT CHAIN 1..293 FT /note="N-acetylneuraminate lyase" FT /id="PRO_1000066938" FT ACT_SITE 137 FT /note="Proton donor" FT /evidence="ECO:0000255|HAMAP-Rule:MF_01237" FT ACT_SITE 165 FT /note="Schiff-base intermediate with substrate" FT /evidence="ECO:0000255|HAMAP-Rule:MF_01237, FT ECO:0000269|PubMed:23418011" FT BINDING 48 FT /ligand="aceneuramate" FT /ligand_id="ChEBI:CHEBI:173083" FT /evidence="ECO:0000305|PubMed:27943302, FT ECO:0007744|PDB:5KZD" FT BINDING 49 FT /ligand="aceneuramate" FT /ligand_id="ChEBI:CHEBI:173083" FT /evidence="ECO:0000305|PubMed:27943302, FT ECO:0007744|PDB:5KZD" FT BINDING 189 FT /ligand="aceneuramate" FT /ligand_id="ChEBI:CHEBI:173083" FT /evidence="ECO:0000305|PubMed:27943302, FT ECO:0007744|PDB:5KZD" FT BINDING 191 FT /ligand="aceneuramate" FT /ligand_id="ChEBI:CHEBI:173083" FT /evidence="ECO:0000305|PubMed:27943302, FT ECO:0007744|PDB:5KZD" FT BINDING 192 FT /ligand="aceneuramate" FT /ligand_id="ChEBI:CHEBI:173083" FT /evidence="ECO:0000305|PubMed:27943302, FT ECO:0007744|PDB:5KZD" FT BINDING 208 FT /ligand="aceneuramate" FT /ligand_id="ChEBI:CHEBI:173083" FT /evidence="ECO:0000305|PubMed:27943302, FT ECO:0007744|PDB:5KZD" FT BINDING 252 FT /ligand="aceneuramate" FT /ligand_id="ChEBI:CHEBI:173083" FT /evidence="ECO:0000305|PubMed:27943302, FT ECO:0007744|PDB:5KZD" FT MUTAGEN 165 FT /note="K->C: 3125-fold decrease in catalytic activity, and FT 3-fold increase in substrate affinity." FT /evidence="ECO:0000269|PubMed:23418011" FT MUTAGEN 192 FT /note="E->N: Increases reaction with fluoropyruvate and the FT alternative substrate FT (2R,3S)-2,3-dihydroxy-4-oxo-N,N-dipropylbutanamide (DHOB)." FT /evidence="ECO:0000269|PubMed:26537532" FT STRAND 7..11 FT /evidence="ECO:0007829|PDB:5LKY" FT HELIX 24..36 FT /evidence="ECO:0007829|PDB:5LKY" FT STRAND 41..47 FT /evidence="ECO:0007829|PDB:5LKY" FT HELIX 48..50 FT /evidence="ECO:0007829|PDB:5LKY" FT HELIX 52..54 FT /evidence="ECO:0007829|PDB:5LKY" FT HELIX 57..70 FT /evidence="ECO:0007829|PDB:5LKY" FT STRAND 75..80 FT /evidence="ECO:0007829|PDB:5LKY" FT HELIX 86..99 FT /evidence="ECO:0007829|PDB:5LKY" FT STRAND 102..107 FT /evidence="ECO:0007829|PDB:5LKY" FT HELIX 116..130 FT /evidence="ECO:0007829|PDB:5LKY" FT STRAND 134..138 FT /evidence="ECO:0007829|PDB:5LKY" FT HELIX 140..143 FT /evidence="ECO:0007829|PDB:5LKY" FT HELIX 149..156 FT /evidence="ECO:0007829|PDB:5LKY" FT STRAND 161..166 FT /evidence="ECO:0007829|PDB:5LKY" FT HELIX 171..180 FT /evidence="ECO:0007829|PDB:5LKY" FT STRAND 184..188 FT /evidence="ECO:0007829|PDB:5LKY" FT HELIX 191..193 FT /evidence="ECO:0007829|PDB:5A8G" FT HELIX 194..199 FT /evidence="ECO:0007829|PDB:5LKY" FT STRAND 203..208 FT /evidence="ECO:0007829|PDB:5LKY" FT HELIX 210..225 FT /evidence="ECO:0007829|PDB:5LKY" FT HELIX 229..249 FT /evidence="ECO:0007829|PDB:5LKY" FT HELIX 251..261 FT /evidence="ECO:0007829|PDB:5LKY" FT HELIX 278..280 FT /evidence="ECO:0007829|PDB:5LKY" FT HELIX 281..291 FT /evidence="ECO:0007829|PDB:5LKY" SQ SEQUENCE 293 AA; 33043 MW; 62D7DEFFDC1AF431 CRC64; MNKDLKGLYA ALLVPFDENG QVNEQGLKQI AQNAIETEEL DGLYVNGSSG ENFLLNTEQK KQVFKVAKEA VGDKVKLIAQ VGSLDLNEAI ELGKYATELG YDALSAVTPF YYPFTFEEIR DYYFDIIEAT QNNMIIYAIP DLTGVNISIE QFSELFNHEK IVGVKYTAPN FFLLERIRKA FPDKLILSGF DEMLVQATIS GVDGAIGSTY NVNGRRARKI FDLARQGQIQ EAYQLQHDSN DIIETVLSMG IYPTLKEILR HRGIDAGLPK RPFKPFNEAH RQTLDQLIAK YDL //