Miho Aoshima

Novel enzyme reactions related to the tricarboxylic acid cycle: phylogenetic/functional implications and biotechnological applications

The tricarboxylic acid (TCA) cycle is an energy-producing pathway for aerobic organisms. However, it is widely accepted that the phylogenetic origin of the TCA cycle is the reductive TCA cycle, which is a non-Calvin-type carbon-dioxide-fixing pathway. Most of the enzymes responsible for the oxidative and reductive TCA cycles are common to the two pathways, the difference being the direction in which the reactions operate. Because the reductive TCA cycle operates in an energetically unfavorable direction, some specific mechanisms are required for the reductive TCA-cycle-utilizing organisms. Recently, the molecular mechanism for the “citrate cleavage reaction” and the “reductive carboxylating reaction from 2-oxoglutarate to isocitrate” in Hydrogenobacter thermophilus have been demonstrated. Both of these reactions comprise two distinct consecutive reactions, each catalyzed by two novel enzymes. Sequence analyses of the newly discovered enzymes revealed phylogenetic and functional relationships between other TCA-cycle-related enzymes. The occurrence of novel enzymes involved in the citrate-cleaving reaction seems to be limited to the family Aquificaceae. In contrast, the key enzyme in the reductive carboxylation of 2-oxoglutarate appears to be more widely distributed in extant organisms. The four newly discovered enzymes have a number of potential biotechnological applications.

A novel enzyme, citryl-CoA lyase, catalysing the second step of the citrate cleavage reaction in Hydrogenobacter thermophilus TK-6.

A novel enzyme catalysing citryl‐CoA cleavage to acetyl‐CoA and oxaloacetate was purified from Hydrogenobacter thermophilus TK‐6, and designated citryl‐CoA lyase (CCL). The citrate cleavage reaction in this organism proceeded by a unique set of two consecutive reactions: (i) citryl‐CoA formation by citryl‐CoA synthetase (CCS) and (ii) citryl‐CoA cleavage by CCL. Purified CCL gave a single 30 kDa band in SDS–PAGE and gel filtration chromatography indicated that the native state of the enzyme exists as a trimer (α3). Citryl‐CoA lyase showed low citrate synthase (CS) activity. Using an oligonucleotide probe, the corresponding gene was cloned and sequenced. The gene was expressed in Escherichia coli and recombinant CCL was also purified. The CCL protein sequence showed similarity to the C‐terminal regions of ATP citrate lyase (ACL) and CS sequences in the database. By further sequence comparisons, the phylogenetic relationship between CCS, CCL, ACL and CS was investigated.

Nondecarboxylating and decarboxylating isocitrate dehydrogenases: oxalosuccinate reductase as an ancestral form of isocitrate dehydrogenase.

Isocitrate dehydrogenase (ICDH) from Hydrogenobacter thermophilus catalyzes the reduction of oxalosuccinate, which corresponds to the second step of the reductive carboxylation of 2-oxoglutarate in the reductive tricarboxylic acid cycle. In this study, the oxidation reaction catalyzed by H. thermophilus ICDH was kinetically analyzed. As a result, a rapid equilibrium random-order mechanism was suggested. The affinities of both substrates (isocitrate and NAD+) toward the enzyme were extremely low compared to other known ICDHs. The binding activities of isocitrate and NAD+ were not independent; rather, the binding of one substrate considerably promoted the binding of the other. A product inhibition assay demonstrated that NADH is a potent inhibitor, although 2-oxoglutarate did not exhibit an inhibitory effect. Further chromatographic analysis demonstrated that oxalosuccinate, rather than 2-oxoglutarate, is the reaction product. Thus, it was shown that H. thermophilus ICDH is a nondecarboxylating ICDH that catalyzes the conversion between isocitrate and oxalosuccinate by oxidation and reduction. This nondecarboxylating ICDH is distinct from well-known decarboxylating ICDHs and should be categorized as a new enzyme. Oxalosuccinate-reducing enzyme may be the ancestral form of ICDH, which evolved to the extant isocitrate oxidative decarboxylating enzyme by acquiring higher substrate affinities.

Analyses of microbial community within a composter operated using household garbage with special reference to the addition of soybean oil.

A commercially available composter was operated using fixed composition of garbage with or without the addition of soybean oil. The composter was operated without adding seed microorganisms or bulking materials. Microflora within the composter were analyzed by denaturing gradient gel electrophoresis (DGGE) in the case of oil addition, or by 16/18 S rRNA gene sequencing of the isolated microorganisms in the case of no oil addition. The results showed that, irrespective of the addition of oil, the bacteria identified were all gram positive, and that lactobacilli seemed to be the key microorganisms. Based on the results, suitable microflora for use in a household composter are discussed.

A novel biotin protein required for reductive carboxylation of 2-oxoglutarate by isocitrate dehydrogenase in Hydrogenobacter thermophilus TK-6

Isocitrate dehydrogenase was purified from Hydrogenobacter thermophilus, and the corresponding gene was cloned and sequenced. The enzyme had similar structural properties to the isocitrate dehydrogenase of Escherichia coli, but differed in its catalytic properties, such as coenzyme specificity, pH dependency and kinetic parameters. Notably, the enzyme catalysed the oxidative decarboxylation of isocitrate, but not the reductive carboxylation of 2‐oxoglutarate. The carboxylation reaction required the addition of cell extract and ATP‐Mg, suggesting the existence of additional carboxylation factor(s). Further analysis of the carboxylation factor(s) resulted in the purification of two polypeptides. N‐terminal amino acid sequencing revealed that the two polypeptides are homologues of pyruvate carboxylase with a biotinylated subunit, but do not catalyse pyruvate carboxylation. Pyruvate carboxylase was also purified, but was not active in stimulating isocitrate dehydrogenase. Isocitrate dehydrogenase, the novel biotin protein, ATP‐Mg and NADH were essential for the reductive carboxylation of 2‐oxoglutarate. These observations indicate that the novel biotin protein is an ATP‐dependent factor, which is involved in the reverse (carboxylating) reaction of isocitrate dehydrogenase.

A novel enzyme, citryl-CoA synthetase, catalysing the first step of the citrate cleavage reaction in Hydrogenobacter thermophilus TK-6: Citryl-CoA synthetase in H. thermophilus

We attempted to purify ATP citrate lyase (ACL) from Hydrogenobacter thermophilus by following the citrate‐, ATP‐ and CoA‐dependent formation of an acyl‐CoA species that was detected as hydroxamate. However, citryl‐CoA rather than acetyl‐CoA was found, indicating that the purified enzyme was a novel citryl‐CoA synthetase (CCS) rather than ACL. Because the reaction catalysed by CCS corresponds to the first half of that mediated by ACL, CCS may be responsible for citrate cleavage in H. thermophilus. Thus, a novel citrate cleavage pathway, which does not involve ACL, appears to exist in this organism. Citryl‐CoA synthetase is composed of two different polypeptides: a large β subunit of 46 kDa and a small α subunit of 36 kDa. The corresponding genes were cloned and sequenced. The deduced amino acid sequences of the two subunits of CCS display significant similarity to those of succinyl‐CoA synthetase (SCS) in the database. As a comparison, SCS was also purified from H. thermophilus and the corresponding genes were cloned and sequenced. Citryl‐CoA synthetase and SCS were homologous, but showed different substrate specificity. The deduced amino acid sequences of the CCS subunits show similarity to part of the ACL sequence. The evolutionary relationship between CCS, SCS and ACL is discussed.

A novel oxalosuccinate-forming enzyme involved in the reductive carboxylation of 2-oxoglutarate in Hydrogenobacter thermophilus TK-6

We have previously demonstrated that the reductive carboxylation of 2‐oxoglutarate in Hydrogenobacter thermophilus TK‐6 is not simply a reversal of the oxidative decarboxylation catalysed by isocitrate dehydrogenase (ICDH). The reaction involves a novel biotin protein (carboxylating factor for ICDH – CFI) and ATP. In this study, we have analysed the ICDH/CFI system responsible for the carboxylation reaction. Sequence analysis revealed a close relationship between CFI and pyruvate carboxylase. Rather unexpectedly, the rate of ATP hydrolysis was greater than that of isocitrate formation or NADH oxidation. Furthermore, ATP hydrolysis catalysed by CFI was dependent on 2‐oxoglutarate but not on ICDH, suggesting that a carboxylated product is formed in the absence of ICDH. The product, which was detectable only at low temperatures, was identified as oxalosuccinate. Thus, CFI was confirmed to be a novel enzyme that catalyses the carboxylation of 2‐oxoglutarate to form oxalosuccinate, which corresponds to the first step of the reductive carboxylation from 2‐oxoglutarate to isocitrate. The CFI‐ICDH system may also be present in mammals, where it could play a significant role in modulating central metabolism.

Cloning and sequencing of a gene encoding 16S ribosomal RNA from a novel hyperthermophilic archaebacterium NC12

A hyperthermophile NC12 was newly isolated from Noboribetsu hot spring. To characterize this organism, a gene coding for 16S rRNA was cloned and sequenced. The 16S rRNA sequence from NC12 shows the highest similarity with those from Pyrodictium occultum and Desulfurococcus mobilis among the sequences in the database, indicating that NC12 belongs to a cluster of extreme thermophiles (Crenarchaeota) in the archaeal domain. However, since the highest identity score was only 91.2%, it is suggested that NC12 may constitute a new genus.

PURIFICATION AND CHARACTERIZATION OF ISOCITRATE DEHYDROGENASE FROM A HYPERTHERMOPHILIC ARCHAEBACTERIUM, CALDOCOCCUS NOBORIBETUS

Isocitrate dehydrogenase from a hyperthermophilic archaebacterium Caldococcus noboribetus produced in Escherichia coli was purified. The purification was performed by heat treatment at 80 degrees C followed by single column chromatography. N-terminal amino acid sequencing analysis revealed that the N-terminal methionine is removed from the purified enzyme. Gel filtration analysis suggests that the enzyme has a homodimeric structure with a molecular weight of 90,000. The isoelectric point of the enzyme was estimated to be 5.6 by isoelectric focusing electrophoresis. The circular dichroism spectrum suggests that the enzyme has a secondary structure consisting of 23% alpha-helix and 34% beta-sheet. Enzymatic activity was observed under neutral pH, and the highest specific activity was obtained using cacodylic acid-KOH (pH 7.0) buffer. MgCl2 or MnCl2 was essential for the activity, and KCl concentrations higher than 0.33 M had an inhibitory effect on it. Apparent Km values were 72 and 43 microM for D,L-isocitrate and NADP, respectively. The enzyme showed extremely high stability against heat treatment, and no activity loss was observed by the treatment at 80 degrees C. The specific activity of the enzyme increased as temperature rose. Nearly no activity was observed at 40 degrees C or lower.