Difference between revisions of "Part:BBa K1640019"

Revision as of 01:59, 19 September 2015

ChlH

Sequence and Features

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

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

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.