Engineering potassium activation into biosynthetic thiolase.

Activation of enzymes by monovalent cations (M+) is a widespread phenomenon in biology. Despite this, there are few structure-based studies describing the underlying molecular details. Thiolases are a ubiquitous and highly conserved family of enzymes containing both K+-activated and K+-independent members. Guided by structures of naturally occurring K+-activated thiolases, we have used a structure-based approach to engineer K+-activation into a K+-independent thiolase. To our knowledge, this is the first demonstration of engineering K+-activation into an enzyme, showing the malleability of proteins to accommodate M+ ions as allosteric regulators. We show that a few protein structural features encode K+-activation in this class of enzyme. Specifically, two residues near the substrate-binding site are sufficient for K+-activation: A tyrosine residue is required to complete the K+ coordination sphere, and a glutamate residue provides a compensating charge for the bound K+ ion. Further to these, a distal residue is important for positioning a K+-coordinating water molecule that forms a direct hydrogen bond to the substrate. The stability of a cation-π interaction between a positively charged residue and the substrate is determined by the conformation of the loop surrounding the substrate-binding site. Our results suggest that this cation-π interaction effectively overrides K+-activation, and is, therefore, destabilised in K+-activated thiolases. Evolutionary conservation of these amino acids provides a promising signature sequence for predicting K+-activation in thiolases. Together, our structural, biochemical and bioinformatic work provide important mechanistic insights into how enzymes can be allosterically activated by M+ ions.

The crystal structure of human mitochondrial 3-ketoacyl-CoA thiolase (T1): insight into the reaction mechanism of its thiolase and thioesterase activities.

Crystal structures of human mitochondrial 3-ketoacyl-CoA thiolase (hT1) in the apo form and in complex with CoA have been determined at 2.0 Å resolution. The structures confirm the tetrameric quaternary structure of this degradative thiolase. The active site is surprisingly similar to the active site of the Zoogloea ramigera biosynthetic tetrameric thiolase (PDB entries 1dm3 and 1m1o) and different from the active site of the peroxisomal dimeric degradative thiolase (PDB entries 1afw and 2iik). A cavity analysis suggests a mode of binding for the fatty-acyl tail in a tunnel lined by the Nß2-Nα2 loop of the adjacent subunit and the Lα1 helix of the loop domain. Soaking of the apo hT1 crystals with octanoyl-CoA resulted in a crystal structure in complex with CoA owing to the intrinsic acyl-CoA thioesterase activity of hT1. Solution studies confirm that hT1 has low acyl-CoA thioesterase activity for fatty acyl-CoA substrates. The fastest rate is observed for the hydrolysis of butyryl-CoA. It is also shown that T1 has significant biosynthetic thiolase activity, which is predicted to be of physiological importance.

The thiolase reaction mechanism: the importance of Asn316 and His348 for stabilizing the enolate intermediate of the Claisen condensation.

The biosynthetic thiolase catalyzes a Claisen condensation reaction between acetyl-CoA and the enzyme acetylated at Cys89. Two oxyanion holes facilitate this catalysis: oxyanion hole I stabilizes the enolate intermediate generated from acetyl-CoA, whereas oxyanion hole II stabilizes the tetrahedral intermediate of the acetylated enzyme. The latter intermediate is formed when the alpha-carbanion of acetyl-CoA enolate reacts with the carbonyl carbon of acetyl-Cys89, after which C-C bond formation is completed. Oxyanion hole II is made of two main chain peptide NH groups, whereas oxyanion hole I is formed by a water molecule (Wat82) and NE2(His348). Wat82 is anchored in the active site by an optimal set of hydrogen bonding interactions, including a hydrogen bond to ND2(Asn316). Here, the importance of Asn316 and His348 for catalysis has been studied; in particular, the properties of the N316D, N316A, N316H, H348A, and H348N variants have been determined. For the N316D variant, no activity could be detected. For each of the remaining variants, the k(cat)/K(m) value for the Claisen condensation catalysis is reduced by a factor of several hundred, whereas the thiolytic degradation catalysis is much less affected. The crystal structures of the variants show that the structural changes in the active site are minimal. Our studies confirm that oxyanion hole I is critically important for the condensation catalysis. Removing either one of the hydrogen bond donors causes the loss of at least 3.4 kcal/mol of transition state stabilization. It appears that in the thiolytic degradation direction, oxyanion hole I is not involved in stabilizing the transition state of its rate limiting step. However, His348 has a dual role in the catalytic cycle, contributing to oxyanion hole I and activating Cys89. The analysis of the hydrogen bonding interactions in the very polar catalytic cavity shows the importance of two conserved water molecules, Wat82 and Wat49, for the formation of oxyanion hole I and for influencing the reactivity of the catalytic base, Cys378, respectively. Cys89, Asn316, and His348 form the CNH-catalytic triad of the thiolase superfamily. Our findings are also discussed in the context of the importance of this triad for the catalytic mechanism of other enzymes of the thiolase superfamily.

The catalytic cycle of biosynthetic thiolase: a conformational journey of an acetyl group through four binding modes and two oxyanion holes.

Biosynthetic thiolase catalyzes the formation of acetoacetyl-CoA from two molecules of acetyl-CoA. This is a key step in the synthesis of many biological compounds, including steroid hormones and ketone bodies. The thiolase reaction involves two chemically distinct steps; during acyl transfer, an acetyl group is transferred from acetyl-CoA to Cys89, and in the Claisen condensation step, this acetyl group is further transferred to a second molecule of acetyl-CoA, generating acetoacetyl-CoA. Here, new crystallographic data for Zoogloea ramigera biosynthetic thiolase are presented, covering all intermediates of the thiolase catalytic cycle. The high-resolution structures indicate that the acetyl group goes through four conformations while being transferred from acetyl-CoA via the acetylated enzyme to acetoacetyl-CoA. This transfer is catalyzed in a rigid cavity lined by mostly hydrophobic side chains, in addition to the catalytic residues Cys89, His348, and Cys378. The structures highlight the importance of an oxyanion hole formed by a water molecule and His348 in stabilizing the negative charge on the thioester oxygen atom of acetyl-CoA at two different steps of the reaction cycle. Another oxyanion hole, composed of the main chain nitrogen atoms of Cys89 and Gly380, complements a negative charge of the thioester oxygen anion of the acetylated intermediate, stabilizing the tetrahedral transition state of the Claisen condensation step. The reactivity of the active site may be modulated by hydrogen bonding networks extending from the active site toward the back of the molecule.

Crystallographic analysis of the reaction pathway of Zoogloea ramigera biosynthetic thiolase.

Biosynthetic thiolases catalyze the biological Claisen condensation of two acetyl-CoA molecules to form acetoacetyl-CoA. This is one of the fundamental categories of carbon skeletal assembly patterns in biological systems and is the first step in many biosynthetic pathways including those which generate cholesterol, steroid hormones and ketone body energy storage molecules. High resolution crystal structures of the tetrameric biosynthetic thiolase from Zoogloea ramigera were determined (i) in the absence of active site ligands, (ii) in the presence of CoA, and (iii) from protein crystals which were flash frozen after a short soak with acetyl-CoA, the enzyme's substrate in the biosynthetic reaction. In the latter structure, a reaction intermediate was trapped: the enzyme was found to be acetylated at Cys89 and a molecule of acetyl-CoA was bound in the active site pocket. A comparison of the three new structures and the two previously published thiolase structures reveals that small adjustments in the conformation of the acetylated Cys89 side-chain allow CoA and acetyl-CoA to adopt identical modes of binding. The proximity of the acetyl moiety of acetyl-CoA to the sulfur atom of Cys378 supports the hypothesis that Cys378 is important for proton exchange in both steps of the reaction. The thioester oxygen atom of the acetylated enzyme points into an oxyanion hole formed by the nitrogen atoms of Cys89 and Gly380, thus facilitating the condensation reaction. The interaction between the thioester oxygen atom of acetyl-CoA and His348 assists the condensation step of catalysis by stabilizing a negative charge on the thioester oxygen atom. Our structure of acetyl-CoA bound to thiolase also highlights the importance in catalysis of a hydrogen bonding network between Cys89 and Cys378, which includes the thioester oxygen atom of acetyl-CoA, and extends from the catalytic site through the enzyme to the opposite molecular surface. This hydrogen bonding network is different in yeast degradative thiolase, indicating that the catalytic properties of each enzyme may be modulated by differences in their hydrogen bonding networks.

A biosynthetic thiolase in complex with a reaction intermediate: the crystal structure provides new insights into the catalytic mechanism.

BACKGROUND: Thiolases are ubiquitous and form a large family of dimeric or tetrameric enzymes with a conserved, five-layered alphabetaalphabetaalpha catalytic domain. Thiolases can function either degradatively, in the beta-oxidation pathway of fatty acids, or biosynthetically. Biosynthetic thiolases catalyze the biological Claisen condensation of two molecules of acetyl-CoA to form acetoacetyl-CoA. This is one of the fundamental categories of carbon skeletal assembly patterns in biological systems and is the first step in a wide range of biosynthetic pathways, including those that generate cholesterol, steroid hormones, and various energy-storage molecules. RESULTS: The crystal structure of the tetrameric biosynthetic thiolase from Zoogloea ramigera has been determined at 2.0 A resolution. The structure contains a striking and novel 'cage-like' tetramerization motif, which allows for some hinge motion of the two tight dimers with respect to each other. The protein crystals were flash-frozen after a short soak with the enzyme's substrate, acetoacetyl-CoA. A reaction intermediate was thus trapped: the enzyme tetramer is acetylated at Cys89 and has a CoA molecule bound in each of its active-site pockets. CONCLUSIONS: The shape of the substrate-binding pocket reveals the basis for the short-chain substrate specificity of the enzyme. The active-site architecture, and in particular the position of the covalently attached acetyl group, allow a more detailed reaction mechanism to be proposed in which Cys378 is involved in both steps of the reaction. The structure also suggests an important role for the thioester oxygen atom of the acetylated enzyme in catalysis.

Biosynthetic thiolase from Zoogloea ramigera. Evidence for a mechanism involving Cys-378 as the active site base.

Biosynthetic thiolase from Zoogloea ramigera was inactivated with a mechanism-based inactivator, 3-pentynoyl-S-pantetheine-11-pivalate (3-pentynoyl-SPP) where K1 = 1.25 mM and kinact = 0.26 min-1, 2,3-pentadienoyl-SPP obtained from nonenzymatic rearrangement of 3-pentynoyl-SPP where K1 = 1.54 mM and kinact = 1.9 min-1 and an affinity labeling reagent, acryl-SPP. The results obtained with the alkynoyl and allenoyl inactivators are taken as evidence that thiolase from Z. ramigera is able to catalyze proton abstraction uncoupled from carbon-carbon bond formation. The inactivator, 3-pentynoyl-SPP and the affinity labeling reagent, acryl-SPP, trap the same active site cysteine residue, Cys-378. To assess if Cys-378 is the active site residue involved in deprotonation of the second molecule of acetyl-CoA, a Gly-378 mutant enzyme was studied. In the thiolysis direction the Gly-378 mutant was more than 50,000-fold slower than wild type and over 100,000-fold slower in the condensation direction. However, the mutant enzyme was still capable of forming the acetyl-enzyme intermediate and incorporated 0.81 equivalents of 14C-label after incubation with [14C]Ac-CoA for 60 min. The reversible exchange of 32P-label from [32P]CoASH into Ac-CoA, catalyzed by the Gly-378 mutant enzyme, proceeded with a Vmax (exchange) 8,000-fold less than the wild type enzyme but at least 10-fold faster than the overall condensation reaction. These data provide evidence that Cys-378 is the active site base.

The reaction of acetyldithio-CoA, a readily enolized analog of acetyl-CoA with thiolase from Zoogloea ramigera.

Acetyldithio-CoA has been shown to be a competent nucleophilic substrate but not an electrophilic substrate for the Claisen condensation catalyzed by thiolase, which normally dimerizes acetyl (Ac)-CoA to acetoacetyl-CoA. Acting as the nucleophile, the kcat/Km for dithioacetyl-CoA is comparable to that of Ac-CoA, the normal substrate. With acetoacetyl-pantetheine acetylating the thiolase to provide the electrophile, the kcat and kcat/Km for the Claisen condensation are 2.1 s-1 and 8.3 X 10(4) M-1 s-1, respectively. The product of the reaction is 3-ketobutyryldithio-CoA. The 3-ketobutyryldithio-CoA has a spectrally determined pKa of 6.55 and the enolate has a lambda max of 357 nm, epsilon 357 = 21,000 cm-1 M-1. Product analysis indicates that acetyldithio-CoA does not serve as the electrophilic partner in the enzymic condensation. This failure is attributed to the inability demonstrated in this study of acetyldithio-CoA to thioacetylate the active site Cys89 of the Zoogloea ramigera thiolase. 1H NMR studies in D2O indicate that thiolase catalyzes the exchange of the alpha-hydrogens, without Cys89 being acetylated, with a rate of 0.63 +/- 0.25 s-1. In the presence of a large excess of acetoacetyl-pantetheine, present to acetylate Cys89 and prevent the thiolytic back reaction, solvent exchange of the alpha-hydrogens can still be detected by observing the isotope-shifted 13C NMR spectrum of [2-13C]acetyldithio-CoA. The exchange of the acetyldithio-CoA alpha-hydrogens with solvent promoted by the acetylated enzyme, must proceed at a rate comparable to that of the condensation reaction.

The NH2-terminal 14-16 amino acids of mitochondrial and bacterial thiolases can direct mature ornithine carbamoyltransferase into mitochondria.

Unlike most mitochondrial matrix proteins, the mitochondrial 3-oxoacyl-CoA thiolase [EC 2.3.1.16] is synthesized with no cleavable presequence and possesses information for mitochondrial targeting and import in the mature protein. This mitochondrial thiolase is homologous with the mature portion of peroxisomal 3-oxoacyl-CoA thiolase and acetoacetyl-CoA thiolase [EC 2.3.1.9] of Zoogloea ramigera along the entire sequence. A hybrid gene encoding the NH2-terminal 16 residues (MALLRGVFIVAAKRTP) of the mitochondrial thiolase fused to the mature portion of rat ornithine carbamoyltransferase [EC 2.1.3.3] (lacking its own presequence) was transfected into COS cells, and subcellular localization of the fusion protein was analyzed. Cell fractionation and immunocytochemical analyses showed that the fusion protein was localized in the mitochondria. These results indicate that the NH2-terminal 16 residues of the mitochondrial thiolase function as a noncleavable signal for mitochondrial targeting and import of this enzyme protein. The fusion protein containing the NH2-terminal 14 residues (MSTPSIVIASARTA) of the bacterial thiolase was also localized in the mitochondria. On the other hand, the fusion protein containing the corresponding portion (MQASASDVVVVHGQRTP) of the peroxisomal thiolase appeared not to be localized to the mitochondria. These results show that the import signal of mitochondrial 3-oxoacyl-CoA thiolase originated from the NH2-terminal portion of the ancestral thiolase. The ancestral enzyme might have already possessed a mitochondrial import activity when mitochondria appeared first, or that it might have acquired the import activity during evolution by accumulation of point mutations in the NH2-terminal portion of the enzyme.

Mechanistic studies on beta-ketoacyl thiolase from Zoogloea ramigera: identification of the active-site nucleophile as Cys89, its mutation to Ser89, and kinetic and thermodynamic characterization of wild-type and mutant enzymes.

Thiolase proceeds via covalent catalysis involving an acetyl-S-enzyme. The active-site thiol nucleophile is identified as Cys89 by acetylation with [14C]acetyl-CoA, rapid denaturation, tryptic digestion, and sequencing of the labeled peptide. The native acetyl enzyme is labile to hydrolytic decomposition with t 1/2 of 2 min at pH 7, 25 degrees C. Cys89 has been converted to the alternate nucleophile Ser89 by mutagenesis and the C89S enzyme overproduced, purified, and assessed for activity. The Ser89 enzyme retains 1% of the Vmax of the Cys89 enzyme in the direction of acetoacetyl-CoA thiolytic cleavage and 0.05% of the Vmax in the condensation of two acetyl-CoA molecules. A covalent acetyl-O-enzyme intermediate is detected on incubation with [14C]acetyl-CoA and isolation of the labeled Ser89-containing tryptic peptide. Comparisons of the Cys89 and Ser89 enzymes have been made for kinetic and thermodynamic stability of the acetyl enzyme intermediates both by isolation and by analysis of [32P]CoASH/acetyl-CoA partial reactions and for rate-limiting steps in catalysis with trideuterioacetyl-CoA.

Fine structural analysis of the Zoogloea ramigera phbA-phbB locus encoding beta-ketothiolase and acetoacetyl-CoA reductase: nucleotide sequence of phbB.

A series of expression plasmids containing either the complete insert from plasmid pUCDBK1 (Peoples et al., 1987) or sub-fragments thereof were constructed in a tac promoter vector. Analysis of protein lysates of induced cultures of these clones identified the gene encoding NADPH-specific acetoacetyl-CoA reductase in the 2.3kb of sequence located downstream from the beta-ketothiolase gene in plasmid pUCDBK1. The complete nucleotide sequence (2.1kb) of this region was determined. An open reading frame was located 88bp downstream from the stop codon of the thiolase gene encoding a potential polypeptide of Mr 25,000, which is in good agreement with that observed for the overexpressed protein on SDS-PAGE. N-terminal protein sequence data obtained by Edman degradation of the purified Mr = 25,000 polypeptide were used to identify the correct start of the NADPH-specific acetoacetyl-CoA reductase gene. Hence in Z. ramigera, the genes encoding beta-ketothiolase (phbA) and NADPH-specific acetoacetyl-CoA reductase (phbB) are organized as phbA-phbB. S1-nuclease analysis of Z. ramigera RNA identified a transcription start site 85 bp upstream from the phbA structural gene locating the promoter region.

Purification and characterization of NADP-linked acetoacetyl-CoA reductase from Zoogloea ramigera I-16-M.

An NADP-linked acetoacetyl-CoA reductase was purified to electrophoretic homogeneity from Zoogloea ramigera I-16-M, a poly(3-hydroxybutyrate)-accumulating bacterium. The purified enzyme showed specific activity of 412 mumol acetoacetyl-CoA reduced per min per mg protein, which constituted an 880-fold purification compared to the crude extract, with a 32% yield. Electrophoretic analysis of the purified enzyme which had been cross-linked with dimethylsuberimidate showed that the native enzyme (Mr 92,000) is a tetramer of four identical subunits (Mr 25,500). Among the various D-(-)- and L-(+)-3-hydroxyacyl-CoAs tested, the purified enzyme oxidized only D-(-)-3-hydroxybutyryl-CoA and to a lesser extent D-(-)-3-hydroxyvaleryl-CoA in the presence of NADP+. The antiserum prepared against the purified enzyme completely inhibited poly(3-hydroxybutyrate) synthesis from acetyl-CoA by a crude extract of Z. ramigera I-16-M cells. These findings indicate that this enzyme plays an indispensable role as the supplier of D-(-)-3-hydroxybutyryl-CoA in poly(3-hydroxybutyrate) synthesis in this bacterium.

Biosynthetic thiolase from zoogloea ramigera. I. Preliminary characterization and analysis of proton transfer reaction.

The biosynthetic thiolase, from Zoogloea ramigera, involved in generation of acetoacetyl-CoA for poly-beta-hydroxybutyrate synthesis, has been prepared pure in quantity for initial structural characterization of this homotetrameric enzyme. Edman degradation provided the sequence of the NH2 terminal 25 residues and an active site cysteine-containing nonapeptide labeled on stoichiometric inactivation by iodoacetamide. Both sequences were used to align the encoding DNA sequence of the cloned gene as described in an accompanying paper. Synthetic analogs of acetoacetyl-S-CoA, modified in the CoA moiety, were prepared and tested, and acetoacetyl-S-pantetheine 11-pivalate 1 was shown to have a kcat/Km of 6.4 X 10(6) M-1 s-1, comparable to the kcat/Km of 2 X 10(7) M-1 s-1 for acetoacetyl-S-CoA. The pantetheine pivalate group facilitates nonaqueous synthetic manipulations and may be generally useful as a CoA replacement. We have also prepared the carba analog of 1, with CH2 replacing S, to yield a beta-diketone analog 10 of acetoacetyl-S-CoA and the corresponding methyl ketone analog 9 of acetyl-S-CoA. These analogs have been used to prove the ability of Z. ramigera thiolase to catalyze proton abstraction from the C-2 methyl group of the acetyl portion of substrate in a transition state separate from C-C bond formation. NMR studies in D2O show exchange only when condensation is possible. Further studies with [2-3H]acetyl-CoA show there is neither pre-equilibrium washout nor detectable kH/kT expressed in turnover and provide no evidence for a discrete acetyl-CoA C-2 carbanion or a nonconcerted reaction.

Biosynthetic thiolase from Zoogloea ramigera. II. Inactivation with haloacetyl CoA analogs.

The thiolase involved in biosynthesis of poly-beta-hydroxybutyrate in Zoogloea ramigera generates an acetyl-enzyme species during catalysis. Up to 0.86 [14C] acetyl eq/subunit of this homotetrameric enzyme is accumulated by acid precipitation in the presence of [14C]acetyl-CoA. Gel filtration of the same solutions produced only 7% acetyl-enzyme suggesting hydrolytic lability of the acetyl-enzyme during the 10-min isolation at 4 degrees C. In an effort to identify active site residues which may function as basic groups to deprotonate at C-2 of acetyl-CoA to generate the required nucleophilic equivalent in carbon-carbon bond formation, we have prepared and tested haloacetyl-thioesters, oxoesters, and amides in the panthetheine pivalate series (Davis, J. T., Moore, R. N., Imperiali, B., Pratt, A. J., Kobayashi, K., Masamune, S., Sinskey, A. J., and Walsh, C. T. (1987) J. Biol. Chem. 262, 82-89). The [14C]bromoacetyl-oxoester alkylatively inactivates thiolase irreversibly with stoichiometric incorporation of four labels/tetramer. Determination of amino acid composition of the radiolabeled tryptic peptide indicated trapping of Cys-89 (Peoples, O. P., Masamune, S., Walsh, C. T., and Sinskey, A. J. (1987) J. Biol. Chem. 262, 97-102), the same residue modified by iodoacetamide. When the bromoacetyl-thioester was used, inactivation was pH-dependent. The data are consistent with the competition of two processes, acylation, and alkylation. Direct (rather than secondary) alkylation of thiolase by the inactivator accounts for the significant 14C incorporation into thiolase with the thioester labeled with [14C] in the pantetheine pivalate moiety. It appears likely that the haloacetyl analogs described herein should be generally useful for affinity labeling other enzymes using acetyl-CoA as a substrate.

Biosynthetic thiolase from Zoogloea ramigera. III. Isolation and characterization of the structural gene.

The gene coding for the biosynthetic thiolase from Zoogloea ramigera has been isolated by using antibody screening methods to detect its expression in Escherichia coli under the transcriptional control of the lac promoter. We have located and determined the nucleotide sequence of the gene. The structural gene is 1173 nucleotides long and codes for a polypeptide of 391 amino acids; 282 nucleotides 5' and 58 nucleotides 3' to the coding sequence are also reported. By comparing the amino acid sequence data predicted from the gene with data determined experimentally, we have derived the complete primary structure of thiolase. A catalytically essential cysteine is located at residue 89. The DNA sequence presented has a very high G/C content, 66.2%, typical of the Z. ramigera genome. In the coding region, this increases to 68.2% and is strongly reflected in the codon usage which demonstrates a strong preference for G or C in the third position. Examination of the 5'-flanking sequence establishes that the NH2-terminal methionine is specified by an ATG codon, 7 nucleotides downstream from a Shine-Dalgarno sequence.

Enzymatic depolymerization of emulsan.

Emulsan, the polyanionic emulsifying agent synthesized by Acinetobacter calcoaceticus RAG-1, was depolymerized by an enzyme obtained from a soil bacterium YUV-1. The extracellular emulsan depolymerase was produced when strains RAG-1 and YUV-1 were grown together on agar medium. The enzyme was extracted from the agar and concentrated by ultrafiltration and ammonium sulfate precipitation. The molecular weight of the enzyme was estimated to be 89,000. Emulsan depolymerase activity was due to an eliminase reaction which split glycosidic linkages within the heteropolysaccharide backbone of emulsan to generate reducing groups and alpha, beta-unsaturated uronides with an absorbance maximum of 233 nm. Deesterified emulsan was degraded by emulsan depolymerase at only 27% of the rate of the native polymer. The treatment of emulsan solutions with emulsan depolymerase for brief periods caused a rapid and parallel drop in viscosity and emulsifying activity. More than 75% of the viscosity and emulsifying activity was lost at a time when less than 0.5% of the glycosidic linkages were broken. These data indicate that (i) emulsan depolymerase is an endoglycosidase and (ii) the higher the molecular weight of emulsan, the greater its emulsifying activity. Exhaustive digestion of emulsan with emulsan depolymerase produced oligosaccharides with a number average molecular weight of about 3,000. The fractionation of the digest on Bio-Gel P-6 yielded four broad peaks. The pooled fractions from each of the peaks contained the same relative amounts of reducing sugar and had an absorbance at 233 nm. The molar ratio of esterified sugar to reducing groups was close to 2 in each fraction.

Purification and characterization of acetoacetyl-CoA synthetase from Zoogloea ramigera I-16-M.

Acetoacetyl-CoA synthetase was purified to electrophoretic homogeneity from Zoogloea ramigera I-16-M, a poly(3-hydroxybutyrate)-accumulating bacterium, which lacks 3-ketoacid CoA-transferase. The purified enzyme had a specific activity of 52.2 mumol acetoacetyl-CoA formed min-1 mg protein-1, which constituted a 680-fold purification compared to the crude extract, with a 5.1% yield. The enzyme absolutely required ATP, CoA, a monovalent cation (K+, Rb+, Cs+ or NH+4) and a divalent cation (Mg2+, Mn2+, Ca2+ or Ni2+) for the activation of acetoacetate, yielding acetoacetyl-CoA, AMP and pyrophosphate in equimolar amounts. The pH optimum of the enzyme reaction was 8.4. The molecular weight of the enzyme was approximately 70 000 as estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, and 72 000 by Sephadex G-200 gel filtration. The enzyme was active only on acetoacetate and to a lesser extent on L(+)-3-hydroxybutyrate, and the Km values for acetoacetate, L(+)-3-hydroxybutyrate, ATP and CoA were 7.6 X 10(-5) M, 1.4 X 10(-3) M, 3.3 X 10(-5) M and 9.1 X 10(-5) M respectively.

Purification and properties of D(-)-3-hydroxybutyrate-dimer hydrolase from Zoogloea ramigera I-16-M.

D(-)-3-Hydroxybutyrate-dimer hydrolase from Zoogloea ramigera I-16-M was purified 7000-fold to electrophoretic homogeneity. The molecular weight of the purified enzyme was 28 000 as determined by Sephadex G-100 gel filtration, and 30 000 as estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The isoelectric point was at pH 5.7. The pH optimum for the enzyme reaction was 8.0. The dimer hydrolase was stereospecific for D(-)-3-[D(-)-3-hydroxybutyryloxy]butyric acid (DD-dimer) but also hydrolyzed D(-)-3-[L(+)-3-hydroxybutyryloxy]butyric acid (DL-dimmer) and L(+)-3-[D(-)-3-hydroxybutyryloxy]butyric acid (LD-dimer) at reduced rates. However, the enzyme did not attack L(+)-3-[L(+)-3-hydroxybutyryloxy]butyric acid (LL-dimer) at all. In addition, the purified hydrolase hydrolyzed several oligomeric esters of D(-)-3-hydroxybutyric acid (DDD-dimer, DDDD-tetramer and DDDDD-pentamer) faster than DD-dimer. Time course experiments with these oligomers and analysis of hydrolytic products of DDD-tetramer methyl ester with the hydrolase indicated that the enzyme attached these substrates from the free hydroxyl terminus releasing monomer units one at a time.

An NAD-linked acetoacetyl-CoA reductase from Zoogloea ramigera I-16-M.

An NAD-linked acetoacetyl-CoA reductase of Zoolgoea ramigera I-16-M was purified to electrophoretic homogeneity. In contrast to the D(-)-3-hydroxybutyryl-CoA-specific NADP-linked acetoacetyl-CoA reductase from the same bacterium [Saito, T. et al (1977) Arch. Microbiol. 114, 211 - 217], the purified enzyme was strictly stereospecific to L(+)-3-hydroxybutyryl-CoA, and was active not only with NAD+ but also with NADP+, although NADP+ was less effective than NAD+ as coenzyme. The enzyme showed a pH optimum at 6.3 for the reduction of acetoacetyl-CoA and at 8.0 for the oxidation of L(+)-3-hydroxybutyryl-CoA. In the reduction reaction, Km values for acetoacetyl-Coa and NADH were 8.8 microM and 6.5 microM, respectively, and in the oxidation reaction, Km values for L(+)-3-hydroxybutyryl-CoA and DNA+ were 7.0 microM and 32 microM, respectively. Among various 3-hydroxyacyl-CoAs tested, L(+)-3-hydroxybutyryl-CoA and L(+)-3-hydroxyvaleryl-CoA were the most active substrates. Poly(3-hydroxybutyrate) synthesis from acetyl-CoA, by a system reconstituted from purified preparations of 3-oxothiolase, acetoacetyl-CoA reductase and poly(3-hydroxybutyrate) synthase, was observed when the NADP-linked but not the NAD-linked reductase was used. These findings indicate that the NAD-linked acetoacetyl-CoA reductase is not directly involved in the biosynthesis of poly(3-hydroxybutyrate).

Purification and properties of D-beta-hydroxybutyrate dehydrogenase from Zoogloea ramigera I-16-M.

D(-)-beta-Hydroxybutyrate dehydrogenase was purified from Zoogloea ramigera I-16-M to electrophoretic homogeneity. The molecular weight of the enzyme as determined by Sephadex G-200 gel filtration was 112,000, and the monomer molecular weight estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate was 28,000, indicating that the native enzyme is a tetramer with four identical subunits. The enzyme showed a pH optimum at 8.0 in the oxidation reaction, and a broad pH optimum (5.5-7.5) in the reduction reaction. The Km values for D(-)-beta-hydroxybutyrate and NAD in the oxidation reaction were 3.2 X 10(-4) M and 5.7 X 10(-5) M, respectively. The Km value for acetoacetate in the reduction reaction was 1.5 X 10(-4) M and that for NADH was 1.5 X 10(-5) M. Acetyl CoA, D-lactate, and 2-hydroxybutyrate were effective inhibitors for the oxidation of D(-)-beta-hydroxybutyrate. The enzyme was sensitive to the inhibitory actions of sulfhydryl reagents such as p-chloromercuribenzoic acid, 5,5'-dithiobis(2-nitrobenzoic acid) and HgCl2.