Cloning, expression, purification and preliminary crystallographic characterization of a shikimate dehydrogenase from<i>Corynebacterium glutamicum</i>

Schoepe, Jan; Niefind, Karsten; Chatterjee, Shivani; Schomburg, Dietmar

doi:10.1107/s1744309106017805

Cited by 5 publications

(4 citation statements)

References 15 publications

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“…The arrangement of these two domains creates a deep groove, in which the cofactor and substrate are located, and forms the active site. The CglQSDH appears to exist as dimer in solution as we reported previously (Schoepe et al, 2006(Schoepe et al, , 2008.…”

Section: Structure Overview Of C Glutamicum Qsdhsupporting

confidence: 76%

Enzyme-substrate complexes of the quinate/shikimate dehydrogenase from Corynebacterium glutamicum enable new insights in substrate and cofactor binding, specificity, and discrimination

Höppner¹,

Schomburg²,

Niefind

2013

Biological Chemistry

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Quinate dehydrogenase (QDH) catalyzes the reversible oxidation of quinate to 3-dehydroquinate by nicotineamide adenine dinucleotide (NADH) and is involved in the catabolic quinate metabolism required for the degradation of lignin. The enzyme is a member of the family of shikimate/quinate dehydrogenases (SDH/QDH) occurring in bacteria and plants. We characterized the dual-substrate quinate/shikimate dehydrogenase (QSDH) from Corynebacterium glutamicum (CglQSDH) kinetically and revealed a clear substrate preference of CglQSDH for quinate compared with shikimate both at the pH optimum and in a physiological pH range, which is a remarkable contrast to closely related SDH/QDH enzymes. With respect to the cosubstrate, CglQSDH is strictly NAD(H) dependent. These substrate and cosubstrate profiles correlate well with the details of three atomic resolution crystal structures of CglQSDH in different functional states we report here: with bound NAD+ (binary complex) and as ternary complexes with NADH plus either shikimate or quinate. The CglQSDH-NADH-quinate structure is the first complex structure of any member of the SDH/QDH family with quinate. Based on this novel structural information and systematic sequence and structure comparisons with closely related enzymes, we can explain the strict NAD(H) dependency of CglQSDH as well as its discrimination between shikimate and quinate.

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Section: Structure Overview Of C Glutamicum Qsdhsupporting

confidence: 76%

Enzyme-substrate complexes of the quinate/shikimate dehydrogenase from Corynebacterium glutamicum enable new insights in substrate and cofactor binding, specificity, and discrimination

Höppner¹,

Schomburg²,

Niefind

2013

Biological Chemistry

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“…The open reading frame Cgl0424 (nomenclature by Ikeda & Nakagawa, 2003) was cloned and overexpressed. The gene product was purified and crystallized and a native X-ray data set was collected as previously described (Schoepe et al, 2006).…”

Section: Methodsmentioning

confidence: 99%

1.6 Å structure of an NAD⁺-dependent quinate dehydrogenase fromCorynebacterium glutamicum

Schoepe

Niefind

Schomburg

2008

Acta Crystallogr D Biol Cryst

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To date, three different functional classes of bacterial shikimate/quinate dehydrogenases have been identified and are referred to as AroE, SDH-L and YdiB. The enzyme AroE and the catalytically much slower SDH-L clearly prefer NADP+/NADPH as the cosubstrate and are specific for (dehydro-)shikimate, whereas in YdiB the differences in affinity for NADP+/NADPH versus NAD+/NADH as well as for (dehydro-)shikimate versus (dehydro-)quinate are marginal. These three subclasses have a similar three-dimensional fold and hence all belong to the same structural class of proteins. In this paper, the crystal structure of an enzyme from Corynebacterium glutamicum is presented that clearly prefers NAD+ as a cosubstrate and that demonstrates a higher catalytic efficiency for quinate rather than shikimate. While the kinetic constants for this enzyme clearly differ from those reported for AroE, SDH-L and YdiB, the three-dimensional structure of this protein is similar to members of these three subclasses. Thus, the enzyme described here belongs to a new functional class of the shikimate/quinate dehydrogenase family. The different substrate and cosubstrate specificities of this enzyme relative to all other known bacterial shikimate/quinate dehydrogenases are discussed by means of analyzing the crystal structure and derived models. It is proposed that in contrast to shikimate, quinate forms a hydrogen bond to the NAD+. In addition, it is suggested that the hydroxyl group of a conserved active-site threonine hydrogen bonds to quinate more effectively than to shikimate. Also, the hydroxyl group of a conserved tyrosine approaches the carboxylate group of quinate more closely than it does the carboxylate group of shikimate. Taken together, these factors most likely lead to a lower Michaelis constant and therefore to a higher catalytic efficiency for quinate. The active site of the dehydrogenase reported here is larger than those of other known shikimate/quinate dehydrogenases, which may explain why quinate is easily accommodated within the catalytic cleft.

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“…Examples of prosperous structure determination however are the shikimate dehydrogenase (SDH or AroE) of Thermus thermophilus (TthSDH), Aquifex aeolicus (AaeSDH) and the recently deposited structures of the SDH of Helicobacter pylori (HpySDH) as well as the bifunctional dehydroquinase-shikimate dehydrogenase (AthDHQ-SDH) from Arabidopis thaliana which were crystallized with its reaction product shikimic acid ( [40][41][42][43]. Similar to that the closely related quinate dehydrogenase (QDH) of Corynebacterium glutamicum (CglQDH) was structurally characterized in four different states: as apo-enzyme and at atomic resolution with bound cofactor NAD + as well as in complex with quinic acid (QA) and the reduced cofactor or shikimic acid (SA) and NADH [44].…”

Section: Enzymatic Products In Protein Structures -How To Crystallize...mentioning

confidence: 99%

Proteins and Their Ligands: Their Importance and How to Crystallize Them

Hoeppner¹,

Schmitt²,

Smits

2013

Advanced Topics on Crystal Growth

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usage of catalytically inactive mutants with bound ligand(s)• creation of an environment (i. g. buffer condition) which shifts the equilibrium constant so that the reaction cannot occurThe most important point concerning preparing co-crystallization trials is the knowledge of the corresponding kinetical parameters. Proteins bind their natural ligand(s) with high affinity, which means in the nM-up to low mM range. To successfully crystallize a protein with the ligand(s) bound, the affinity needs to be determined. There are numerous biophysical techniques to achieve this, for example Intrinsic Tryptophan Fluorescence, Isothermal Calorimetry, Surface Plasmon Resonance and many others. In principle the affinity is determined by the size of a ligand as well as the property of the binding site of the protein. As first approximation, one can state that affinity increases with a decrease in ligand size.

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Cloning, expression, purification and preliminary crystallographic characterization of a shikimate dehydrogenase fromCorynebacterium glutamicum

Cited by 5 publications

References 15 publications

Enzyme-substrate complexes of the quinate/shikimate dehydrogenase from Corynebacterium glutamicum enable new insights in substrate and cofactor binding, specificity, and discrimination

Enzyme-substrate complexes of the quinate/shikimate dehydrogenase from Corynebacterium glutamicum enable new insights in substrate and cofactor binding, specificity, and discrimination

1.6 Å structure of an NAD⁺-dependent quinate dehydrogenase fromCorynebacterium glutamicum

Proteins and Their Ligands: Their Importance and How to Crystallize Them

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Cloning, expression, purification and preliminary crystallographic characterization of a shikimate dehydrogenase fromCorynebacterium glutamicum

Cited by 5 publications

References 15 publications

Enzyme-substrate complexes of the quinate/shikimate dehydrogenase from Corynebacterium glutamicum enable new insights in substrate and cofactor binding, specificity, and discrimination

Enzyme-substrate complexes of the quinate/shikimate dehydrogenase from Corynebacterium glutamicum enable new insights in substrate and cofactor binding, specificity, and discrimination

1.6 Å structure of an NAD+-dependent quinate dehydrogenase fromCorynebacterium glutamicum

Proteins and Their Ligands: Their Importance and How to Crystallize Them

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1.6 Å structure of an NAD⁺-dependent quinate dehydrogenase fromCorynebacterium glutamicum