Part:BBa_K1640019
ChlH
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 856
Illegal BglII site found at 1606
Illegal BglII site found at 2229
Illegal BglII site found at 2308
Illegal BglII site found at 3615 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1150
Illegal AgeI site found at 35
Illegal AgeI site found at 65
Illegal AgeI site found at 959
Illegal AgeI site found at 1127
Illegal AgeI site found at 2645
Illegal AgeI site found at 2699
Illegal AgeI site found at 2918 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 3044
Illegal SapI.rc site found at 401
Illegal SapI.rc site found at 2974
Overview
Biology & Literature
ChlH is the catalytic subunit of Magnesium chelatase. This oligomeric enzyme initiates the first committed step of the chlorophyll-a biosynthesis pathway via insertion of an Mg2+ ion into protoporphyrin IX to generate Mg-protoporphyrin IX. Specifically, ChlH is the subunit known to bind porphyrin, and potentially also the Mg2+ ion. During this process, ChlH interacts with two AAA ATPase-like subunits of Mg-chelatase (ChlI and ChlD) to catalyse the ATP-dependent insertion of Mg2+ into protoporphyrin IX (Adhikari et al., 2011).
The 4166 bp ChlH gene was engineered synthetically by Integrated DNA Technologies (IDT) in 3 gene blocks (Table 1). The original gene sequence was taken from Chlamydomonas Reinhardtii and subsequently codon optimized for expression in Escherichia coli. Integrity of the protein sequence was closely maintained throughout this optimisation process, but translation of the original clone and the synthesised sequences has revealed one mutation (‘E’ → ‘D’; ‘GAG’ → ‘GAT’).
Table 1: Gene blocks
| 1(G13) | 1678 bp |
| 2 (P2) | 980 bp |
| 3 (3-6) | 1508 bp |
ChlH and the pSB1C3_001 KAN plasmid were successfully assembled in two parts.
1. Assembled G13, the CAM vector and 3 - 6 via double restriction digest with EcoRI and EcoRI + PstI and ligation reaction
2. Cloned P2 into the vector with the other parts via Gibson Assembly and then performed a restriction digest (EcoRI and EcoRI + PstI) on the assembly product to check for correct assembly (Figure 1).
Protein information
Magnesium chelatase sits at the branch point of the common tetrapyrrole pathway and inserts Mg2+ into Proto to produce Mg-Proto, the first unique intermediate of the chlorophyll biosynthetic pathway. It is known that the BchH/ChlH subunit binds the substrate and, for this reason, is thought to be the catalytic component of the enzyme. The ChlH subunit makes conformational changes upon binding its porphyrin substrate.
A study done on Rhodobacter capsulatus has demonstrated the apo structure to contain three major lobe-shaped domains connected at a single point, with additional densities at the tip of two lobes termed the “thumb” and “finger” (figure: 2). This independent reconstruction of a substrate-bound ChlH complex permitted insight into substrate-induced conformational changes (Sirijovski et al., 2008).
Number of amino acids: 1378
Molecular weight: 152265.2
Theoretical pI: 5.34
Amino acid composition:
Ala (A) 117 8.5%
Arg (R) 70 5.1%
Asn (N) 79 5.7%
Asp (D) 82 6.0%
Cys (C) 13 0.9%
Gln (Q) 46 3.3%
Glu (E) 94 6.8%
Gly (G) 101 7.3%
His (H) 15 1.1%
Ile (I) 52 3.8%
Leu (L) 142 10.3%
Lys (K) 79 5.7%
Met (M) 35 2.5%
Phe (F) 51 3.7%
Pro (P) 70 5.1%
Ser (S) 85 6.2%
Thr (T) 69 5.0%
Trp (W) 14 1.0%
Tyr (Y) 49 3.6%
Val (V) 115 8.3%
Pyl (O) 0 0.0%
Sec (U) 0 0.0%
(B) 0 0.0% (Z) 0 0.0% (X) 0 0.0%
Total number of negatively charged residues (Asp + Glu): 176
Total number of positively charged residues (Arg + Lys): 149
Atomic composition:
Carbon C 6769
Hydrogen H 10680
Nitrogen N 1836
Oxygen O 2059
Sulfur S 48
Formula: C6769H10680N1836O2059S48
Total number of atoms: 21392
Extinction coefficients:
Extinction coefficients are in units of M-1 cm-1, at 280 nm measured in water.
Ext. coefficient 150760
Abs 0.1% (=1 g/l) 0.990, assuming all pairs of Cys residues form cystines
Ext. coefficient 150010
Abs 0.1% (=1 g/l) 0.985, assuming all Cys residues are reduced
Estimated half-life:
The N-terminal of the sequence considered is L (Leu).
The estimated half-life is: 5.5 hours (mammalian reticulocytes, in vitro).
3 min (yeast, in vivo).
2 min (Escherichia coli, in vivo).
Instability index:
The instability index (II) is computed to be 33.06
This classifies the protein as stable.
Aliphatic index: 87.60
Grand average of hydropathicity (GRAVY): -0.262
References
Adhikari, N.D., Froehlich, J.E., Strand, D.D., Buck, S.M., Kramer, D.M., Larkin, R.M. (2011) GUN4-Porphyrin Complexes Bind the ChlH/GUN5 Subunit of Mg-Chelatase and Promote Chlorophyll Biosynthesis in Arabidopsis. Plant Cell 23: 1449-1467.
Chen, X., Pu, H., Fang, Y., Wang, X., Zhao, S., Lin, Y., Zhang, M., Dai, H-E., Gong, W., Liu, L. (2015). Crystal Structure of the catalytic subunit of magnesium chelatase. Nature Plants, 1, doi:10.1038/nplants.2015.125.
Sirijovski, N., Lunqvist, J., Rosenback, M., Elmlund, H., Al-Karadaghi, S., Willows, R.D., Hansson, M. (2008). Substrate-binding Model of the Chlorophyll Biosynthetic Magnesium Chelatase BchH Subunit. Journal of Biological Chemistry, 283, 11652-11660.
| None |