Characterization of bacterial homocitrate synthase involved in lysine biosynthesis

Wulandari, Asri Peni; Miyazaki, Junichi; Kobashi, Nobuyuki; Nishiyama, Makoto; Hoshino, Takayuki; Yamane, Hisakazu

doi:10.1016/s0014-5793(02)02877-6

Cited by 44 publications

(44 citation statements)

References 26 publications

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“…Steady state kinetic analysis of the Zn(II)-bound form of WT SpHCS yielded k cat and K m values for AcCoA and 2-OG (Fig. 4, A and B, and (11,32). We then measured the kinetic parameters for conservative and alanine substitutions of the active site residues that interact with 2-OG (Fig.…”

Section: Volume 284 • Number 51 • December 18 2009mentioning

confidence: 99%

Crystal Structure and Functional Analysis of Homocitrate Synthase, an Essential Enzyme in Lysine Biosynthesis

Bulfer¹,

Scott²,

Couture³

et al. 2009

Journal of Biological Chemistry

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Homocitrate synthase (HCS) catalyzes the first and committed step in lysine biosynthesis in many fungi and certain Archaea and is a potential target for antifungal drugs. Here we report the crystal structure of the HCS apoenzyme from Schizosaccharomyces pombe and two distinct structures of the enzyme in complex with the substrate 2-oxoglutarate (2-OG). The structures reveal that HCS forms an intertwined homodimer stabilized by domain-swapping between the N-and C-terminal domains of each monomer. The N-terminal catalytic domain is composed of a TIM barrel fold in which 2-OG binds via hydrogen bonds and coordination to the active site divalent metal ion, whereas the C-terminal domain is composed of mixed ␣/␤ topology. In the structures of the HCS apoenzyme and one of the 2-OG binary complexes, a lid motif from the C-terminal domain occludes the entrance to the active site of the neighboring monomer, whereas in the second 2-OG complex the lid is disordered, suggesting that it regulates substrate access to the active site through its apparent flexibility. Mutations of the active site residues involved in 2-OG binding or implicated in acid-base catalysis impair or abolish activity in vitro and in vivo. Together, these results yield new insights into the structure and catalytic mechanism of HCSs and furnish a platform for developing HCSselective inhibitors.

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Section: Volume 284 • Number 51 • December 18 2009mentioning

confidence: 99%

Crystal Structure and Functional Analysis of Homocitrate Synthase, an Essential Enzyme in Lysine Biosynthesis

Bulfer¹,

Scott²,

Couture³

et al. 2009

Journal of Biological Chemistry

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“…In the case of the enzyme from Penicillium chrysogenum, the inhibition by lysine was shown to be competitive with respect to ␣-ketoglutarate and pH-dependent with only 20% inhibition by 1 mM lysine at pH 6.6 -7.0 (10). It has also been reported that HCS from Thermus thermophilus is highly sensitive to lysine (11). Regulation of HCS activity by CoA and divalent metal ions has also been reported (12).…”

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confidence: 95%

Regulatory Mechanism of Histidine-tagged Homocitrate Synthase from Saccharomyces cerevisiae

Andi

West

Cook

2005

Journal of Biological Chemistry

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Homocitrate synthase (HCS) catalyzes one of the regulated steps of the ␣-aminoadipate pathway for lysine biosynthesis in fungi. The kinetic mechanism of regulation of HCS from Saccharomyces cerevisiae by Na ؉ and the feedback inhibitor lysine was studied by measuring the initial rate in the absence and presence of the effectors. The data suggest that Na ؉ is an activator at low concentrations and an inhibitor at high concentrations and that these effects occur as a result of the monovalent ion binding to two different sites in the free enzyme. Inhibition and activation by Na ؉ can occur simultaneously, with the net rate of the enzyme determined by Na ؉ /K iNa ؉ and Na ؉ /K act , where K iNa ؉ and K act are the inhibition and activation constants, respectively. The inhibition by Na ؉ was eliminated at high concentrations of acetyl-CoA, the second substrate bound, but the activation remained. Fluorescence binding studies indicated that lysine bound with high affinity to its binding site as an inhibitor. The inhibition by lysine was competitive versus ␣-ketoglutarate and linear in the physiological range of lysine concentrations up to 5 mM. The effects of Na ؉ and lysine were independent of one another. A model is developed for regulation of HCS that takes into account all of the effects discussed above.

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“…We have shown that in an extremely thermophilic bacterium, Thermus thermophilus HB27, lysine is synthesized through ␣-aminoadipate, although lysine is synthesized through diaminopimelate in most bacteria (13,18,20,22,23,31). We have previously characterized HICDH from T. thermophilus HB27 (TtHICDH) in detail and have shown that the enzyme has four unique features (19).…”

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Crystal Structure of Tetrameric Homoisocitrate Dehydrogenase from an Extreme Thermophile, Thermus thermophilus : Involvement of Hydrophobic Dimer-Dimer Interaction in Extremely High Thermotolerance

et al. 2005

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The crystal structure of homoisocitrate dehydrogenase involved in lysine biosynthesis from Thermus thermophilus (TtHICDH) was determined at 1.85-Å resolution. Arg85, which was shown to be a determinant for substrate specificity in our previous study, is positioned close to the putative substrate binding site and interacts with Glu122. Glu122 is highly conserved in the equivalent position in the primary sequence of ICDH and archaeal 3-isopropylmalate dehydrogenase (IPMDH) but interacts with main-and side-chain atoms in the same domain in those paralogs. In addition, a conserved Tyr residue (Tyr125 in TtHICDH) which extends its side chain toward a substrate and thus has a catalytic function in the related ␤-decarboxylating dehydrogenases, is flipped out of the substrate-binding site. These results suggest the possibility that the conformation of the region containing Glu122-Tyr125 is changed upon substrate binding in TtHICDH. The crystal structure of TtHICDH also reveals that the arm region is involved in tetramer formation via hydrophobic interactions and might be responsible for the high thermotolerance. Mutation of Val135, located in the dimer-dimer interface and involved in the hydrophobic interaction, to Met alters the enzyme to a dimer (probably due to steric perturbation) and markedly decreases the thermal inactivation temperature. Both the crystal structure and the mutation analysis indicate that tetramer formation is involved in the extremely high thermotolerance of TtHICDH.Homoisocitrate dehydrogenase (HICDH) is the third enzyme involved in lysine biosynthesis through ␣-aminoadipate (19) and is a member of a family of ␤-decarboxylating dehydrogenases that includes isocitrate dehydrogenase (ICDH) in the tricarboxylic acid cycle, 3-isopropylmalate dehydrogenase (IPMDH) in leucine biosynthesis, and tartrate dehydrogenase in vitamin production (2). It has been suggested that these enzymes diverged from a common ancestral ␤-decarboxylating dehydrogenase (4, 9, 34).We have shown that in an extremely thermophilic bacterium, Thermus thermophilus HB27, lysine is synthesized through ␣-aminoadipate, although lysine is synthesized through diaminopimelate in most bacteria (13,18,20,22,23,31). We have previously characterized HICDH from T. thermophilus HB27 (TtHICDH) in detail and have shown that the enzyme has four unique features (19). The first is its substrate specificity. Although TtHICDH is essential for lysine biosynthesis in the bacterium, TtHICDH can catalyze the reaction with isocitrate as a substrate; isocitrate is the substrate of ICDH at a 20-fold-higher efficiency than toward the native substrate, homoisocitrate. This feature contrasts with HICDH from Saccharomyces cerevisiae, which utilizes only homoisocitrate as a substrate (19). Moreover, the substrate specificity is easily altered by a single mutation, Arg85Val, to lose activity for isocitrate but to acquire activity for 3-isopropylmalate, a substrate of IPMDH. The second feature is the unique subunit organization. TtHICDH is a homotetramer, while mo...

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Characterization of bacterial homocitrate synthase involved in lysine biosynthesis

Cited by 44 publications

References 26 publications

Crystal Structure and Functional Analysis of Homocitrate Synthase, an Essential Enzyme in Lysine Biosynthesis

Crystal Structure and Functional Analysis of Homocitrate Synthase, an Essential Enzyme in Lysine Biosynthesis

Regulatory Mechanism of Histidine-tagged Homocitrate Synthase from Saccharomyces cerevisiae

Crystal Structure of Tetrameric Homoisocitrate Dehydrogenase from an Extreme Thermophile, Thermus thermophilus : Involvement of Hydrophobic Dimer-Dimer Interaction in Extremely High Thermotolerance

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