The genetic context in bacterial genomes and screening for potential substrates can help identify the biochemical functions of bacterial enzymes. The Gram-negative, strictly anaerobic bacterium Veillonella ratti possesses a gene cluster that appears to be related to l-fucose metabolism and contains a putative dihydrodipicolinate synthase/N-acetylneuraminate lyase protein (FucH). Here, screening of a library of 2-keto-3-deoxysugar acids with this protein and biochemical characterization of neighboring genes revealed that this gene cluster encodes enzymes in a previously unknown “route I” nonphosphorylating l-fucose pathway. Previous studies of other aldolases in the dihydrodipicolinate synthase/N-acetylneuraminate lyase protein superfamily used only limited numbers of compounds, and the approach reported here enabled elucidation of the substrate specificities and stereochemical selectivities of these aldolases and comparison of them with those of FucH. According to the aldol cleavage reaction, the aldolases were specific for (R)- and (S)-stereospecific groups at the C4 position of 2-keto-3-deoxysugar acid but had no structural specificity or preference of methyl groups at the C5 and C6 positions, respectively. This categorization corresponded to the (Re)- or (Si)-facial selectivity of the pyruvate enamine on the (glycer)aldehyde carbonyl in the aldol-condensation reaction. These properties are commonly determined by whether a serine or threonine residue is positioned at the equivalent position close to the active site(s), and site-directed mutagenesis markedly modified C4-OH preference and selective formation of a diastereomer. I propose that substrate specificity of 2-keto-3-deoxysugar acid aldolases was convergently acquired during evolution and report the discovery of another l-2-keto-3-deoxyfuconate aldolase involved in the same nonphosphorylating l-fucose pathway in Campylobacter jejuni. The genetic context in bacterial genomes and screening for potential substrates can help identify the biochemical functions of bacterial enzymes. The Gram-negative, strictly anaerobic bacterium Veillonella ratti possesses a gene cluster that appears to be related to l-fucose metabolism and contains a putative dihydrodipicolinate synthase/N-acetylneuraminate lyase protein (FucH). Here, screening of a library of 2-keto-3-deoxysugar acids with this protein and biochemical characterization of neighboring genes revealed that this gene cluster encodes enzymes in a previously unknown “route I” nonphosphorylating l-fucose pathway. Previous studies of other aldolases in the dihydrodipicolinate synthase/N-acetylneuraminate lyase protein superfamily used only limited numbers of compounds, and the approach reported here enabled elucidation of the substrate specificities and stereochemical selectivities of these aldolases and comparison of them with those of FucH. According to the aldol cleavage reaction, the aldolases were specific for (R)- and (S)-stereospecific groups at the C4 position of 2-keto-3-deoxysugar acid but had no structural specificity or preference of methyl groups at the C5 and C6 positions, respectively. This categorization corresponded to the (Re)- or (Si)-facial selectivity of the pyruvate enamine on the (glycer)aldehyde carbonyl in the aldol-condensation reaction. These properties are commonly determined by whether a serine or threonine residue is positioned at the equivalent position close to the active site(s), and site-directed mutagenesis markedly modified C4-OH preference and selective formation of a diastereomer. I propose that substrate specificity of 2-keto-3-deoxysugar acid aldolases was convergently acquired during evolution and report the discovery of another l-2-keto-3-deoxyfuconate aldolase involved in the same nonphosphorylating l-fucose pathway in Campylobacter jejuni. Microorganisms, including bacteria, archaea, yeasts, and fungi, utilize not only hexoses but also pentoses (d-xylose, l-arabinose, and d-arabinose) and 6-deoxyhexose sugars (l-rhamnose and l-fucose) as their sole carbon source. The metabolism of these sugars is classified into two pathways with or without phosphorylated intermediates. The former pathways consisting of isomerases (EC 5.3.1.-), kinases (EC 2.7.1.-), epimerases (EC 5.1.3.-), and/or aldolase (EC 4.1.2.-) have been extensively examined (such as l-fucose; Fig. 1A) (1Kanehisa M. Sato Y. Kawashima M. Furumichi M. Tanabe M. KEGG as a reference resource for gene and protein annotation.Nucleic Acids Res. 2016; 44 (26476454): D457-D46210.1093/nar/gkv1070Crossref PubMed Scopus (3609) Google Scholar). The latter pathways are (partially) analogous to the nonphosphorylative Entner–Doudoroff pathway from archaea (2Bräsen C. Esser D. Rauch B. Siebers B. Carbohydrate metabolism in Archaea: current insights into unusual enzymes and pathways and their regulation.Microbiol. Mol. Biol. Rev. 2014; 78 (24600042): 89-17510.1128/MMBR.00041-13Crossref PubMed Scopus (2) Google Scholar) and have been classified into three pathways, in which the sugar is commonly converted into a 2-keto-3-deoxysugar acid intermediate through the participation of aldose 1-dehydrogenase (EC 1.1.1.-), sugar lactonase (EC 3.1.1.-), and sugar acid dehydratase (EC 4.2.1.-). In the “route I” pathway (such as l-rhamnose; Fig. 1D), the 2-keto-3-deoxysugar acid intermediate is cleaved through an aldolase reaction into the appropriate aldehyde and pyruvate, which is completely homologous to the nonphosphorylative Entner–Doudoroff pathway from archaea. The “route II” pathway corresponds to an alternative pathway of pentoses, in which the 2-keto-3-deoxypentonate (KDP) 2The abbreviations used are: KDP2-keto-3-deoxypentonateKDGlu2-keto-3-deoxygluconateKDGal2-keto-3-deoxygalactonateKDR2-keto-3-deoxyrhamnonateKDF2-keto-3-deoxyfuconateDHDPSdihydrodipicolinate synthaseNALN-acetylneuraminate lyaseDRdiastereomer rate. intermediate is converted to α-ketoglutarate via α-ketoglutaric semialdehyde by KDP dehydratase (EC 4.2.1.141(43)) and α-ketoglutaric semialdehyde dehydrogenase (EC 1.2.1.26) (3Watanabe S. Shimada N. Tajima K. Kodaki T. Makino K. Identification and characterization of l-arabonate dehydratase, l- 2-keto-3-deoxyarabonate dehydratase and l-arabinolactonase involved in an alternative pathway of l-arabinose metabolism: novel evolutionary insight into sugar metabolism.J. Biol. Chem. 2006; 281 (16950779): 33521-3353610.1074/jbc.M606727200Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 4Brouns S.J. Walther J. Snijders A.P. van de Werken H.J. Willemen H.L. Worm P. de Vos M.G. Andersson A. Lundgren M. Mazon H.F. van den Heuvel R.H. Nilsson P. Salmon L. de Vos W.M. Wright P.C. et al.Identification of the missing links in prokaryotic pentose oxidation pathways: evidence for enzyme recruitment.J. Biol. Chem. 2006; 281 (16849334): 27378-2738810.1074/jbc.M605549200Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). On the other hand, in the “route III” pathway (such as l-fucose; Fig. 1C), 2-keto-3-deoxysugar acid intermediates are converted to pyruvate and α-keto acid by the sequential actions of dehydrogenase and hydrolase (5Yew W.S. Fedorov A.A. Fedorov E.V. Rakus J.F. Pierce R.W. Almo S.C. Gerlt J.A. Evolution of enzymatic activities in the enolase superfamily: l- fuconate dehydratase from Xanthomonas campestris.Biochemistry. 2006; 45 (17144652): 14582-1459710.1021/bi061687oCrossref PubMed Scopus (74) Google Scholar, 6Watanabe S. Makino K. Novel modified version of non-phosphorylated sugar metabolism: an alternative l-rhamnose pathway of Sphingomonas sp.FEBS J. 2009; 276 (19187228): 1554-156710.1111/j.1742-4658.2009.06885.xCrossref PubMed Scopus (27) Google Scholar, 7Watanabe S. Fukumori F. Nishiwaki H. Sakurai Y. Tajima K. Watanabe Y. Novel non-phosphorylative pathway of pentose metabolism from bacteria.Sci. Rep. 2019; 9 (30655589): 15510.1038/s41598-018-36774-6Crossref PubMed Scopus (33) Google Scholar). Metabolic genes related to these pathways often cluster together with putative sugar transporter genes and a transcriptional regulator gene in the genomes (Fig. 1G), and the encoding enzymes are classified into limited numbers of the known protein superfamilies: cluster of orthologous groups of proteins (COG). These insights facilitate estimations of potential substrates and/or metabolic pathways. 2-keto-3-deoxypentonate 2-keto-3-deoxygluconate 2-keto-3-deoxygalactonate 2-keto-3-deoxyrhamnonate 2-keto-3-deoxyfuconate dihydrodipicolinate synthase N-acetylneuraminate lyase diastereomer rate. Among these metabolic enzymes, 2-keto-3-deoxysugar acid aldolases are classified into two classes. Class I aldolases are characterized by a covalent intermediate, which is a protonated Schiff base formed between a lysine residue and the carbonyl carbon of the substrate. The DHDPS/NAL protein superfamily (COG0329) contains the enzymes for d-2-keto-3-deoxygluconate (d-KDGlu) (8Lamble H.J. Heyer N.I. Bull S.D. Hough D.W. Danson M.J. Metabolic pathway promiscuity in the archaeon Sulfolobus solfataricus revealed by studies on glucose dehydrogenase and 2-keto-3-deoxygluconate aldolase.J. Biol. Chem. 2003; 278 (12824170): 34066-3407210.1074/jbc.M305818200Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar), l-2-keto-3-deoxyrhamnonate (l-KDR) (9Watanabe S. Saimura M. Makino K. Eukaryotic and bacterial gene clusters related to an alternative pathway of non-phosphorylated l-rhamnose metabolism.J. Biol. Chem. 2008; 283 (18505728): 20372-2038210.1074/jbc.M801065200Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), l-2-keto-3-deoxygalactonate (l-KDGal) (10Hilditch S. Berghäll S. Kalkkinen N. Penttilä M. Richard P. The missing link in the fungal d-galacturonate pathway: identification of the l-threo-3-deoxy-hexulosonate aldolase.J. Biol. Chem. 2007; 282 (17609199): 26195-26120110.1074/jbc.M704401200Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar), and d-2-keto-3-deoxypentonate (d-KDP) (11Bhaskar V. Kumar M. Manicka S. Tripathi S. Venkatraman A. Krishnaswamy S. Identification of biochemical and putative biological role of a xenolog from Escherichia coli using structural analysis.Proteins. 2011; 79 (21294156): 1132-114210.1002/prot.22949Crossref PubMed Scopus (6) Google Scholar) from archaea, fungi, and/or bacteria together with the archetypical DHDPS (EC 4.2.1.52) and NAL (EC 4.1.3.3) (Fig. 2A). Only l-KDR aldolase from bacteria has been reported in class II aldolases (COG3836; HpcH) with the absolute requirement of a divalent metal cofactor (9Watanabe S. Saimura M. Makino K. Eukaryotic and bacterial gene clusters related to an alternative pathway of non-phosphorylated l-rhamnose metabolism.J. Biol. Chem. 2008; 283 (18505728): 20372-2038210.1074/jbc.M801065200Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). On the other hand, three genetically unidentified bacterial aldolases for l-KDP (12Dahms A.S. Anderson R.L. 2-Keto-3-deoxyl-l-arabonate aldolase and its role in a new pathway of l-arabinose degradation.Biochem. Biophys. Res. Commun. 1969; 36 (5808295): 809-81410.1016/0006-291X(69)90681-0Crossref PubMed Scopus (36) Google Scholar), d-KDP (13Dahms A.S. 3-Deoxy-d-pentulosonic acid aldolase and its role in a new pathway of d-xylose degradation.Biochem. Biophys. Res. Commun. 1974; 60 (4423285): 1433-143910.1016/0006-291X(74)90358-1Crossref PubMed Scopus (65) Google Scholar), and d-2-keto-3-deoxyfuconate (d-KDF) (14Dahms A.S. Anderson R.L. d-Fucose metabolism in a pseudomonad: IV. Cleavage of 2-keto-3-deoxy-d-fuconate to pyruvate and d-lactaldehyde by 2-keto-3-deoxy-l-arabonate aldolase.J. Biol. Chem. 1972; 247 (5016652): 2238-2241Abstract Full Text PDF PubMed Google Scholar) may also belong to this type because of their similar metal dependence. Physiological route I pathways are generally only considered to be found in d-glucose, d-galacturonate, l-rhamnose, d-fucose, d-xylose, and l-arabinose metabolism. (Reverse) aldol condensation is more useful for the synthesis of biologically, pharmaceutically, and/or agrochemically significant compounds than (forward) physiological aldol cleavage (15Windle C.L. Müller M. Nelson A. Berry A. Engineering aldolases as biocatalysts.Curr. Opin. Chem. Biol. 2014; 19 (24780276): 25-3310.1016/j.cbpa.2013.12.010Crossref PubMed Scopus (73) Google Scholar). This reaction enables the formation of a carbon–carbon bond between two carbonyl compounds, during which two new stereogenic centers are made. Among several reported class I aldolase enzymes, d-KDGlu aldolase (KdgA) from the hyperthermophilic Archaeon Sulfolobus solfataricus (as described above) has been well-studied biochemically and structurally, and the catalytic mechanism containing eight active-site residues at least is elucidated significantly (8Lamble H.J. Heyer N.I. Bull S.D. Hough D.W. Danson M.J. Metabolic pathway promiscuity in the archaeon Sulfolobus solfataricus revealed by studies on glucose dehydrogenase and 2-keto-3-deoxygluconate aldolase.J. Biol. Chem. 2003; 278 (12824170): 34066-3407210.1074/jbc.M305818200Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 16Theodossis A. Walden H. Westwick E.J. Connaris H. Lamble H.J. Hough D.W. Danson M.J. Taylor G.L. The structural basis for substrate promiscuity in 2-keto-3-deoxygluconate aldolase from the Entner–Doudoroff pathway in Sulfolobus solfataricus.J. Biol. Chem. 2004; 279 (15265860): 43886-4389210.1074/jbc.M407702200Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar) (Fig. 2A). In the aldol condensation reaction, the ω-amino functionality of Lys155 (site 4) forms a Schiff base with the carbonyl C2 of pyruvate, which tautomerizes to afford an enamine intermediate that may attack the (Re)- or (Si)-face of the carbonyl of d-glyceraldehyde (Fig. 2B). This reaction is mediated by the phenolic substituent of Tyr130 (active site 2), which plays a key catalytic role in shuttling protons between reactive enzyme-bound intermediates to yield two diastereomers: d-KDGlu and d-KDGal. In the aldol cleavage reaction, their C4-OH groups are also in a position to interact with this tyrosyl oxygen, which mediates proton extraction by the substrate, and the C5- and C6-OH groups make bridging water-mediated hydrogen-bonding interactions with (the main and/or side chains of) active sites 3, 6, 7, and 8 (Fig. 2C). The carboxylate group is recognized by the main and side chains of Thr43 and Thr44 in a characteristic Gly-Xaa-Xaa-Gly-Glu motif (active site 1). In the present study, I focused on the EO124_RS00050-RS00025 gene cluster from Veillonella ratti ATCC 17746, a strictly anaerobic bacterium. Although gene components are partially homologous with those of known l-fucose pathways, a putative DHDPS/NAL-like protein (EO124_RS00030; FucH) was also present. The library screening of the 2-keto-3-deoxysugar acids of this protein (and biochemical characterization of neighboring components) revealed that the gene cluster was responsible for (unidentified) route I of the nonphosphorylative l-fucose pathway. This approach also enabled more detailed comparisons of the substrate specificities and stereochemical selectivities of not only FucH as a l-KDF aldolase but also other 2-keto-3-deoxysugar acid aldolases in the DHDPS/NAL protein superfamily. I previously characterized gene clusters related to nonphosphorylative sugar metabolism from Herbaspirillum huttiense and Acidovorax avenae, in which an altronate dehydratase-like gene (protein) (COG2721; UxaA) functions as a dehydratase for d-arabinonate, l-xylonate, d-altronate, d-idonate, l-gluconate, and/or l-fuconate (7Watanabe S. Fukumori F. Nishiwaki H. Sakurai Y. Tajima K. Watanabe Y. Novel non-phosphorylative pathway of pentose metabolism from bacteria.Sci. Rep. 2019; 9 (30655589): 15510.1038/s41598-018-36774-6Crossref PubMed Scopus (33) Google Scholar, 17Watanabe S. Fukumori F. Watanabe Y. Substrate and metabolic promiscuities of d-altronate dehydratase family proteins involved in non-phosphorylative d-arabinose, sugar acid, l- galactose and l-fucose pathways from bacteria.Mol. Microbiol. 2019; 112 (30985034): 147-16510.1111/mmi.14259Crossref PubMed Scopus (6) Google Scholar). On the other hand, the homologous gene from V. ratti ATCC 17746 (EO124_RS00050; FucC) clustered with several genes related to putative l-fucose metabolism: EO124_RS00040, l-fucose/H+ symporter (TC 2.A.1.7; FucP); the C-terminal half of EO124_RS00045, l-fucose mutarotase (EC 5.1.3.29; FucM); EO124_RS00045, l-fucose 1-dehydrogenase (EC 1.1.1.122; FucA); the N-terminal half of EO124_RS00045, l-fuconolactonase (FucB; EC 3.1.1.-); EO124_RS00025, l-lactaldehyde reductase (EC 1.1.1.77; FucO); and EO124_RS00050; function unknown DHDPS/NAL-like gene (FucH) (Fig. 1G). To date, the metabolic pathways of l-fucose in microorganisms (bacteria) have been classified into two routes. The final products of the “phosphorylative route” are dihydroxyacetone phosphate and l-lactaldehyde, with the latter being aerobically oxidized to l-lactate (by l-lactaldehyde dehydrogenase (EC 1.2.1.22); AldA) or anaerobically reduced to (S)-1,2-propanediol (by FucO) (Fig. 1A) (18Baldomà L. Aguilar J. Metabolism of l-fucose and l-rhamnose in Escherichia coli: aerobic-anaerobic regulation of l-lactaldehyde dissimilation.J. Bacteriol. 1988; 170 (3275622): 416-42110.1128/JB.170.1.416-421.1988Crossref PubMed Google Scholar). The nonphosphorylative pathway corresponds to route III, and the metabolic fate of the l-KDF intermediate is pyruvate and l-lactate via l-2,4-diketo-3-deoxyfuconate (Fig. 1C) (5Yew W.S. Fedorov A.A. Fedorov E.V. Rakus J.F. Pierce R.W. Almo S.C. Gerlt J.A. Evolution of enzymatic activities in the enolase superfamily: l- fuconate dehydratase from Xanthomonas campestris.Biochemistry. 2006; 45 (17144652): 14582-1459710.1021/bi061687oCrossref PubMed Scopus (74) Google Scholar, 7Watanabe S. Fukumori F. Nishiwaki H. Sakurai Y. Tajima K. Watanabe Y. Novel non-phosphorylative pathway of pentose metabolism from bacteria.Sci. Rep. 2019; 9 (30655589): 15510.1038/s41598-018-36774-6Crossref PubMed Scopus (33) Google Scholar). FucH was not present within gene clusters related to these l-fucose pathways (Fig. 1G). Therefore, to elucidate the physiological role(s) of this type of gene cluster, I initially characterized FucH from V. ratti (referred to as VrFucH). Limited numbers of 2-keto-3-deoxysugar acids are generally used for functional characterization because of the difficulties associated with their preparation (not commercially available) (8Lamble H.J. Heyer N.I. Bull S.D. Hough D.W. Danson M.J. Metabolic pathway promiscuity in the archaeon Sulfolobus solfataricus revealed by studies on glucose dehydrogenase and 2-keto-3-deoxygluconate aldolase.J. Biol. Chem. 2003; 278 (12824170): 34066-3407210.1074/jbc.M305818200Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 9Watanabe S. Saimura M. Makino K. Eukaryotic and bacterial gene clusters related to an alternative pathway of non-phosphorylated l-rhamnose metabolism.J. Biol. Chem. 2008; 283 (18505728): 20372-2038210.1074/jbc.M801065200Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 10Hilditch S. Berghäll S. Kalkkinen N. Penttilä M. Richard P. The missing link in the fungal d-galacturonate pathway: identification of the l-threo-3-deoxy-hexulosonate aldolase.J. Biol. Chem. 2007; 282 (17609199): 26195-26120110.1074/jbc.M704401200Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 11Bhaskar V. Kumar M. Manicka S. Tripathi S. Venkatraman A. Krishnaswamy S. Identification of biochemical and putative biological role of a xenolog from Escherichia coli using structural analysis.Proteins. 2011; 79 (21294156): 1132-114210.1002/prot.22949Crossref PubMed Scopus (6) Google Scholar, 12Dahms A.S. Anderson R.L. 2-Keto-3-deoxyl-l-arabonate aldolase and its role in a new pathway of l-arabinose degradation.Biochem. Biophys. Res. Commun. 1969; 36 (5808295): 809-81410.1016/0006-291X(69)90681-0Crossref PubMed Scopus (36) Google Scholar, 13Dahms A.S. 3-Deoxy-d-pentulosonic acid aldolase and its role in a new pathway of d-xylose degradation.Biochem. Biophys. Res. Commun. 1974; 60 (4423285): 1433-143910.1016/0006-291X(74)90358-1Crossref PubMed Scopus (65) Google Scholar, 14Dahms A.S. Anderson R.L. d-Fucose metabolism in a pseudomonad: IV. Cleavage of 2-keto-3-deoxy-d-fuconate to pyruvate and d-lactaldehyde by 2-keto-3-deoxy-l-arabonate aldolase.J. Biol. Chem. 1972; 247 (5016652): 2238-2241Abstract Full Text PDF PubMed Google Scholar). Alternatively, I enzymatically prepared nine 2-keto-3-deoxysugar acid(s) from sugar acids, as described under “Experimental procedures.” The potential enzyme activity of VrFucH was initially assayed by HPLC (Fig. S1). When each 2-keto-3-deoxysugar acid was incubated with the purified recombinant protein, a novel peak with a later retention time appeared in a time-dependent manner and was identical to lactaldehyde (∼12.6 min for l-KDF, d-KDF, and l-KDR), glycolaldehyde (∼12.4 min for d-KDP and l-KDP), and glyceraldehyde (∼11.3 min for d-KDGlu, l-KDGlu, d-KDGal, and l-KDGal). When catalyzed with KDGlu(s) and KDGal(s), novel peaks that differed from glyceraldehyde were commonly observed and found to be identical to pyruvate (∼9.8 min) and the alternative diastereomer. l-KDF and d-KDP were the best substrates (Fig. 3A). These results indicated that VrFucH catalyzes the reversible retro-aldolase reaction for 2-keto-3-deoxysugar acid(s) to afford pyruvate and aldehyde. To investigate substrate specificity in more detail, the activity of the aldol cleavage reaction was spectrophotometrically assessed using l-lactate dehydrogenase as a coupling enzyme. Kinetic parameters from the Lineweaver–Burk plot are shown in Table 1 and Fig. 3F. The kcat/Km values for l-KDF and d-KDP were similar (6570 and 6340 min−1 mm−1, respectively), whereas those for the other substrates were reduced by ∼1–3 orders of magnitude and caused by very small kcat values. Among the substrates tested, clear structural preferences were detected at C4-(R)-OH and C6-CH3 but not at C5-OH; for example, the ratios of l-KDF to l-KDR (C4) and l-KDF to l-KDGal (C6) were 29 and 14, respectively (red bars in Fig. 4A).Table 1Kinetic parameters of EO124_RS00030 from (VrFucH)EnzymeaA His6-tagged recombinant enzyme was used.SubstratebEight different substrate concentrations between 0.1 and 1 mm were used.C4-OHKmkcatkcat/KmR/ScThe ratio of kcat/Km.mmmin−1min−1 mm−1foldWTl-KDFR0.327 ± 0.0042150 ± 86570 ± 6529l-KDRS1.28 ± 0.07289 ± 12226 ± 3d-KDRRNPNPNPNPd-KDFS0.535 ± 0.1334.39 ± 0.688.37 ± 0.71d-KDPR0.430 ± 0.0752690 ± 2346340 ± 5004.3l-KDPS0.923 ± 0.1831340 ± 2101470 ± 56d-KDGluR0.692 ± 0.223107 ± 21161 ± 1847d-KDGalS1.85 ± 0.386.21 ± 0.953.39 ± 0.23l-KDGalR1.47 ± 0.43208 ± 48144 ± 99.5l-KDGluS1.16 ± 0.0417.5 ± 0.415.2 ± 0.2T164Sl-KDFR0.583 ± 0.0741260 ± 972180 ± 12011l-KDRS2.54 ± 0.21503 ± 35198 ± 2d-KDRRNPNPNPNPd-KDFS1.08 ± 0.01163 ± 1151 ± 0d-KDPR0.345 ± 0.023293 ± 8853 ± 332.7l-KDPS1.88 ± 0.13599 ± 39319 ± 7d-KDGluR0.570 ± 0.05115.0 ± 1.026.4 ± 0.61.3d-KDGalS0.900 ± 0.11617.9 ± 1.320.1 ± 1.1l-KDGalR3.10 ± 0.50158 ± 2351.1 ± 0.82.5l-KDGluS2.11 ± 0.1642.7 ± 2.620.3 ± 0.3a A His6-tagged recombinant enzyme was used.b Eight different substrate concentrations between 0.1 and 1 mm were used.c The ratio of kcat/Km. Open table in a new tab In the aldol condensation reaction, the HPLC analysis showed that l-lactaldehyde, d-lactaldehyde, and glycolaldehyde were rapidly consumed in a time-dependent manner, and the reaction reached equilibrium within 6 h (Fig. 5A and Fig. S2). However, glyceraldehyde(s) were suitable for estimating selectivity because of similar retention between KDF, KDR, KDP, and pyruvate. During their reactions, both peaks for glyceraldehyde and pyruvate decreased in parallel, and two product peaks corresponding to KDGlu and KDGal were clearly visible throughout the time course of the reaction, affording 96:4 and 95:5 mixtures of d-KDGlu:d-KDGal and l-KDGal:l-KDGlu, respectively (Fig. 5B), suggesting strict (Re)-facial selectivity (Fig. 2B). Aliphatic aldehydes, including acetaldehyde and propionaldehyde, were not acceptable for VrFucH. As described in the introduction, the DHDPS/NAL protein superfamily contains three 2-keto-3-deoxysugar acid aldolases. l-KDR aldolase (LRA4) converts l-KDR (a C4-diastereomer of l-KDF) into pyruvate and l-lactaldehyde (the same products as VrFucH) (Fig. 2B), and the encoding gene is located within a unique fungal gene cluster related to route I of the nonphosphorylative l-rhamnose pathway (Fig. 1, D and G) (9Watanabe S. Saimura M. Makino K. Eukaryotic and bacterial gene clusters related to an alternative pathway of non-phosphorylated l-rhamnose metabolism.J. Biol. Chem. 2008; 283 (18505728): 20372-2038210.1074/jbc.M801065200Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). l-KDGal aldolase (EC 4.1.2.54; LGA1) is involved in fungal d-galacturonate metabolism as the fourth enzyme, in which l-KDGal is converted into pyruvate and l-glyceraldehyde (10Hilditch S. Berghäll S. Kalkkinen N. Penttilä M. Richard P. The missing link in the fungal d-galacturonate pathway: identification of the l-threo-3-deoxy-hexulosonate aldolase.J. Biol. Chem. 2007; 282 (17609199): 26195-26120110.1074/jbc.M704401200Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). On the other hand, YagE is more closely related phylogenetically to VrFucH (31% sequence identity) than any DHDPS/NAL members (∼25%) (Fig. 6) and functions as d-KDP aldolase in d-xylonate metabolism (Fig. 1, E and G) (11Bhaskar V. Kumar M. Manicka S. Tripathi S. Venkatraman A. Krishnaswamy S. Identification of biochemical and putative biological role of a xenolog from Escherichia coli using structural analysis.Proteins. 2011; 79 (21294156): 1132-114210.1002/prot.22949Crossref PubMed Scopus (6) Google Scholar, 19Shimada T. Momiyama E. Yamanaka Y. Watanabe H. Yamamoto K. Ishihama A. Regulatory role of XynR (YagI) in catabolism of xylonate in Escherichia coli K-12.FEMS Microbiol. Lett. 2017; 364 (29087459): fnx22010.1093/femsle/fnx220Crossref Scopus (12) Google Scholar). Therefore, LRA4, LGA1, and YagE from Pichia stipites (also named Scheffersomyces stipitis), Hypocrea jecorina (also named Trichoderma reesei), and Escherichia coli were enzymatically characterized in detail using a library of 2-keto-3-deoxysugar acids (referred to as PsLRA4, HjLGA1, and EcYagE, respectively). In the aldol cleavage reaction, l-KDR (the physiological substrate) and l-KDP were the best substrates, and the kcat/Km values of the other substrates were reduced by 1–3 orders of magnitude because of very small kcat values (Fig. 3, B and G, and Table S1). The C4-(S)-OH preference was completely opposite to that of VrFucH (for example, the ratio of l-KDR to l-KDF in kcat/Km was 48), whereas the C6-CH3 preference was similar (red bars in Fig. 4B). In the aldol condensation reaction, PsLRA4 catalyzed the selective formation of d-KDGal and l-KDGlu in 83 and 98% diastereomer rates (DRs), respectively (Fig. 5D). Therefore, PsLRA4 showed (Se)-facial selectivity (Fig. 2B). l-KDF was the best substrate in the aldol cleavage reaction, whereas the kcat/Km value for l-KDGal (the physiological substrate) was 21-fold lower than that for l-KDF because of a high Km value; 69.0 and 1430 min−1 mm−1, respectively (Fig. 3, C and H, and Table S2). The C4-(S)-OH and C6-CH3 preferences were similar to those of VrFucH; for example, the ratios of l-KDR to l-KDF (C4) and l-KDF to l-KDGal (C6) in kcat/Km were 24 and 21 (red bars in Fig. 4C). In the aldol condensation reaction, l-glyceraldehyde was the only active substrate, affording a 83:17 mixture of l-KDGal:l-KDGlu (Fig. 5F). The reaction with d-glyceraldehyde using 10-fold amounts of the enzyme gave a 83:17 mixture of d-KDGlu and d-KDGal. Therefore, HjLGA1 showed (Re)-facial selectivity (Fig. 2B). d-KDP was the best substrate in the aldol cleavage reaction, whereas the kcat/Km value was 4.8-fold higher than that for l-KDF (Fig. 3, D and I, and Table S3). In the aldol condensation reaction using d-glyceraldehyde, EcYagE catalyzed the selective formation of d-KDGlu in 88% DR (Fig. 5H). On the other hand, when catalyzing with l-glyceraldehyde, this enzyme exhibited no diastereocontrol, which gave a 53:47 mixture of l-KDGal and l-KDGlu. This property is consistent that there is no C4-(R)-OH preference between the two diastereomers (red bars in Fig. 4D). Therefore, the structural preference of C4-OH and facial selectivity in the reversible aldol reaction were clearly linked, suggesting a common amino acid residue(s) responsible for these stereochemical specificities. The catalytic mechanism of VrFucH as a l-KDF aldolase (and PsLRA4, HjLGA1, and EcYagE) may be typical of DHDPS/NAL members, particularly KdgA from S. solfataricus (SsKdgA), as described in the introduction; Lys162 and Tyr133 correspond to two lysine and tyrosine residues at active sites 4 and 2, respectively (Fig. 2B) (16Theodossis A. Walden H. Westwick E.J. Connaris H. Lamble H.J. Hough D.W. Danson M.J. Taylor G.L. The structural basis for substrate promiscuity in 2-keto-3-deoxygluconate aldolase from the Entner–Doudoroff pathway in Sulfolobus solfataricus.J. Biol. Chem. 2004; 279 (15265860): 43886-4389210.1074/jbc.M407702200Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). On the other hand, SsKdgA utilizes not only d-glyceraldehyde (a natural substrate) but also l-glyceraldehyde as acceptable aldehydes and exhibits poor diastereocontrol, which gives a 50:50 mixture of d-KDGlu:d-KDGal or l-KDGal:l-KDGlu (8Lamble H.J. Heyer N.I. Bull S.D. Hough D.W. Danson M.J. Metabolic pathway promiscuity in the archaeon Sulfolobus solfataricus revealed by studies on glucose dehydrogenase and 2-keto-3-deoxygluconate aldolase.J. Bi