Molecular cloning and functional analysis of (R)-3-hydroxyacyl-acyl carrier protein:coenzyme A transacylase from Pseudomonas mendocina LZ.
An inactive (R)-3-hydroxyacyl-acyl carrier protein:coenzyme A transacylase (PhaG(Pm)) was cloned from a newly isolated Proteobacteria Pseudomonas mendocina LZ. It is the first characterized native inactive PhaG protein. Sequence analysis indicated that there were only two sites where the amino acid sequence differed between this inactive protein and the functional PhaG(Pp) from P. putida. The differences were located at position 78 and in the region 109-113 in the amino acid sequence. Mutagenesis was carried out to investigate these two sites. A recombinant strain harboring a S78C PhaG(Pp) mutant accumulated polyhydroxyalkanoates (PHA) at 11.9% of the cellular dry weight, as compared to the 21.6% PHA produced by the recombinant harboring the wild-type PhaG(Pp). On the other hand, the changes in the amino acid region 109-113 of PhaG(Pp) to its corresponding region of PhaG(Pm) resulted in negligible PHA accumulation. This demonstrated that region 109-113 in PhaG is relatively important for transacylase activity, while position 78 just plays a supporting role for the enzyme. Furthermore, 3-D structural models of PhaG(Pp) and PhaG(Pm) developed by computational prediction revealed that the variation in amino acids at 109-113 leads to the destruction of the PhaG catalytic center, resulting in the loss of enzyme activity.
Zheng
Leo Zhong
LZ
MOE laboratory of Protein Science, Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing, China.
Li
Zhi
Z
Tian
Hong-Lei
HL
Li
Ming
M
Chen
Guo-Qiang
GQ
D000217:Acyltransferases / Q000737:chemistry* / Q000235:genetics*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000235:genetics; D020134:Catalytic Domain; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D005798:Genes, Bacterial; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D011487:Protein Conformation; D044203:Pseudomonas mendocina / Q000201:enzymology* / Q000235:genetics; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D000217:Acyltransferases; C114682:3-hydroxyacyl-CoA-acyl carrier protein transferase