Cloning, sequence analysis, and overexpression of Escherichia coli folK, the gene coding for 7,8-dihydro-6-hydroxymethylpterin-pyrophosphokinase

Abstract

The gene coding for the Escherichia coli enzyme 7,8-dihydro-6-hydroxymethylpterin-pyrophosphokinase has been cloned and sequenced. This gene, designated folK, codes for a protein of 159 amino acids, including an amino-terminal methionine. The protein was overexpressed in E. coli MC4100 by cloning the gene behind the lacUVS promoter in a high-copy-number plasmid. The enzyme was purified to homogeneity. Amino-terminal analysis of the purified protein showed that the amino-terminal methionine had been removed. The compositional molecular mass (17,945 Da) was identical to the molecular mass determined by mass spectrometry. The enzyme was observed to have a large number of proline residues and migrated anomalously in sodium dodecyl sulfate-polyacrylamide gels, with an apparent molecular mass of 23,000 Da.

2.0 Å X‐ray structure of the ternary complex of 7,8‐dihydro‐6‐hydroxymethylpterinpyrophosphokinase from Escherichia coli with ATP and a substrate analogue

“…4. The pterin-based hydrogen bond interactions as well as the hydrophobic interactions are similar to those described for Table 2 Comparison of active site residues for E. coli PPPK (Ec) [3] and other PPPKs…”

“…The cloning, expression and puri¢cation of E. coli PPPK have been described previously [3]. The enzyme was crystallised under a number of di¡erent conditions.…”

“…PPPK from Escherichia coli is a monomeric 158 residue protein [3]. Amongst the PPPKs cloned and sequenced from both prokaryotic and eukaryotic organisms [3^7], there is an overall sequence conservation of approximately 30%, indicating considerable similarity of three-dimensional structures.…”

A cluster of four genes encoding enzymes for five steps in the folate biosynthetic pathway of Streptococcus pneumoniae

“…Inasmuch as one of the protein products, SulD, carries two of these activities, it is conceivable that one of the other proteins may harbor another activity, such as the phosphatase needed to convert the product of GTPCH action to dihydroneopterin. The syncretion of two activities in SulD that are coded for as separate polypeptides in B. subtilis (37) and E. coli (40) is carried even further in the case of P. carinii, in which the polypeptide product of a single gene, fas, also contains a DHPS region and an additional segment of unknown function in addition to the two activities of SulD (44). It was shown that the fas product can express its multiple activities, like SulD, without prior cleavage (43).…”

“…On the basis of these findings, putative genes encoding DHPS, PPPK, and DHNA were identified in Bacillus subtilis (37) and Pneumocystis carinii (43,44). Later, genes for PPPK and DHPS were independently identified in E. coli (7,8,40).…”