The Contribution of Adjacent Subunits to the Active Sites ofd-3-Phosphoglycerate Dehydrogenase

The cyanobacterium Microcystis aeruginosa is widely known for its production of the potent hepatotoxin microcystin. This cyclic heptapeptide is synthesized non-ribosomally by the thiotemplate function of a large modular enzyme complex encoded within the 55-kb microcystin synthetase gene (mcy) cluster. The mcy gene cluster also encodes several stand-alone enzymes, putatively involved in the tailoring and export of microcystin. This study describes the characterization of the 2-hydroxy-acid dehydrogenase McyI, putatively involved in the production of D-methyl aspartate at position 3 within the microcystin cyclic structure. A combination of bioinformatics, molecular, and biochemical techniques was used to elucidate the structure, function, regulation, and evolution of this unique enzyme. The recombinant McyI enzyme was overexpressed in Escherichia coli and enzymatically characterized. The hypothesized native activity of McyI, the interconversion of 3-methyl malate to 3-methyl oxalacetate, was demonstrated using an in vitro spectrophotometric assay. The enzyme was also able to reduce ␣-ketoglutarate to 2-hydroxyglutarate and to catalyze the interconversion of malate and oxalacetate. Although NADP(H) was the preferred cofactor of the McyI-catalyzed reactions, NAD(H) could also be utilized, although rates of catalysis were significantly lower. The combined results of this study suggest that hepatotoxic cyanobacteria such as M. aeruginosa PCC7806 are capable of producing methyl aspartate via a novel glutamate mutase-independent pathway, in which McyI plays a pivotal role.The hepatotoxic microcystins compose the largest and most structurally diverse group of cyanobacterial toxins. Over 65 isoforms of microcystin varying by degree of methylation, hydroxylation, epimerization, peptide sequence, and toxicity have been identified (1). Underlying the extraordinary heterogeneity present among the microcystins is their common cyclic structure and possession of several rare nonproteinogenic amino acid moieties. Collectively, the microcystins may be described as monocyclic heptapeptides containing both D-and L-amino acids plus N-methyldehydroalanine and a unique ␤-amino acid side group, 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid (Fig. 1) (2). Although various amino acid substitutions can occur within the microcystin cyclic structure, the most common toxin isoform, microcystin LR, contains lysine and arginine at positions 2 and 4, respectively (Fig. 1).The microcystin biosynthesis (mcy) gene cluster was the first complex metabolite gene cluster to be fully sequenced from a cyanobacterium. In Microcystis aeruginosa PCC7806, the mcy gene cluster spans 55 kb and comprises 10 genes arranged in two divergently transcribed operons, mcyA-C and mcyD-J. Although most open reading frames within the mcy gene cluster have been assigned a function based on homologous entries in the data bases, no obvious function could be assigned to the 1014-bp gene mcyI. Preliminary sequence analysis of the inferred primary peptide seq...

Section: Discussionmentioning

confidence: 66%

Characterization of the 2-Hydroxy-acid Dehydrogenase McyI, Encoded within the Microcystin Biosynthesis Gene Cluster of Microcystis aeruginosa PCC7806

Pearson

Barrow

Neilan

2007

“…Subunit dissociation results in a shift in fluorescence from 340 to 360 nm (8,12) and a loss of serine binding (16). All mutants in this study maintained an emission maximum at 340 nm and retained their ability to bind serine, indicating an intact association of subunits.…”

Section: Methodsmentioning

confidence: 62%

“…This linkage also comprises the locus at which the substrate binding domain and the nucleotide binding domain join to form the active site cleft. Amino acid residues within the active site cleft that seem to play a role in substrate binding and potentially in the regulation of the enzymatic activity have been identified previously (8). In order for the active site to close around the substrate during catalysis, it would seem that some flexibility is required in the vicinity of this Gly-Gly sequence.…”

mentioning

confidence: 99%

Amino Acid Residue Mutations Uncouple Cooperative Effects inEscherichia coli d-3-Phosphoglycerate Dehydrogenase

Grant

2001

Self Cite

D-3-Phosphoglycerate dehydrogenase from Escherichia coli contains two Gly-Gly sequences that occur at junctions between domains. A previous study (Grant, G. A., Xu, X. L., and Hu, Z. (2000) Biochemistry 39, 7316 -7319) determined that the Gly-Gly sequence at the junction between the regulatory and substrate binding domain functions as a hinge between the domains. Mutations in this area significantly decrease the ability of serine to inhibit activity but have little effect on the K m and k cat . Conversely, the present study shows that mutations to the Gly-Gly sequence at the junction of the substrate and nucleotide binding domains, which form the active site cleft, have a significant effect on the k cat of the enzyme without substantially altering the enzyme's sensitivity to serine. In addition, mutation of Gly-294, but not Gly-295, has a profound effect on the cooperativity of serine inhibition. Interestingly, even though cooperativity of inhibition can be reduced significantly, there is little apparent effect on the cooperativity of serine binding itself. An additional mutant, G336V,G337V, also reduces the cooperativity of inhibition, but in this case serine binding also is reduced to the point at which it cannot be measured by equilibrium dialysis. The double mutant G294V,G336V demonstrates that strain imposed by mutation at one hinge can be relieved partially by mutation at the other hinge, demonstrating linkage between the two hinge regions. These data show that the two cooperative processes, serine binding and catalytic inhibition, can be uncoupled. Consideration of the allowable torsional angles for the side chains introduced by the mutations yields a range of values for these angles that the glycine residues likely occupy in the native enzyme. A comparison of these values with the torsional angles found for the inhibited enzyme from crystal coordinates provides potential beginning and ending orientations for the transition from active to inhibited enzyme, which will allow modeling of the dynamics of domain movement.

“…DsbA is also monomeric but has an additional helical insert, which, together with the thioredoxin domain, provides a much more extended hydrophobic surface than that of thioredoxin for substrate binding, and therefore DsbA shows low isomerase activity of about 5% of that PDI (25) and 20% of DsbC (3). In this connection, it is known that the active sites of some enzymes are shared between different subunits (26,27). For DsbA and the monomeric domain a of PDI with 14% isomerase activity (28) of PDI, their small size may allow two enzyme molecules to attack two disulfide bonds of a substrate simultaneously.…”

Section: Figmentioning

confidence: 99%

The N-terminal Sequence (Residues 1–65) Is Essential for Dimerization, Activities, and Peptide Binding of Escherichia coli DsbC

Sun

Wang

2000

Limited proteolysis of DsbC with trypsin resulted in a compact and stable C-terminal fragment (residues 66 -216), fDsbC, which retains the active site sequence, -Cys 98 -Gly-Tyr-Cys 101 -, and shows only minor differences in conformation compared with that of the intact molecule. The pK a of active site thiol and the K SS with glutathione are very close to that of DsbC, respectively; however, fDsbC is inactive as an isomerase in catalyzing the formation of correct disulfide bonds in scrambled RNase A and denatured and reduced bovine pancreatic trypsin inhibitor and shows only 13% thiol-protein oxidoreductase activity (TPOR) of DsbC. In contrast to the dimeric DsbC, fDsbC exists as a monomer and has no chaperone activity in assisting the reactivation of denatured D-glyceraldehyde-3-phosphate dehydrogenase. The heterodimer of DsbC with the inactive DsbC carboxymethylated at both active site thiols shows about 50% TPOR activity of DsbC but no isomerase activity, indicating that the DsbC subunit in the heterodimer displays full TPOR activity but little, if any, isomerase activity. It is concluded that the N-terminal sequence (residues 1-65) is essential for dimer formation and chaperone activity of DsbC. The active sites in both subunits of the dimeric DsbC appear to be essential for its isomerase activity.The Dsb protein family in bacterial periplasm has been recently characterized to be responsible for the formation of native disulfide bonds of newly synthesized polypeptides (1). At least six proteins, DsbA, DsbB, DsbC, DsbD, DsbE, and DsbG, have been identified, and they work in conjunction with one another (2). DsbC is a soluble protein catalyzing the rearrangement of disulfide bonds (3) and therefore considered as the prokaryotic counterpart of the eukaryotic protein-disulfide isomerase (PDI) 1 (3, 4). PDI has recently been shown to have not only enzyme activities but also chaperone activity (5-7).Compared with PDI, DsbC shows lower isomerase activity but even more marked chaperone activity (8).As a member of the thioredoxin superfamily, DsbC shares 24% sequence identity with DsbG (9) but no apparent sequence homology with other members of the Dsb family or with other members of the thioredoxin superfamily except for the motif of Phe-X-X-X-X-Cys-X-X-Cys in the active site (10). DsbC is a homodimer (3), and each of its 23.4-kDa subunits is composed of 216 amino acid residues with four cysteines, two in the active site sequence of -Cys 98 -Gly-Tyr-Cys 101 -and the other two at positions 141 and 163, believed to form a disulfide bond with a structural function (3). It was predicted that the molecule appeared to consist of C-and N-terminal domains of similar length, with the C-terminal half recognized as another variation on the thioredoxin theme (10). The crystal structure of oxidized DsbC shows that the molecule has a V-shape with each arm for one subunit, and each subunit consists of a Cterminal thioredoxin-like domain connected by a hinged linker helix to an N-terminal domain responsible for dimerization (9).Li...