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1vl6

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Table of contents
  1. 1. Protein Summary
  2. 2. Ligand Summary
  3. 3. References

Title Crystal structure of NAD-dependent malic enzyme (TM0542) from Thermotoga maritima at 2.61 A resolution. To be published
Site JCSG
PDB Id 1vl6 Target Id 282415
Molecular Characteristics
Source Thermotoga maritima msb8
Alias Ids TPS1211,TM0542, 84943 Molecular Weight 41041.13 Da.
Residues 376 Isoelectric Point 5.32
Sequence mdaleihrflkgkirtalpvekvdretlsllytpgvadvaracaedpektyvytsrwntvavvsdgsav lglgnigpygalpvmegkaflfkafadidafpiclseseeekiisivkslepsfgginledigapkcfr ilqrlseemnipvfhddqqgtavvvsaaflnalkltekkieevkvvvngigaagynivkflldlgvknv vavdrkgilnendpetclneyhleiaritnperlsgdletalegadffigvsrgnilkpewikkmsrkp vifalanpvpeidpelareagafivatgrsdhpnqvnnllafpgimkgavekrskitknmllsaveaia rscepeperiipeafdmkvhlnvytavkgsa
  BLAST   FFAS

Structure Determination
Method XRAY Chains 1
Resolution (Å) 2.61 Rfree 0.23929
Matthews' coefficent 2.85 Rfactor 0.19189
Waters 44 Solvent Content 56.87

Pathway

Reactions found in Metabolic Reconstruction for TM0542

Name: malic enzyme (NAD)
Metabolic Subsystem: Pyruvate Met
Reaction: : mal-L + nad --> co2 + nadh + pyr
Classification: EC:1.1.1.38
 

Ligand Information
Ligands
Metals

Jmol

 
Google Scholar output for 1vl6
1. The Buccaneer software for automated model building. 1. Tracing protein chains
K Cowtan - Acta Crystallographica Section D: Biological , 2006 - scripts.iucr.org
 

Protein Summary

Malate oxidoreductase (malic enzyme, TM0542) from Thermotoga maritima catalyzes the oxidative decarboxylation of L-malate to pyruvate with the concomitant reduction of the cofactor NAD+ or NADP+. N-terminal domian belongs to Pfam00390, while C-terminal domain to Pfam03949).

TM0542 has two structural domains: N-terminal domain with aminoacid dehydrogenase-like fold (SCOP sunid:53222) core: 3 layers: ?/?/?; parallel ?-sheet of 4 strands; this domain is decorated with additional structures in family of malic enzyme N-terminal domain (SCOP sunid:53240). C-terminal domain has NAD(P)-binding Rossmann-fold (SCOP sunid:51734) core: 3 layers, ?/?/?; parallel ?-sheet of 6 strands, order 321456. C-terminal domain belongs to aminoacid dehydrogenase-like family (SCOP sunid:51883) with extra N-terminal helix displacing the C-terminal helix (following strand 6) from its usual position creating a family nicotineamide-binding site.

TM0542 has strong similarity to sequence of proteins from COG0281 (SfcA, malic enzymes) and to several structures of know or predicted malic enzymes (i.e.:2haeA, 1ww8A, 2a9fA, 2aw5A, 1efkA, 1llqA, and 1gq2A).


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Functional Assay

Based on structure and sequence similarities, TM0542 is hypothesized to be an NAD(P)-dependent malic enzyme (ME), which converts (S)-malate to pyruvate. Using a direct assay, TM0542 demonstrated malic enzyme activity (Figure 1 and Table 1). The decrease in activity at concentrations above 5 mM malate suggests substrate inhibition. In terms of turnover, TM0542 did not demonstrate selectivity for cofactor (NAD+ vs. NADP).

Structural and sequence comparisons indicate that TM0542 is highly similar to other bacterial MEs (PDB IDs: 2DVM, 2HAE), yet there is no kinetic data for these other bacterial enzymes with which to draw a meaningful activity based comparison. TM0542 is also similar to human NAD(P) ME (PDB ID: 1PJ3), which is well-characterized and provides data for comparison to TM0542 (Table 1). The Km values for malate and NAD are 100-fold lower for TM0542 compared to human ME1,2; however, the human ME turnover values and overall catalytic efficiencies are many orders of magnitude higher than that for TM0542, indicating that TM0542 is far less efficient than the human enzyme. However, for direct comparison, the kinetic parameters should be determined with Mg2+ instead of Mn2+ at pH 7.4.

Table 1: Comparison of TM0542 and human ME kinetic parameters

 

malate

NAD+

NADP

 

Km (mM)

kcat

(s-1)

kcat/Km (mM-1s-1)

Km (mM)

kcat

(s-1)

kcat/Km (mM-1s-1)

Km (mM)

kcat

(s-1)

TM0542*

1.2

0.013

0.011

0.31

0.0077

0.025

ND

0.0046

Human ME#

131

1301

101

0.572

1202

2102

1.62

6.72

*50 mM Tris (pH 8.0), 1 mM EDTA and 5 mM MnCl2; #50 mM Tris (pH 7.4) and 10 mM MgCl2, Not determined due to sigmoidal rather than hyperbolic dependence of activity on NADP concentration

 

Additional Findings

Thetm0542 gene is predicted to be part of a three-gene transcriptional unit (Fig 2). The other two genes, tm0540 and tm0541, encode the N- and C-terminal subunits of fumarate hydratase, respectively. Fumarate hydratase converts (S)-malate to fumarate (and the reverse reaction, when required by metabolic flux). In human ME, a fumarate-binding site that acts as an allosteric activator is present at one of the dimeric interfaces. Although TM0542 and human ME share high structural and sequence similarity, the N-terminal dimer interface and fumarate binding site (consisting of Q64, R67, R91) are not conserved. Attempts at identifying a potential binding site through structural alignments and manual inspection of the dimer interface for the binding motif did not yield promising candidates. Thus, there is no structural evidence for a fumarate allosteric site in TM0542.

 

BioLEd Contributors: Joseph Breheny, Kanishk Jain, Michael Billet, John Bui, Huy Do, Golda Harris, Jung Woo Hong, Joo Ho Kim, Cameron Mura, Carol Price, Linda Columbus. Funded by NSF DUE 1044858.

References

1 Hung HC, Kuo MW, Chang GG, Liu GY. Characterization of the functional role of allosteric site residue Asp102 in the regulatory mechanism of human mitochondrial NAD(P)+-dependent malate dehydrogenase (malic enzyme). Biochem J. 2005 392:39-45.
 2Hsieh JY, Liu GY, Chang GG, Hung HC. Determinants of the dual cofactor specificity and substrate cooperativity of the human mitochondrial NAD(P)+-dependent malic enzyme: functional roles of glutamine 362. J Biol Chem. 2006 281:23237-45.

Ligand Summary



References

Reviews

References

 

No references found.

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Files (2)

FileSizeDateAttached by 
 Figure 1.png
Michaelis-Menten Kinetics of TM0542
39.51 kB15:47, 24 Jan 2012kj3kvActions
 Figure2.png
Transcriptional Unit
20.81 kB15:48, 24 Jan 2012kj3kvActions
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