Part:BBa_K1080000

ChlI1
Magnesium chelatase subunit I - Catalyzes the insertion of magnesium ion into protoporphyrin IX to yield Mg-protoporphyrin IX. Forms an ATP dependent hexameric ring complex and a complex with the ChlD subunit. Transcript is light regulated and may be diurnal and/or circadian.

Usage and Biology

In the early stages of chlorophyll biosynthesis is the chelation of magnesium into the protoprophyrin-IX complex. The ATP hydrolysis by CHLI subunit of magnesium chelatase is essential for the insertion of Mg. CHLI1 is an isoform of CHLI that encodes the . It has the action of an ATPase and is reduced by thioredoxin. Magnesium chelatase is sensitive to thiol groups and it has been found that the insertion of Mg2+ increases as CHLI1 is reduced [1].

Figure 1: Reaction catalysed by Mg-chelatase, inserting a magnesium ion into Protoporphyrin IX to make Mg-protoporphyrin IX.

Reaction: ATP + protoporphyrin IX + Mg2+ + H2O = ADP + phosphate + Mg-protoporphyrin IX + 2 H+.

Operon usage

Figure 2: This gene has been used in an operon with other genes responsible for the synthesis pathway from Protoporphyrin IX to MG-protoporphyrin IX. ChlI1 and ChlD form an Mg-chelatase complex, which acts upon protoporphyrin, catalysing the insertion of a Magnesium ion into the centre of the protoporphyrin IX. GUN4 aids this reaction by activating the Mg-chelatase enzyme complex.
The plasmid is under the control of the lac promoter.
The entire operon can be found at BBa_K1326008.
Future plans involve adding ChlH to this operon, which binds Mg-chelatase with GUN4 to protoporphyrin.

Biobrick Design:

Source Genbank accession: [http://www.ncbi.nlm.nih.gov/nuccore/NW_001843537.1?report=genbank&from=707985&to=715043 NW_001843537.1]

Source Uniprot reference: [http://www.uniprot.org/uniprot/A8IMZ5 A8IMZ5]

cDNA gene sequence from Chlamydomonas reinhardtii was sourced from NCBI database, chloroplast targeting sequence was removed. EcoRI/XbaI/SpeI/PstI restriction sites were removed via codon adjustment, biobrick prefix and RBS were added to start of gene, biobrick suffix added to end of gene.

Biobrick construction: Gibson assembly of 2 synthesised DNA fragments into BB vector.

Protein Structure

Figure 3: ChlI protein crystal structure.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 231
Illegal BglII site found at 887
Illegal BglII site found at 1082
Illegal BamHI site found at 279
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

Experimental validation

Figure 4 SDS-PAGE gel showing expression of Plastocyanin and ChlI1 protein.

Figure 5 Western blot of Plastocyanin and ChlI1. Florescence in the last 5 lanes confirms expression of ChlI1.

Amino Acid Sequence

MAATEVKAAE GRTEKELGQA RPIFPFTAIV GQDEMKLALI LNVIDPKIGG VMIMGDRGTG KSTTIRALAD LLPEMQVVAN DPFNSDPTDP ELMSEEVRNR
VKAGEQLPVS SKKIPMVDLP LGATEDRVCG TIDIEKALTE GVKAFEPGLL AKANRGILYV DEVNLLDDHL VDVLLDSAAS GWNTVEREGI SISHPARFIL
VGSGNPEEGE LRPQLLDRFG MHAQIGTVKD PRLRVQIVSQ RSTFDENPAA FRKDYEAGQM ALTQRIVDAR KLLKQGEVNY DFRVKISQIC SDLNVDGIRG
DIVTNRAAKA LAAFEGRTEV TPEDIYRVIP LCLRHRLRKD PLAEIDDGDR VREIFKQVFG ME

References and documentation are available. Please note the modified algorithm for extinction coefficient.

Number of amino acids: 362

Molecular weight: 39952.7

Theoretical pI: 5.11

Amino acid composition:
Ala (A) 31 8.6%
Arg (R) 28 7.7%
Asn (N) 12 3.3%
Asp (D) 29 8.0%
Cys (C) 3 0.8%
Gln (Q) 13 3.6%
Glu (E) 29 8.0%
Gly (G) 28 7.7%
His (H) 4 1.1%
Ile (I) 25 6.9%
Leu (L) 34 9.4%
Lys (K) 19 5.2%
Met (M) 10 2.8%
Phe (F) 12 3.3%
Pro (P) 19 5.2%
Ser (S) 14 3.9%
Thr (T) 17 4.7%
Trp (W) 1 0.3%
Tyr (Y) 4 1.1%
Val (V) 30 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): 58 Total number of positively charged residues (Arg + Lys): 47

Atomic composition:

Carbon C 1752 Hydrogen H 2857 Nitrogen N 499 Oxygen O 539 Sulfur S 13

Formula: C1752H2857N499O539S13 Total number of atoms: 5660

Extinction coefficients:

Extinction coefficients are in units of M-1 cm-1, at 280 nm measured in water.

Ext. coefficient 11585 Abs 0.1% (=1 g/l) 0.290, assuming all pairs of Cys residues form cystines

Ext. coefficient 11460 Abs 0.1% (=1 g/l) 0.287, assuming all Cys residues are reduced

Estimated half-life:

The N-terminal of the sequence considered is M (Met).

The estimated half-life is:

                            30 hours (mammalian reticulocytes, in vitro).
                           >20 hours (yeast, in vivo).
                           >10 hours (Escherichia coli, in vivo).

Instability index:

The instability index (II) is computed to be 29.73 This classifies the protein as stable.

Aliphatic index: 96.16

Grand average of hydropathicity (GRAVY): -0.250

Source

Chlamydomonas reinhardtii