An aldolase and dehydrogenase complex from the polychlorinated biphenyl degradation pathway of the bacterium Burkholderia xenovorans LB400 was purified. The aldolase, BphI, had the highest activity with Mn(2+) as the cofactor and was able to transform 4-hydroxy-2-oxopentanoate and 4-hydroxy-2-oxohexanoate to pyruvate and acetaldehyde or propionaldehyde with similar specificity constants. Aldolase activity was competitively inhibited by the pyruvate enolate analogue, oxalate, with a K(ic) of 0.93 microM. The pH-rate profiles suggested the involvement of a pK(a) 7.7 catalytic base in the reaction mechanism. BphI activity was activated 15-fold when substrate turnover was occurring in the dehydrogenase, BphJ, which can be attributed partially to nicotinamide coenzyme binding to BphJ. BphJ had similar specificity constants for acetaldehyde or propionaldehyde and was able to utilize aliphatic aldehydes from two to five carbons in length as substrates, although K(m) values for these aldehyes were >20 mM. When 4-hydroxy-2-oxopentanoate was provided as a substrate to the BphI-BphJ complex in a coupled enzyme assay, no lag in the progress curve of BphJ was observed. When 1 mM propionaldehyde was added exogenously to a reaction mixture containing 0.1 mM 4-hydroxy-2-oxopentanoate, 95% of the CoA esters produced was acetyl CoA. Conversely, 99% of the CoA esters produced was propionyl CoA when a 10-fold molar excess of exogenous acetaldehyde was added in a reaction mixture containing 4-hydroxy-2-oxohexanoate. These results demonstrate that acetaldehyde and propionaldehyde, products of the BphI reaction, are not released in the bulk solvent but are channeled directly to the dehydrogenase.

HpaI, a class II pyruvate-specific aldolase involved in the catabolic pathway of hydroxyphenylacetate, is overexpressed and purified. A previous suggestion that phosphate is involved in proton transfer of pyruvate, based on the crystal structure of the homologous 2-dehydro-3-deoxygalactarate aldolase, is not substantiated from biochemical studies with HpaI. Thus, specific activities of the enzyme for the substrate 4-hydroxy-2-ketopentanoate in sodium HEPES and Tris-acetate buffers are higher than in sodium phosphate buffer. The enzyme also catalyzed the partial reaction of pyruvate proton exchange with an initial rate of 0.77 mmol min(-)(1) mg(-)(1) in phosphate-free buffer, as monitored by nuclear magnetic resonance. Steady-state kinetic analysis shows that the enzyme is also able to catalyze the aldol cleavage of 4-hydroxy-2-ketohexanoate and 3-deoxy-d-manno-oct-2-ulosonic acid (KDO). The enzyme exhibits significant oxaloacetate decarboxylase activity, with a k(cat) value 2.4-fold higher than the corresponding value for the aldol cleavage of 4-hydroxy-2-ketopentanoate. Sodium oxalate, an analogue of the enolate intermediate of the enzyme-catalyzed reaction, is a competitive inhibitor of the enzyme, with a K(i) value of 5.5 microM. Replacement of an active site arginine residue (R70) with alanine by site-specific mutagenesis resulted in an enzyme that lacks both aldolase and decarboxylase activities. The mutant enzyme is also unable to catalyze pyruvate proton exchange. The dissociation constant for pyruvate in the R70A mutant, determined by fluorescence titration, is similar to that of the wild-type enzyme, indicating that pyruvate binding is not affected by this mutation. Together, the results show that R70 influences catalysis in HpaI, particularly at the pyruvate proton exchange step.

Cellulases play important roles in the dietary fibre digestion in pigs, and have multiple industrial applications. The porcine intestinal microbiota display a unique feature in rapid cellulose digestion. Herein, we have expressed a cellulase gene, p4818Cel5_2A, which singly encoded a catalytic domain belonging to glycoside hydrolase family 5 subfamily 2, and was previously identified from a metagenomic expression library constructed from porcine gut microbiome after feeding grower pigs with a cellulose-supplemented diet. The activity of purified p4818Cel5_2A was maximal at pH 6.0 and 50 °C and displayed resistance to trypsin digestion. This enzyme exhibited activities towards a wide variety of plant polysaccharides, including cellulosic substrates of avicel and solka-Floc®, and the hemicelluloses of β-(1 → 4)/(1 → 3)-glucans, xyloglucan, glucomannan and galactomannan. Viscosity, reducing sugar distribution and hydrolysis product analyses further revealed that this enzyme was a processive endo-β-(1 → 4)-glucanase capable of hydrolyzing cellulose into cellobiose and cellotriose as the primary end products. These catalytic features of p4818Cel5_2A were further explored in the context of a three-dimensional homology model. Altogether, results of this study report a microbial processive endoglucanase identified from the porcine gut microbiome, and it may be tailored as an efficient biocatalyst candidate for potential industrial applications.

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HpaI and BphI are two pyruvate class II aldolases found in aromatic meta-cleavage degradation pathways that catalyze similar reactions but are not related in sequence. Steady-state kinetic analysis of the aldol addition reactions and product inhibition assays showed that HpaI exhibits a rapid equilibrium random order mechanism while BphI exhibits a compulsory order mechanism, with pyruvate binding first. Both aldolases are able to utilize aldehyde acceptors two to five carbons in length; however, HpaI showed broader specificity and had a preference for aldehydes containing longer linear alkyl chains or C2-OH substitutions. Both enzymes were able to bind 2-keto acids larger than pyruvate, but only HpaI was able to utilize both pyruvate and 2-ketobutanoate as carbonyl donors in the aldol addition reaction. HpaI lacks stereospecific control producing racemic mixtures of 4-hydroxy-2-oxopentanoate (HOPA) from pyruvate and acetaldehyde while BphI synthesizes only (4S)-HOPA. BphI is also able to utilize acetaldehyde produced by the reduction of acetyl-CoA catalyzed by the associated aldehyde dehydrogenase, BphJ. This aldehyde was directly channeled from the dehydrogenase to the aldolase active sites, with an efficiency of 84%. Furthermore, the BphJ reductive deacylation reaction increased 4-fold when BphI was catalyzing the aldol addition reaction. Therefore, the BphI-BphJ enzyme complex exhibits unique bidirectionality in substrate channeling and allosteric activation.

4-Hydroxy-4-methyl-2-oxoglutarate/4-carboxy-4-hydroxy-2-oxoadipate (HMG/CHA) aldolase from Pseudomonas putida F1 catalyzes the last step of the bacterial protocatechuate 4,5-cleavage pathway. The preferred substrates of the enzyme are 2-keto-4-hydroxy acids with a 4-carboxylate substitution. The enzyme also exhibits oxaloacetate decarboxylation and pyruvate ␣-proton exchange activity. Sodium oxalate is a competitive inhibitor of the aldolase reaction. The pH dependence of k cat /K m and k cat for the enzyme is consistent with a single deprotonation with pK a values of 8.0 ؎ 0.1 and 7.0 ؎ 0.1 for free enzyme and enzyme substrate complex, respectively. The 1.8 Å x-ray structure shows a four-layered ␣-␤-␤-␣ sandwich structure with the active site at the interface of two adjacent subunits of a hexamer; this fold resembles the RNase E inhibitor, RraA, but is novel for an aldolase. The catalytic site contains a magnesium ion ligated by Asp-124 as well as three water molecules bound by Asp-102 and Glu-199. A pyruvate molecule binds the magnesium ion through both carboxylate and keto oxygen atoms, completing the octahedral geometry. The carbonyl oxygen also forms hydrogen bonds with the guanadinium group of Arg-123, which site-directed mutagenesis confirms is essential for catalysis. A mechanism for HMG/CHA aldolase is proposed on the basis of the structure, kinetics, and previously established features of other aldolase mechanisms.

Background: HpaI is a metal-dependent pyruvate aldolase bridging aromatic hydrocarbon degradation and central metabolism.Results: Crystal structures of enzyme⅐substrate complexes reveal the catalytic mechanism. Conclusion: Two water molecules, one of them Co 2ϩ -bound, are acid-base catalysts, whereas an arginine residue stabilizes transient negative charges. Significance: Results provide a framework for engineering pyruvate aldolases with improved biocatalysis of C-C bond formation.

BackgroundAcetylated 4-O-(methyl)glucuronoxylan (GX) is the main hemicellulose in deciduous hardwood, and comprises a β-(1→4)-linked xylopyranosyl (Xylp) backbone substituted by both acetyl groups and α-(1→2)-linked 4-O-methylglucopyranosyluronic acid (MeGlcpA). Whereas enzymes that target singly acetylated Xylp or doubly 2,3-O-acetyl-Xylp have been well characterized, those targeting (2-O-MeGlcpA)3-O-acetyl-Xylp structures in glucuronoxylan have remained elusive.ResultsAn unclassified carbohydrate esterase (FjoAcXE) was identified as a protein of unknown function from a polysaccharide utilization locus (PUL) otherwise comprising carbohydrate-active enzyme families known to target xylan. FjoAcXE was shown to efficiently release acetyl groups from internal (2-O-MeGlcpA)3-O-acetyl-Xylp structures, an activity that has been sought after but lacking in known carbohydrate esterases. FjoAcXE action boosted the activity of α-glucuronidases from families GH67 and GH115 by five and nine times, respectively. Moreover, FjoAcXE activity was not only restricted to GX, but also deacetylated (3-O-Araf)2-O-acetyl-Xylp of feruloylated xylooligomers, confirming the broad substrate range of this new carbohydrate esterase.ConclusionThis study reports the discovery and characterization of the novel carbohydrate esterase, FjoAcXE. In addition to cleaving singly acetylated Xylp, and doubly 2,3-O-acetyl-Xylp, FjoAcXE efficiently cleaves internal 3-O-acetyl-Xylp linkages in (2-O-MeGlcpA)3-O-acetyl-Xylp residues along with densely substituted and branched xylooligomers; activities that until now were missing from the arsenal of enzymes required for xylan conversion.Electronic supplementary materialThe online version of this article (10.1186/s13068-018-1074-3) contains supplementary material, which is available to authorized users.